U.S. patent application number 13/075039 was filed with the patent office on 2011-07-21 for implantable tissue compositions and method.
This patent application is currently assigned to Pathak Holdings, LLC. Invention is credited to Chandraskekhar P. Pathak, Sanjav M. Thigle.
Application Number | 20110177150 13/075039 |
Document ID | / |
Family ID | 36000568 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110177150 |
Kind Code |
A1 |
Pathak; Chandraskekhar P. ;
et al. |
July 21, 2011 |
IMPLANTABLE TISSUE COMPOSITIONS AND METHOD
Abstract
Novel implantable tissue fixation methods and compositions are
disclosed. Methods and compositions of tissue, fixed using
polymeric and/or variable length crosslinks, and di- or
polymercapto compounds are described. Also described are the
methods and compositions wherein the tissue is fixed using
biodegradable crosslinkers. Methods and compositions for making
radio-opaque tissue are also described. Methods and compositions to
obtain a degradable implantable tissue-synthetic biodegradable
polymer composite are also described. Compositions and methods of
incorporating substantially water-insoluble bioactive compounds in
the implantable tissue are also disclosed. The use of membrane-like
implantable tissue to make an implantable drug delivery patch are
also disclosed. Also described are the compositions and methods to
obtain a coated implantable tissue. Medical applications
implantable tissue such as heart valve bioprosthesis, vascular
grafts, meniscus implant, drug delivery patch are also
disclosed.
Inventors: |
Pathak; Chandraskekhar P.;
(Phoenix, AZ) ; Thigle; Sanjav M.; (Maharashtra,
IN) |
Assignee: |
Pathak Holdings, LLC
|
Family ID: |
36000568 |
Appl. No.: |
13/075039 |
Filed: |
March 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11661062 |
Feb 22, 2007 |
7919112 |
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PCT/US05/30187 |
Aug 24, 2005 |
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13075039 |
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60604737 |
Aug 26, 2004 |
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Current U.S.
Class: |
424/422 ;
424/569; 427/2.24 |
Current CPC
Class: |
A61P 29/00 20180101;
A61L 27/58 20130101; A61L 27/34 20130101; A61L 27/3687 20130101;
A61L 2420/08 20130101; A61L 27/3604 20130101; A61L 2300/608
20130101; A61L 2430/34 20130101; A61L 27/3608 20130101; A61L 27/18
20130101; A61L 27/3641 20130101; A61L 27/38 20130101; A61L 27/3612
20130101; A61L 27/52 20130101; A61L 2430/20 20130101; A61P 43/00
20180101; A61L 2300/406 20130101; A61L 27/54 20130101; A61L
2300/604 20130101; A61P 21/02 20180101; A61K 35/12 20130101; A61L
2400/18 20130101; A61L 2420/02 20130101; A61L 27/16 20130101; A61L
2300/404 20130101; A61P 25/24 20180101; A61P 3/00 20180101; A61L
2430/40 20130101; A61P 35/00 20180101; A61L 27/24 20130101; A61L
27/3633 20130101; A61L 27/3683 20130101; A61L 2300/606 20130101;
A61P 25/04 20180101; A61L 27/3625 20130101; A61P 31/00
20180101 |
Class at
Publication: |
424/422 ;
424/569; 427/2.24 |
International
Class: |
A61F 2/00 20060101
A61F002/00; A61K 35/34 20060101 A61K035/34; A61P 43/00 20060101
A61P043/00; A61P 31/00 20060101 A61P031/00; A61P 35/00 20060101
A61P035/00; A61P 3/00 20060101 A61P003/00; A61P 29/00 20060101
A61P029/00; A61P 21/02 20060101 A61P021/02; A61P 25/24 20060101
A61P025/24; A61P 25/04 20060101 A61P025/04; A61L 33/00 20060101
A61L033/00 |
Claims
1. A bioprosthetic article comprising an implantable tissue coated
with a composition selected from a group consisting of covalently
crosslinked hydrogel and non-polymeric coating materials.
2. The article of claim 1 wherein the implantable tissue, before
coating, is chosen from the group consisting of decellularized
tissue, chemically modified tissue, and crosslinked tissue.
3. The article of claim 2 wherein the crosslinked tissue is
obtained by a process chosen from the group consisting of
glutaraldehyde crosslinking, ethyl-3-(3-dimethylaminopropyl
carbodiimide (EDC) crosslinking, dye mediated photooxidation
crosslinking, and hexamethylene diisocyanate crosslinking.
4. The article of claim 1 wherein the hydrogel comprises polymers
formed on the tissue by condensation polymerization.
5. The article of claim 1 wherein the hydrogel comprises a reaction
product of reactive precursor species that comprise nucleophilic
functional groups and reactive precursor species that comprise
electrophilic functional groups.
6. The article of claim 1 wherein the hydrogel comprises polymers
formed on the tissue by free radical polymerization.
7. The article of claim 6 wherein the hydrogel comprises a polymer
polymerized from a monomer chosen from the group consisting of
acrylates, methacrylates, acrylamides, methacrylamide vinyl ethers,
and n-vinyl pyrrolidinone.
8. The article of claim 1 wherein the hydrogel is
biodegradable.
9. The article of claim 8 wherein the hydrogel comprises a polymer
polymerized from a monomer chosen from the group consisting of
lactide, glycolide, and caprolactone.
10. The article of claim 1 wherein the hydrogel is formed by
crosslinking and polymerization of polyethylene glycol based water
soluble macromonomers.
11. The article of claim 1 wherein the tissue is has two opposing
sides joined by a thickness, with the coating being on each of the
two opposing sides.
12. The article of claim 1 wherein the coating has a thickness of
between about 5 micron to about 2 nm thick.
13. The article of claim 1 wherein the tissue comprises bovine
pericardium, porcine pericardium, equine pericardium, porcine
submucosa arterial tissue, vein tissue, ureter tissue, corneal
tissue, an aortic root, meniscus tissue, ligaments, basal membrane,
bladder tissue, tendons, cartilage, heart valve tissue,
demineralized bone, placenta, umbilical cord tissue, skin tissue,
cadaver tissue, and dura mater.
14. The article of claim 1 further comprising a drug for release
after implantation.
15. The article of claim 14 wherein a drug is selected from the
group consisting of antiviral agents; antiinfectives, antibiotics;
antipruritics; antipsychotics; cholesterol- or lipid-reducing
agents; cell cycle inhibitors; anticancer agents; antiparkinsonism
drugs; HMG-CoA inhibitors; antirestenosis agents; antiinflammatory
agents; antiasthmatic agents; anthelmintic; immunosuppressives;
muscle relaxants; antidiuretic agents; vasodilators; nitric oxide;
nitric oxide-releasing compounds; beta-blockers; hormones;
antidepressants; decongestants; calcium channel blockers; growth
factors, bone growth factors, bone morphogenic proteins; wound
healing agents; analgesics and analgesic combinations; local
anesthetic agents; antihistamines; sedatives;
angiogenesis-promoting agents; angiogenesis-inhibiting agents; and
tranquilizers.
16. The article of claim 1 wherein the tissue comprises a groove or
hole, wherein said groove and/or said hole comprises a drug
delivery composition.
17. The article of claim 16 wherein the drug delivery composition
comprises a member chosen from the group consisting of a
bioerodible polymer, a biodegradable polymer, a solid drug matrix,
and a liquid drug.
18. The article of claim 1 wherein the coating coats only a portion
of the tissue.
19. A process of preparing a bioprosthetic article comprising
applying monomers or polymers to a surface of an implantable tissue
and reacting the monomers or polymers on the surface to form a
hydrogel.
20. The process of claim 19 wherein the implantable tissue, before
coating, is chosen from the group consisting of decellularized
tissue, chemically modified tissue and crosslinked tissue.
21. The process of claim 19 wherein the reaction comprises
condensation polymerization of the polymers.
22. The process of claim 19 wherein the reaction comprises free
radical polymerization of the monomers.
23. The process of claim 19 wherein the hydrogel is
biodegradable.
24. The process of claim 19 wherein the hydrogel is formed by
crosslinking and polymerization of polyethylene glycol based water
soluble macromonomers.
25. The article of claim 1 wherein the non-polymeric material is
solid or liquid.
26. The article of claim 1 wherein the non-polymeric material is
selected from the group consisting of bone wax, fatty acids,
stearic acid. oleic acid, sugar derivatives, sucrose acetate,
mineral oil, peanut oil, cotton seed oil, tocopherol, vitamin E,
vitamin E acetate and polyethylene glycol.
27. A method of treating a patient comprising applying the
bioprosthetic article of claim 1 to the patient for post operative
adhesion prevention or as a hernia patch.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/661,062 filed Feb. 22, 2007 which is a
national stage entry of PCT Application No. PCT/US05/030187 filed
Aug. 24, 2005, which claims benefit of priority to U.S. provisional
application No. 60/604,737, filed Aug. 26, 2004, each of which are
hereby incorporated by reference herein.
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The present invention relates to methods and compositions
for the preparation of biological tissues or extracellular matrices
and their medical applications.
BACKGROUND OF THE INVENTION
[0004] The use of human or animal tissue for medical or surgical
use is a rapidly growing therapeutic field. Many uses of processed
biological tissues for implantation into humans have been reported.
The commercial products or products under development include wound
healing dressings, tissue heart valves, ligament substitutes,
pericardial patches and membranes, vascular grafts and the like.
The use of animal tissue offers an inexpensive source of materials
to fabricate tissue-based medical products. The problems with the
animal tissue transplantation include inflammation, unwanted
degradation, control over the degradation process, calcification,
inability to release bioactive compounds in a controlled manner,
and rejection of the transplanted tissue.
[0005] The primary component of many biological tissues is a
protein called collagen. Collagen-based biomaterials generally
induce a mild inflammatory response, which results in degradation
of the protein. This degradation can be prevented by chemical
modification or crosslinking of tissue proteins and is achieved by
reacting difunctional and polyfunctional crosslinkers capable of
forming irreversible and stable intermolecular chemical
crosslinking between two collagen chains. Chemical crosslinking may
also increase strength and durability of the tissue. Many heart
valve bioprosthesis manufacturers use glutaraldehyde as a
crosslinking agent for stabilization of the bioprosthesis tissue.
The chemistry of glutaraldehyde is complex but well documented.
Glutaraldehyde reacts with free amine groups from lysine residues
on collagen and forms Schiff base addition products. Although
glutaraldehyde is the most commonly used chemical fixative for
biological tissues, there are a number of drawbacks associated with
its use in the production of bioprosthetic devices. For example,
the long term durability of glutaraldehyde-fixed bioprostheses is
not well established. Another drawback to glutaraldehyde fixation
of bioprostheses relates to the release of cytotoxic glutaraldehyde
on the tissue surface thereby hindering the growth of cells,
especially endothelial cells, on the surface of the tissue.
Glutaraldehyde fixed tissue is also susceptible to calcification
which leads to device failure.
[0006] To overcome limitations of glutaraldehyde crosslinking,
other chemical crosslinking agents capable of reacting with amine,
carboxyl and hydroxyl group have been explored. Tissue crosslinking
chemistry has been recently reviewed. However, none of alternative
chemistries have resulted into a commercial clinical heart valve
product. Tissue crosslinking chemistry is challenging due to a
variety of reasons. From the chemistry point of view, the
crosslinking reaction is a heterogeneous reaction, where the
reactant (tissue) is always in a separate phase (solid state phase)
as compared to the crosslinker (solution, oiled or liquid phase).
This limits the accessibility of tissue functional groups for
crosslinking reaction. The solid state nature of tissue also makes
it difficult for large crosslinker molecules such as, by way of
example, and not limitation, polymers to penetrate inside the
tissue matrix and crosslink the reactive sites. Generally, the
crosslinking reaction must be done without denaturing the protein.
The denatured tissue/collagen (gelatin) is more susceptible to
enzymatic degradation and denatured proteins have inferior
mechanical properties as compared to non-denatured tissue. To
prevent denaturing of tissue, the use of aggressive organic
solvents and high temperatures in tissue crosslinking is generally
avoided. It is generally believed that an aqueous medium with
physiological conditions (pH 7.2, 37.degree. C.) is best suited for
tissue crosslinking. Fixation under physiological conditions is
most likely to preserve the natural conformation of proteins
present in the tissue. Glutaraldehyde is one of the few
crosslinking agents capable of reacting with the tissue in water
under physiological conditions.
[0007] In known approaches, most of the tissue crosslinking is
restricted to di- or polyfunctional small compounds such as, by way
of example, and not limitation, glutaraldehyde. Small compounds can
easily penetrate solid tissue matrix and can crosslink surface as
well as bulk components of the tissue matrix. In order for
crosslinking to occur, two or more reactive functional groups must
react with two polymeric chains to form an interchain crosslinked
moiety. Most tissue crosslinkers are single chemical entities and
therefore have fixed molecular length. The fixed length crosslinker
can only react with those sites which are within the close
proximity of its reactive functional groups. Therefore, it cannot
crosslink the tissue if the reactive sites present on the tissue
are at a shorter or longer distance than the length of crosslinker.
Also, during the crosslinking reaction, one of the crosslinking
functional group reacts with the crosslinkable moiety such as, by
way of example, and not limitation, collagen. After the reaction,
the other functional group must react with other reactive site on
the collagen to complete the crosslinking reaction. This often may
not be possible due to limited length and mobility of crosslinker.
This results in a number of dangling bonds with incomplete
crosslinking. Therefore, the length of a crosslinker serves as a
major limitation in achieving a high degree of crosslinking. Thus,
there is a need for tissue crosslinking methods wherein the
crosslinks formed may have variable lengths.
[0008] Polymeric crosslinkers can be useful in crosslinking the
tissue due to high molecular flexibility of polymeric molecular
coil and polymer's ability to impart additional properties to the
tissue matrix. However, polymeric crosslinkers are large molecules
which cannot diffuse/penetrate inside the tissue matrix and react
with sites present in the bulk of the tissue. This limits the
ability of polymeric crosslinker to surface crosslinking only.
Known techniques generally do not teach the successful use of
polymeric crosslinkers in tissue crosslinking. There is a need for
methods and compositions that permit the incorporation and
crosslinking of tissue using polymers or that generate polymeric
crosslinks.
[0009] Shape memory biomaterials have the ability to change to a
predetermined shape when subjected to an appropriate energy
stimulus. Nitinol alloy is one of the well-known shape memory
biomaterials. Many applications of Nitinol materials have been
commercialized. These applications include peripheral vascular
stent and stent grafts, vena cava filters, etc. Bioprosthetic
tissues having shape memory properties can be extremely useful in
making novel medical devices. There is a need for tissue-based
biomaterials that can remember shape maintained during fixation or
stabilization and tissue-based materials with the ability to
remember and recover the shape when deformed by a mechanical
force.
[0010] Unfixed or non-crosslinked animal tissue undergoes enzymatic
degradation when implanted in human/animal body. Usually such
degradation is followed after a moderate to severe inflammatory
response; presumably due to an immunological reaction to the
foreign biological materials in the host body. Non-crosslinked
animal tissue such as, by way of example, and not limitation,
porcine small-intestinal submucosa has been commercialized as a
wound dressing material. In many medical applications, it is
desirable to have a biological degradable tissue with no or little
inflammatory response and control over its degradation profile and
properties. Known techniques generally do not teach methods and
compositions that will affect the degradation behavior of
biological tissue. Therefore, there is also a need for methods and
compositions that can reduce the inflammatory response to the
animal or human tissue. Compositions and methods that will control
the degradation time of the implanted tissue are also needed.
[0011] Animal tissue used in commercial bioprostheses such as heart
valve, vascular graft and vascular patch is limited by tissue
thickness, size and protein (chemical) composition. For example,
bovine pericardium, a widely used animal tissue has a thickness
ranging 1 to 2 mm which may too thick for some medical applications
such as low profile stent graft application. The useful tissue
recovered from one animal is also limited in size. Typical area of
bovine pericardial or porcine pericardial tissue may range from 50
to 150 square inches. This size and thickness limitation may limit
the use of tissue in making large medical device such as tissue
based dialysis catheters. The tissue size limits also increases
production costs due to lower yields. The higher size of
implantable tissue may permit to manufacture more devices per
tissue and reduce manufacturing costs. Therefore there is need for
tissue, especially membrane like tissue, which can be made in wide
ranges of size, thickness and with different chemical compositions
for bioprosthesis applications.
[0012] Synthetic biodegradable polymers have received considerable
interest in the medical and pharmaceutical field at least because
they can perform temporary therapeutic functions and are eliminated
from the body once their therapeutic function has been
accomplished. Some of the well-known applications of biodegradable
polymers include surgical sutures, staples, or other wound closure
devices, as a carrier for bioactive substances for controlled drug
delivery, etc. Several types of biodegradable polymers have been
reported in the subject literature, however, polymers prepared from
hydroxy acids have received much attention due to their
degradability and toxicological safety. Homopolymers and copolymers
based on the l-lactic acid, dl-lactic acid and glycolic acid are
among the most widely used polymers for medical applications. These
polymers can be formulated into variety of physical forms such as,
by way of example, and not limitation, fibers or filaments with
acceptable mechanical properties and degradation profile and
nontoxic degradation products. Synthetic biodegradable polymers
such as, by way of example, and not limitation, polyanhydrides,
polylactones, and polyhydroxyacids have been extensively
investigated for controlled drug delivery applications as well as
for a scaffold for tissue engineering. These polymers can release a
bioactive compound upon bioerosion and thus permit localized
controlled therapeutic delivery. There is a need for biological
tissue, preferably degradable biological tissue, which can release
a bioactive compound in a controlled manner, preferably using a
hydrolysis or bioerosion mechanism. There is also a need for
materials which can provide properties of synthetic biodegradable
material and biological tissues.
[0013] Polyethylene oxide (PEO) or polyethylene glycol (PEG) is a
water soluble biocompatible polymer which is being used in several
commercially available pharmaceutical and medical products. PEG is
water soluble and non-ionic in nature. When injected in human or
animal body, it is rapidly cleared by the body. When it is
immobilized either physically or chemically on a polymer surface,
it renders the surface highly resistant to protein adsorption. The
resistance to protein adsorption is believed to be responsible for
reduced bacterial and cell adhesion to PEG-rich surfaces. The
reduction in protein adsorption also increases the biocompatibility
of blood- and tissue-contacting materials. Hydrated PEG chain is
not recognized by the immune system, therefore it is used to reduce
the immunogenicity and antigenicity of proteins and hence increase
their circulation time. Nonionic hydrogels, such as, by way of
example, and not limitation, the poly(ethylene glycol) (PEG)-based
hydrogels, are biocompatible and are non-cell adhesive.
Tissue-based bioprostheses which combine the properties of PEG and
biological tissue may be useful for many medical applications.
[0014] In view of the foregoing, there is a need for compositions
and methods that provide biostable implantable tissue. There is
also a need for biodegradable biological tissue with control over
its degradation time and with the ability to release bioactive
compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The present invention is illustrated by way of example, and
not by way of limitation, in the figures of the accompanying
drawings and in which like reference numerals refer to similar
elements and in which:
[0016] FIG. 1 is a schematic representation of exemplary steps
involved in tissue crosslinking by free radical polymerization, in
accordance with an embodiment of the present invention;
[0017] FIG. 2 is an exemplary reaction scheme for tissue
crosslinking using acrylic acid n-hydroxysuccinimide (NHS) ester,
in accordance with an embodiment of the present invention;
[0018] FIG. 3 is an exemplary reaction scheme for tissue
crosslinking using di- or polyunsaturated acid n-hydroxysuccinimide
(NHS) ester, in accordance with an embodiment of the present
invention;
[0019] FIG. 4 is an exemplary reaction scheme for tissue
crosslinking using unsaturated aldehydes, in accordance with an
embodiment of the present invention;
[0020] FIG. 5 is a schematic representation of the exemplary
preparation of tissue with patterns of biostable and biodegradable
regions within the tissue, in accordance with an embodiment of the
present invention;
[0021] FIG. 6 is a schematic representation of an exemplary
shape-preserving tissue fixation method, in accordance with an
embodiment of the present invention.
[0022] FIG. 7 is a schematic representation of exemplary steps
involved in tissue crosslinking using mercapto compounds, in
accordance with an embodiment of the present invention;
[0023] FIG. 8 is a schematic representation of an exemplary tissue
crosslinking using biodegradable tissue crosslinker, in accordance
with an embodiment of the present invention;
[0024] FIG. 9 is a schematic representation of an exemplary
biodegradable uncrosslinked tissue modified using polyethylene
glycol, in accordance with an embodiment of the present
invention;
[0025] FIG. 10 is an exemplary reaction scheme for tissue or
collagen modification with polyhydroxyacid or polylactones, in
accordance with an embodiment of the present invention.
[0026] FIG. 11 is a schematic representation of an exemplary
membrane-like tissue modifications (A-F), in accordance with an
embodiment of the present invention;
[0027] Unless otherwise indicated illustrations in the figures are
not necessarily drawn to scale.
SUMMARY OF THE INVENTION
[0028] To achieve the forgoing and other objects and in accordance
with the purpose of the invention, a variety of implantable tissue
compositions and methods thereof are described.
[0029] One embodiment of the present invention provides a
composition of matter, comprising an uncrosslinked biological
tissue, wherein the tissue is chemically modified with unsaturated
polymerizable groups
[0030] Another embodiment of the present invention provides a
composition of matter comprising a biological tissue modified with
unsaturated groups, wherein unsaturated groups are used in chemical
crosslinking of the tissue.
[0031] Another embodiment of the present invention provides a
composition of matter comprising a biological tissue modified with
unsaturated groups, wherein the cross-linked biological tissue is
produced by treating the tissue under effective cross-linking
condition comprising a free radical initiator or photoinitiator.
Preferably the crosslinking is done in presence of a mono or
polyunsaturated compound capable of copolymerizing with the
unsaturated groups in the tissue.
[0032] Another embodiment of the present invention provides a
composition of matter comprising a biological tissue modified with
unsaturated groups, wherein unsaturated groups are copolymerized
with free radical polymerizable comonomers. The comonomers may
include functional monomers with reactive functional groups such as
epoxide or isocyanate; monomers with charged groups; monomers that
undergo crosslinking and biodegradation; monomers that produce
thermosensitive polymers; monomers with long alkyl chains; monomers
that produce crystalline or semicrystalline polymers; monomers that
produce functional polymers upon hydrolysis such as polyvinyl
alcohol and monomers that have radio-opaque moieties.
[0033] Another embodiment of the present invention provides a
composition of matter comprising a biological tissue modified and
crosslinked with unsaturated groups, wherein unsaturated modified
groups and/or crosslinks with unsaturated groups are copolymerized
with free radical polymerizable comonomers.
[0034] Another embodiment of the present invention provides a
composition of matter comprising a biological tissue modified with
unsaturated groups, wherein unsaturated groups are copolymerized
with free radical polymerizable comonomers that have biodegradable
or hydrolizable groups. The biodegradable monomers may be
hydrophilic or hydrophobic.
[0035] Another embodiment of the present invention provides a
composition of matter comprising a biological tissue modified with
unsaturated groups, wherein a cross-linked biological tissue is
produced by treating the unsaturated groups modified tissue under
effective cross-linking conditions with an organic di or
poly-mercapto compounds. Preferably, the di or poly-mercapto
organic compound is a solute in a fluid comprising a solvent.
[0036] Another embodiment of the present invention provides a
composition of matter comprising a membrane like biological tissue
and a surgical adhesive. The membrane like tissue and surgical
adhesive are formulated to form a "surgical adhesive patch". The
surgical adhesive patch could be biodegradable.
[0037] Yet another embodiment of the present invention relates to a
cross-linked biological tissue produced by treating the tissue
under effective cross-linking conditions with a biodegradable
crosslinker. Preferably, the biodegradable crosslinker is a solute
in a fluid comprising a solvent.
[0038] Yet another embodiment of the present invention provides a
composition comprising a radio-opaque implantable animal
tissue.
[0039] Yet another embodiment of the present invention relates to a
biological tissue having shape memory properties.
[0040] Yet another embodiment of the present invention relates to a
biological tissue wherein certain parts or regions of the tissue
are made biostable while the remaining parts of the tissue are made
biodegradable. The biostable and biodegradable regions with in the
tissue can be of any geometry.
[0041] Yet another embodiment of the present invention relates to a
non-crosslinked degradable biological tissue produced by treating
the tissue under effective treatment conditions with a
monofunctional regent capable of reacting with primary amine groups
on the tissue. Preferably, the monofunctional reagent is: a
polyether derivative or an activated acid derivative such as
n-hydroxysuccinimide derivative, or a cyclic lactide such as
glycolide or lactide or an isocyanate derivative or an anhydride
derivative.
[0042] Yet another embodiment of the present invention relates to
the substantially biostable or biodegradable tissue produced by
treating the tissue under effective treatment conditions with a
cyclic lactone to produce a tissue-polylactone graft copolymer.
[0043] Yet another embodiment of the present invention relates to a
biological tissue/synthetic biodegradable polymer composite
produced by treating the tissue with a fluid comprising synthetic
biodegradable polymer.
[0044] Yet another embodiment of the present invention relates to a
biological tissue synthetic biodegradable polymer composite wherein
synthetic biodegradable polymer is chemically bonded to the
biological tissue.
[0045] Another embodiment of the present invention relates to a
degradable biological tissue-synthetic biodegradable polymer
composite produced by treating a non-crosslinked tissue under
effective treatment conditions with a synthetic biodegradable
polymer. Preferably, the synthetic biodegradable polymer is
polylactone or polyhydroxyacid derivative in a fluid comprising a
solvent. The synthetic biodegradable polymer can also be a
crosslinked polymer.
[0046] Yet another embodiment of the present invention relates to a
biological tissue produced by treating the tissue with a fluid
comprising synthetic biodegradable polymer and a bioactive
compound.
[0047] Yet another embodiment of the present invention provides a
composition of matter that promotes the localized controlled
delivery of at least one drug.
[0048] Yet another embodiment of the present invention provides an
animal tissue based controlled drug delivery patch that releases at
least one bioactive compound.
[0049] Yet another embodiment of the present invention relates to a
method of cross-linking a tissue is provided. The method comprises:
linking the free radical polymerizable groups on the tissue with a
covalent bond; crosslinking the free radical polymerizable groups
using free radical mechanism or cyclic dimerization.
[0050] Yet another embodiment of the present invention relates to a
method of cross-linking a tissue is provided. The method comprises:
covalently linking the compounds containing at least one free
radical polymerizable group on the tissue; crosslinking the free
radical polymerizable group using a di or poly-mercapto organic
compounds.
[0051] Yet another embodiment of the present invention relates to a
method of cross-linking a tissue is provided. The method comprises:
crosslinking the tissue with compounds containing at least one free
radical polymerizable group; further crosslinking the free radical
polymerizable group using free radical chemistry such as free
radical dimerization and polymerization, free radical crosslinking
or free radical copolymerization with monomer.
[0052] In still another embodiment of the present invention, a
method for making a biodegradable biological tissue is provided.
The method comprises: treating the tissue under effective
cross-linking conditions with a fluid comprising a biodegradable
crosslinker.
[0053] In still another embodiment of the present invention, a
method for incorporating a biodegradable polymer in a biological
tissue is provided. The method comprises: dehydrating the
biological tissue; treating the dehydrated tissue with a solution
of biodegradable polymer in an organic solvent; removing the
solvent from the treated tissue.
[0054] In still another embodiment of the present invention, a
method for incorporating a biodegradable polymer and a bioactive
compound in a biological tissue is provided. The method comprises:
dehydrating the biological tissue; treating dehydrated tissue with
a solution of biodegradable polymer and bioactive compound in an
organic solvent; removing the solvent from the treated tissue.
[0055] In still another embodiment of the present invention, a
method for making a drug delivery patch from a membrane like tissue
is provided. The method comprises: dehydrating the membrane like
biological tissue; treating dehydrated membrane like tissue with a
solution of biodegradable polymer and a bioactive compound in an
organic solvent; removing the solvent from the treated tissue;
releasing the compound from the biodegradable polymer. Preferably
the bioactive compound is a cell cycle inhibitor such as Lovastatin
(HMG-CoA inhibitor or statin), paclitaxel, and Rapamycin. The
biodegradable polymer may be hydrophobic or hydrophilic. The
biodegradable polymer can be a crosslinked polymer.
[0056] In still another embodiment of the present invention, a
method of coating a biological tissue with biodegradable polymer is
provided. The method comprises: dehydrating the biological tissue;
spraying a coating solution comprising biodegradable polymer in a
solvent; removing the solvent from the treated tissue.
[0057] In still another embodiment of the present invention, a
method of coating a biological tissue with biodegradable polymer is
provided. The method comprises: dehydrating the biological tissue;
dipping the dehydrated tissue in a coating solution comprising
biodegradable polymer in a solvent; removing the solvent from the
treated tissue.
[0058] In still another embodiment of the present invention, a
method for making a radio-opaque implantable tissue is provided.
The method comprises: treating a biological tissue with a
radio-opaque compound under effective treatment conditions to
covalently bond the radio-opaque compound to the tissue. The
preferred radio-opaque compound is iodinated organic compound.
[0059] In still another embodiment of the present invention, a
method treating the tissue under effective cross-linking conditions
with a di or polymercapto organic compound is provided.
[0060] In still another embodiment of the present invention, a
method of coating a biological implantable tissue with a
biodegradable hydrogel is provided. The method comprises: treating
a tissue with a precursor or biodegradable hydrogel components;
crosslinking the precursors to produce a biodegradable hydrogel
coating on the surface of the tissue.
[0061] In still another embodiment of the present invention, a
method of coating a biological implantable tissue with
biodegradable hydrogel comprising cells/bioactive compound is
provided. The method comprises: treating a tissue with a precursor
or biodegradable hydrogel components comprising cells and/or
bioactive compounds; crosslinking the precursors to produce a
biodegradable hydrogel coating with entrapped cells/drug in the
coating on the surface of the tissue.
[0062] In still another embodiment of the present invention, a
method of coating a biological tissue with non-crosslinked
biodegradable hydrogel is provided. The method comprises:
dehydrating the biological tissue; treating the dehydrated tissue
with a solution of biodegradable polymer in an organic solvent;
removing the solvent from the treated tissue; exposing the tissue
to a biological environment to hydrate the tissue and biodegradable
polymer.
[0063] In still another embodiment of the present invention, a
method for incorporating a biodegradable polymer and bioactive
substance in biological tissue is provided. The method comprises;
forming groves or holes on tissue surface; filling the grooves or
holes with a biodegradable polymer and bioactive compound;
releasing the bioactive compound in a controlled manner.
[0064] Another embodiment of the present invention provides a
degradable animal tissue coated with or incorporated with,
Demineralized Bone Matrix (DBM) and/or purified Bone Marrow
Proteins (BMP)'s. This mixture provides a matrix that allows the
cellular components of the body to migrate into it and thus produce
osteoinduction where needed. The matrix composition, enzymes (such
as thrombin and plasmin), BMPs, growth factors and DBM and their
concentrations, calcium salts such as calcium phosphates are
adequately formulated to optimize the longevity of this temporal
scaffolding structure and the osteoinduction which needs to occur.
All of the animal tissue components are biodegradable, but during
osteogenesis the mixture provides a non-collapsible scaffold that
can determine the shape and location of the newly formed bone.
[0065] Another embodiment of the present invention provides a
composition of matter comprising a degradable tissue coated with a
biodegradable polymer comprising at least one growth factor and/or
a drug.
[0066] Yet another embodiment of the present invention provides a
composition of matter that promotes wound healing, comprising: a
biodegradable implantable animal tissue coated with biodegradable
polymer and an effective concentration of at least one growth
factor, wherein the concentration of growth factor is effective in
promoting wound healing.
[0067] Another embodiment of the present invention provides a
composition of matter that promotes the growth of cells,
comprising: a degradable animal tissue; a hydrogel coating on the
surface of degradable tissue; and an effective concentration of at
least one growth factor, wherein the concentration of the growth
factor is effective in promoting the directed migration of the
animal cells. In another embodiment, genetically altered cells
and/or other cells may also be included in the tissue coated
hydrogels of this invention.
[0068] Yet another embodiment of the present invention provides a
composition of matter that promotes the proliferation and/or
differentiation of animal cells, comprising: an implantable animal
tissue, a hydrogel; and an effective concentration of at least one
growth factor, wherein the concentration is effective in promoting
proliferation and/or differentiation of animal cells.
[0069] Yet another embodiment of the present invention provides a
composition of matter that promotes the localized delivery of at
least one growth factor. Preferably the growth factor is vascular
endothelial growth factor (VEGF) or BMP or mixtures thereof.
[0070] Yet another embodiment of the present invention provides a
process for promoting the healing of wounds, comprising applying to
the wound, a composition that contains a non-crosslinked animal
degradable animal tissue modified with a synthetic polymer and an
effective concentration of at least one growth factor or one small
molecule therapeutic, wherein the concentration is effective to
promote wound healing.
[0071] Another embodiment of the present invention provides a
degradable implantable animal tissue based composition that
promotes the localized delivery of a poorly water soluble form of a
bioactive compound, such as chlorhexidene; chlorhexidene diacetate
monohydrate or chlorhexidene dihydrochloride; chlorhexidene
gluconate, silver salts such as silver chloride, silver iodide,
silver acetate, silver lactate, cell cycle inhibitor such as
paclitaxel, lovastatin, rapamycin, simvastatin, rifampin; or
anti-arrhythmic agent such as amiodarone.
[0072] In still another embodiment of the present invention, a
method for tissue crosslinking or fixation is provided. The method
comprises; linking the free radical polymerizable groups on the
tissue with a covalent bond; crosslinking the free radical
polymerizable groups using free radical polymerizable monomers
comprising primary amine group. Further crosslinking the primary
amine groups using di or polyfunctional crosslinker such as
glutaraldehyde.
[0073] In yet another embodiment of the present invention, method
for making a degradable tissue matrix comprising substantially
water insoluble drug or bioactive compound is provided. The method
comprises: dehydrating the membrane like biological tissue;
treating dehydrated membrane like tissue with a solution of a
substantially water insoluble bioactive compound in an organic
solvent; removing the solvent from the treated tissue.
[0074] In still another embodiment of the present invention, a
method for making a tissue capable of remembering the shape is
provided.
[0075] In another embodiment, aIn another embodiment of the present
invention, a method for making a tissue is provided wherein certain
parties of the tissue are biostable and/or biodegradable.
[0076] Other features, advantages, and object of the present
invention will become more apparent and be more readily understood
from the following detailed description, which should be read in
conjunction with the accompanying drawings.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0077] The present invention is best understood by reference to the
detailed figures and description set forth herein.
[0078] Embodiments of the invention are discussed below with
reference to the Figures. However, those skilled in the art will
readily appreciate that the detailed description given herein with
respect to these figures is for explanatory purposes as the
invention extends beyond these limited embodiments.
[0079] In order to clarify the terminology present invention, the
following definitions are provided. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
is commonly understood by one who is skilled in the art.
[0080] "Biodegradable" denotes a material that will degrade in a
biological environment by either a biologically assisted mechanism,
such as, by way of example, and not limitation, an enzyme catalyzed
reaction, or by a chemical mechanism which can occur in a
biological medium, such as, by way of example, and not limitation,
hydrolysis.
[0081] "Biostable" denotes a high chemical stability of a compound
in an aqueous environment, which is similar to the environment,
found in the human body, such as, by way of example, and not
limitation, phosphate buffered saline (pH 7.2).
[0082] "Bioactive" refers to one or all of the activities of a
compound that show pharmacological or biological activity in human
or animal body. Such biological activity is preferred to have
therapeutic effect. Substances or compounds that are bioactive are
referred to as "drugs" or "bioactive compounds." The bioactive
compounds that can be used include, but are not limited to,
antiviral agents; antiinfectives such as, by way of example, and
not limitation, antibiotics; antipruritics; antipsychotics;
cholesterol- or lipid-reducing agents; cell cycle inhibitors;
anticancer agents; antiparkinsonism drugs; HMG-CoA inhibitors;
antirestenosis agents; antiinflammatory agents; antiasthmatic
agents; anthelmintic; immunosuppressives; muscle relaxants;
antidiuretic agents; vasodilators; nitric oxide; nitric
oxide-releasing compounds; beta-blockers; hormones;
antidepressants; decongestants; calcium channel blockers; growth
factors such as, by way of example, and not limitation, bone growth
factors or bone morphogenic proteins; wound healing agents;
analgesics and analgesic combinations; local anesthetic agents;
antihistamines; sedatives; angiogenesis-promoting agents;
angiogenesis-inhibiting agents; tranquilizers and the like.
Cellular elements which can be used for therapeutic use include,
but are not limited to mammalian cells including stem cells,
cellular components or fragments, enzymes, DNA, RNA, and genes may
also be included as bioactive components.
[0083] The term "minimally invasive surgery" or (MIS) is used
herein includes, but is not limited to, surgical techniques such
as, by way of example, and not limitation, laparoscopy,
thoracoscopy, arthroscopy, intraluminal endoscopy, endovascular
techniques, catheter-based cardiac techniques (such as, by way of
example, and not limitation, balloon angioplasty), and
interventional radiology.
[0084] The term "dehydration" broadly refers to any method that
removes the water from the tissue without denaturing the tissue.
This includes, but is not limited to processes such as, by way of
example, and not limitation, lyophilization, vacuum drying, air
drying, or solvent-based drying such as, by way of example, and not
limitation, exposing the tissue to various alcohol-based
solutions.
[0085] "Sustained release" or "long term release or deliveries" are
phrases used interchangeably herein, to denote longer than the
expected delivery of a bioactive compound from the tissue matrix.
In some embodiments, delivery will be at least a day or more, and
may extend to weeks, months, or a few years. However, alternate
embodiments encompass shorter term release. The long term release
can be achieved by any of a number of mechanisms.
[0086] A "hydrogel" refers to a semisolid composition constituting
a substantial amount of water, and in which polymers or mixtures
thereof are dissolved or dispersed. The hydrogels may be physically
or chemically crosslinked.
[0087] The term "fluid" generally refers to solutions, emulsions,
and suspensions.
[0088] The term "exposing" refers to soaking the tissue in a fluid
comprising the treatment agent for a period of time sufficient to
treat the tissue. The soaking may be performed by, but is not
limited to, incubation, swirling, immersion, mixing, or
vortexing.
[0089] Polymeric nomenclature variations such as, by way of
example, and not limitation, poly(lactic acid), polylactic acid, or
polylacticacid refer to the same polymer, unless or otherwise
stated clearly. This is also true for all others polymers referred
to herein.
[0090] The term "macromonomer or "macromer" refers to oligomeric or
polymeric materials capable of undergoing fee radical
polymerization.
[0091] The term "activated" means increasing the chemical
reactivity of a given functional group so that it can react with
the target molecule under mild conditions. By way of example and
not limitation, the acid functionality in acrylic acid is not
reactive enough to react with amine groups of the tissue in water
at pH 7.2 at room temperature. The reactivity of acid group can be
increased sufficiently so that it can react with proteins or tissue
in water at pH 7.2 at room temperature. This may be achieved by
forming n-hydroxy succinimide ester of acid functional group. Many
activation chemistries are known in the peptide synthesis or
protein modification art and will be apparent to one skilled in the
art, in light of the teachings of the present invention. Preferred
activating moieties include, but are not limited to, disuccinimidyl
moieties, n-hydroxy disuccinimidyl moieties, sulfo-disuccinimidyl
moieties, and mixtures thereof.
[0092] The phrase "effective cross-linking condition" generally
refers to, but is not limited to, exemplary conditions such as
treating a biological tissue like bovine pericardium tissue (size 2
cm by 2 cm) with 20 ml 0.4% glutaraldehyde solution in distilled
water or in 20 mM phosphate buffered saline (PBS, pH 7.2) for 24 to
48 hours at room temperature (around 25.degree. C.).
[0093] "In situ" denotes at a local surgical site, especially
within or in contact with living organisms, tissue, organs, or the
body.
[0094] "Biodegradable polymer" denotes those polymers or
macromolecules which degrade or hydrolyze inside the human or
animal body without producing harmful degradation products.
Polylacticacid or poly(lactic acid) or poly(lactide) or PLA is term
used for a polymer which is made from lactide or lactic acid.
Similarly, PGA is term used for polyglycolic acid or polyglycolate.
Such polymers are generally referred to as polylactones or
polyhydroxyacids.
[0095] The term "hydrophobic" is defined as materials or polymers
having a low degree of water absorption or attraction.
[0096] The term "hydrophilic" is defined as materials or polymers
having a strong affinity for water.
[0097] The term "Tissue Engineering" is defined as the use of a
combination of cells, engineering materials, and suitable
biochemical factors to synthesize biological tissues.
[0098] The term "tissue" or "extracellular matrix" (ECM) includes
human or animal tissue suitable for human or animal implantation or
medical use. The tissue defined herein includes but is not limited
to items such as, by way of example, and not limitation, ligaments;
meniscus; basal membrane; bladder tissue, tendons; cartilage
tissue; tubular tissue such as, by way of example, and not
limitation, arterial tissue and vein tissue; heart valve tissue;
demineralized bone tissue; tissues used to construct heart valves
such as, by way of example, and not limitation, dura mater and
pericardium tissue; transparent issue such as, by way of example,
and not limitation, cornea and lens tissue; membrane-like tissue
such as, by way of example, and not limitation, porcine
pericardium; bovine pericardium; porcine intestine tissue and lung
tissue, more specifically porcine sub-mucosa tissue; bladder
tissue; human tissue that is generated and discarded during human
childbirth, e.g., human placenta and umbilical cord tissue
generated during child birth; amniotic membrane tissue; and the
like. Among the tubular tissue, tissue such as, by way of example,
and not limitation, bovine mesenteric vein, bovine thoracic artery,
bovine ureter, sheep carotid arteries are used in preferred
embodiments. The tissue can be derived from, but is not limited to,
sources such as, by way of example, and not limitation, bovine,
human, porcine, horse, sheep, kangaroo, rat, mouse, dog, cat, or
rabbit. Tissue derived from human source is generally preferred.
Human processed tissue such as AlloDerm.RTM. marketed by LifeCell
Corporation, N.J. may also be used. If the tissue used is from an
animal source, the animal may be genetically modified to yield a
uniform tissue type or tissue that has similar biological
properties as human tissue. For example, the tissue may be obtained
from cloned animals. Many cloning technologies including those that
have already developed or yet to be developed may, for example, and
without limitation, be used to obtain a cloned animal tissue. The
animals may also be genetically modified so that the antigens which
are recognized by the human immune system are suppressed. The
tissue may also be obtained from technologies employed in the
tissue engineering art. In one embodiment using a tissue
engineering approach, a tissue is generated by growing animal or
human derived cells on a biodegradable scaffold. For example,
Apligraf.TM. is a commercially available human tissue engineering
product that may also be used in certain applications. In one
embodiment, an engineered tissue that is provided as a sheet or
membrane is used. In some embodiments, tissue that is not denatured
may be preferred. Tissue that is acellular or processed to remove
cells or cell debris is used in other embodiments. In some
embodiments, it may be preferable to use animal tissue that is
substantially free from infectious or harmful animal viruses or
proteins such as, by way of example, and not limitation, BSE.
[0099] "Bioprosthesis" is defined to include any prosthesis which
is derived in whole or part from animal or other organic tissue and
which is suitable for human or animal implantation. Thus, the term
generally includes, but is not limited to, devices such as animal
tissue based heart valves, vascular grafts, annuloplasty rings and
other medical devices such as, by way of example, and not
limitation, vascular grafts or patch, heart replacements devices,
urinary tract and bladder replacements, bowel and tissue resections
in general and the like. In general, though not required, the type
of tissue utilized as the starting material as well as its
modification will depend on the intended use. For example, in the
heart valve application, a biostable mechanically durable tissue
may, for example, and without limitation, be used. In such an
application, a bovine pericardium or porcine aortic root that is
crosslinked with a biostable crosslinker may, for example, and
without limitation, be used. If the tissue is to be used as a
biodegradable drug delivery patch or degradable tissue engineering
scaffold, then a non-crosslinked or biodegradable tissue may, for
example, and without limitation, be used.
[0100] "Crosslink" is defined as understood by those skilled in the
bioprosthesis or polymer chemistry art. In general, cross-linking
refers to the method of forming covalent bonds or crosslinks
between polymeric/macromolecular molecules. The crosslinking
process also generally refers to a fixation process which
stabilizes the tissue by making the tissue less antigenic and thus
less susceptible to enzymatic degradation. A "crosslinking agent"
is defined as a compound capable of forming the crosslinking. For
example, glutaraldehyde is generally known in the art as
crosslinking agent for the tissue.
[0101] PEG or PEO is a term used for polymer containing ethylene
oxide repeat units.
[0102] The term "polymerizable" denotes that molecules have the
capacity to form additional covalent bonds resulting in monomer
interlinking to oligomer or polymer formation, for example,
molecules contain carbon-carbon double bonds of acrylate-type
molecules. Such polymerization is characteristically initiated by
free-radical formation, for example, resulting from photon
absorption of certain dyes and chemical compounds to ultimately
produce free-radicals. The term polymerizable is also applicable to
compounds which can undergo condensation polymerization and form a
linear or crosslinked polymer.
[0103] The following examples are provided as a means of
illustrating some embodiments of the present invention and are in
no way considered limiting of the present invention.
Methods and Compositions for the Preparation of Biostable
Tissues.
[0104] An exemplary tissue crosslinking method embodiment of the
present invention is herein provided for biological tissues to be
used in, by way of example, and not limitation, bioprosthetic
medical devices such as, by way of example, and not limitation,
heart valves, vascular grafts, surgical patch, ligament and
meniscus implants.
[0105] One embodiment of the present invention provides for a
method of crosslinking a tissue comprising steps of treating the
tissue under effective cross-linking conditions with a crosslinker
comprising unsaturated polymerizable groups and further
crosslinking the unsaturated polymerizable groups using free
radicals. An alternate embodiment of the present invention provides
for a method of crosslinking a tissue comprising steps of treating
the tissue under effective cross-linking conditions with a
crosslinker comprising unsaturated polymerizable groups and further
crosslinking the unsaturated polymerizable groups by free radical
copolymerization. The present invention also provides for a method
for forming a crosslinked tissue comprising the steps of providing
a tissue suitable for human implantation; exposing the tissue to a
fluid comprising a functional monomer capable of reacting with a
tissue until a portion of the tissue functional groups are
covalently bonded to the monomer; and copolymerizing the functional
monomer-treated tissue under effective cross-linking conditions
with a compound having at least one free radical polymerizable
group. Yet another embodiment of the present invention uses a
cross-linked biological tissue produced by treating the tissue
under effective cross-linking conditions with a biodegradable
crosslinker. In one embodiment, the biodegradable crosslinker may
be a solute in a fluid comprising a solvent. It is to be understood
that in some applications of the present invention may, for
example, and without limitation, be used to produce a general
composition of matter and/or a bioactive tissue.
[0106] As will be set forth in some detail below, one aspect of the
present invention provides for a composition of matter that
promotes the localized controlled delivery of at least one
drug.
[0107] FIG. 1 is a schematic representation of exemplary steps
involved in tissue crosslinking by free radical polymerization, in
accordance with an embodiment of the present invention. Such
crosslinking is achieved in two steps. In the first step, the
biological tissue of interest is contacted with one or more of the
disclosed compounds under conditions effective to introduce
unsaturated polymerizable or dimerizable groups in the tissue,
i.e., modified with unsaturated polymerizable groups. In the second
step, the unsaturated groups are used to form crosslinks between
components of the tissue, i.e., the unsaturated groups on the
modified tissue are copolymerized with free radical polymerizable
monomer and free radical initiator to form a crosslinked tissue
with a polymeric crosslink.
[0108] The first step shown by way of example in the FIG. 1 for the
preparation of tissue with unsaturated groups will next be
described in some detail. The unsaturated group modifying agents
that may, for example, and without limitation, be used in
accordance with embodiments of the present invention comprise an
organic functional small molecule, typically, but not limited to, a
monomer with functional groups capable of reacting with tissue.
Thus, exemplary compounds suitable for use with the described
embodiments of the present invention can be generally represented
by the following structural formula:
F--X--U
[0109] Where F is a functional group reactive with proteins present
in extra cellular matrix such as, by way of example, and not
limitation, collagen, elastin, keratin and the like; U is an
unsaturated group capable of undergoing free radical
copolymerization, cyclic dimerization or reaction with thiol group;
and X is an organic molecule or radical covalently linking F and U.
X may comprise Carbon-Carbon, Carbon-Hydrogen, Carbon-Nitrogen,
Carbon-Oxygen, Carbon-Sulfur, Nitrogen-Hydrogen and Oxygen-Hydrogen
covalent bonds. X may be polymeric or non-polymeric. F is a
functional group that is sufficiently reactive with the components
of the tissue such as, by way of example, and not limitation,
collagen or elastin molecules present in the biological tissue. For
example, functional groups reactive with collagen and suitable for
use may include, but not limited to, anhydride, isocyanate, epoxy,
n-hydroxysuccinimide, n-hydroxysulfosuccinimide, aldehyde or other
protein reactive functionalities known in the art or yet to be
developed. It is preferred to have one F per molecule if the tissue
crosslinking is not desired. If tissue crosslinking is desired,
then the number of F moieties can be two or more per molecule. If
the number of F moieties is two or more, the F moieties can be the
same functional groups (homo-functional) or different functional
groups (hetero-functional). U is an unsaturated group capable of
undergoing free radical polymerization or copolymerization. U also
can undergo a dimerization reaction. U can have at least one
carbon-carbon double bond or triple bond capable of undergoing free
radical polymerization. The preferred unsaturated groups include,
but not limited to, acrylamides, methacrylamides, acrylates,
diacrylates, oligoacrylates, acrolein, methacrolein, fumarates,
maleates, methacrylates, dimethacrylates, oligomethoacrylates,
itaconates, cinnamic acid derivatives or other biologically
acceptable free radical polymerizable groups. The number of U
moieties may be one or more per molecule. The arrangement of X, F
and U in the molecule may vary in any order according to various
embodiments of the present invention. There may be more than one F
or U group per molecule.
[0110] In various embodiments of the present invention, molecules
which are capable of modifying the tissue with unsaturated groups
include, but not limited to, glycidyl methacrylate, glycidyl
acrylate, acrylic acid N-hydroxysuccinimide ester, methacrylic acid
N-hydroxysuccinimide ester; fumaric acid N-hydroxysuccinimide
ester; maleic acid N-hydroxysuccinimide ester; itaconic acid
N-hydroxysuccinimide ester; acrylic anhydride, methacrylic
anhydride, acryloyl chloride, methacryloyl chloride,
2-isocyanatoethyl methacrylate, 2-hydroxyethyl methacrylate, maleic
anhydride, fumaric anhydride, cinnamic acid NHS ester, cinnamoyl
chloride and the like. Compounds like activated unsaturated di- or
polyfunctional acids such as, by way of example, and not
limitation, fumaric acid N-hydroxysuccinimide ester; maleic acid
N-hydroxysuccinimide ester; itaconic acid N-hydroxysuccinimide
ester may crosslink the tissue as well as introduce unsaturated
groups. Other dual function compounds (i.e., capable of
crosslinking and introducing unsaturated groups) will be apparent
to one skilled in the art, in light of the teachings of the present
invention.
[0111] The tissue modification using functional monomers taught by
way of example in the present detailed description can be made
using any synthetic methodologies known to the skilled in the art
of synthetic polymer chemistry, in light of the teachings of the
present invention. In one embodiment, another aspect of the present
invention is directed to a method of introducing an unsaturated
group tissue. The method comprises treating the tissue under
effective treatment conditions with a polymerizable organic
compound. In one preferred approach, bovine pericardium tissue is
modified with an activated unsaturated acid compound. The
modification method comprises exposing the tissue to a fluid
comprising activated unsaturated acids that can react with amino
groups in tissue under mild conditions. As used herein, the term
"activated" is applied to an acid moiety-containing compound
containing an additional moiety such that the activated acid can
react with amino groups under mild conditions. Preferred activating
moieties include, but are not limited to, disuccinimidyl moieties,
n-hydroxy disuccinimidyl moieties, sulfo-disuccinimidyl moieties,
and mixtures thereof. In one embodiment, bovine pericardium tissue
is modified using acrylic acid succinimide ester (ANHS). Briefly,
bovine pericardium pieces, cut from a freshly obtained bovine
pericardial sac, are treated acrylic acid succinimide ester (ANHS)
dissolved in dimethyl sulfoxide. The reaction is carried out, for
example, in aqueous medium buffered with PBS (pH 7.2). The
modification reaction is, for example, carried out for 6 hours at
ambient temperature (25.degree. C.) and then for 12 hours at
4.degree. C. with gentle shaking. ANHS is sparingly soluble in
water, therefore a solubility enhancing compound such as, by way of
example, and not limitation, dimethyl sulfoxide may, for example,
and without limitation, be used to facilitate the reaction between
activated acid groups and primary amine groups on the tissue. In
place of dimethyl sulfoxide, other solvents may also be used. Water
soluble solvents are more preferred. Preferred solvents include but
not limited to ethanol, methanol, isopropanol, n-methyl
pyrrolidinone, dimethyl acetamide, dimethyl formamide,
tetrahydrofuran, dixoane, acetone, methyl ethyl ketone and the
like. The reaction occurs in mild conditions, in water (PBS, pH
7.2) and is usually complete in about 24 hours, more preferably
within about six hours. Acidic or alkaline pH conditions may be
used to alter reaction kinetics, but mild reaction conditions are
generally preferred. The reaction may be carried out in pH ranging
from 9 to 6, more preferably 6 to 8 and even more preferably at pH
6.5 to 7.5. Many buffering agents may be used to control the pH.
Sodium bicarbonate buffer may be used to achieve pH around 9,
phosphate buffer, triethanol amine buffer,
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid Sodium salt
(MES) buffer, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
(HEPES) buffer and the like may be used to control the pH around 7.
Acetate buffer may be used to maintain the pH around 4. The tissue
modification can also be substantially completed in about 10
minutes and therefore may permit the use of this method during a
surgical procedure to modify an autologous tissue. In another
embodiment of this method, the modification reaction is carried out
under mild acidic conditions; for example, without limitation, a
MES buffer, (pH 6.5). In this case, the ANHS is added every 1.5
hours for example, without limitation, to replace the hydrolyzed
ANHS. ANHS is generally deactivated by reaction with water
generating acrylic acid. The NHS groups of ANHS react with primary
amines groups of the tissue forming stable amide bonds. In another
embodiment, the modification reaction is carried out, for example,
without limitation, in organic solvent such as, though not limited
to, dimethyl sulfoxide. Tissue is exposed to, for example, without
limitation, 0.2% solution methacrylic anhydride and 1.0%
triethylamine in dimethyl sulfoxide for 24-48 hours. The treated
tissue is then removed and washed, for example, without limitation,
with PBS 3 times to remove solvents and stored until further use.
Triethyl amine is used as a base catalyst in modification
reactions. Other organic base catalyst such as pyridine, trimethyl
amine, N,N-diisopropylethylamine (DIPEA) and the like may also be
used. The concentration of methacrylic anhydride and activated NHS
ester may be used to control the number of unsaturated groups in
the tissue. The concentration of methacrylic anhydride and
activated NHS ester during the modification reaction may vary from
0.01% to 25%, most preferably 0.5% to 5% even more preferably 0.2%
to 2%. Room temperature reaction conditions (20-25.degree. C.) are
most preferred. Temperature higher than 60.degree. C. (above shrink
temperature of tissue) is least preferred. The ANHS or methacrylic
anhydride reacts with primary amines groups on the tissue. The
reaction variables such as, by way of example, and not limitation,
time, temperature, concentration, pressure may be controlled in
such a way that about 1 to about 100 percent primary amine groups
on the tissue are modified. More preferably, about 10 to about 95
percent amine groups are modified, even more preferably about 40 to
about 95 percent amine groups are modified. FIG. 2 is an exemplary
reaction scheme for tissue crosslinking using acrylic acid
n-hydroxysuccinimide (NHS) ester, in accordance with an embodiment
of the present invention; and, FIG. 3 is an exemplary reaction
scheme for tissue crosslinking using di- or polyunsaturated acid
n-hydroxysuccinimide (NHS) ester, in accordance with an embodiment
of the present invention. Those skilled in the art, in light of the
teachings of the present invention, will recognize that many
changes could be made to the tissue modification procedure
exemplified above and such changes are considered to be within the
scope of the present invention.
[0112] In another embodiment, polymerizable groups are introduced
using glycidyl methacrylate as a tissue modification agent (see
Example 37, step 1 described below). The glycidyl group can react
with a number of reactive functional groups such as, by way of
example, and not limitation, primary and secondary amines,
carboxyl, hydroxyl, and thiol groups. This reactivity with multiple
functional groups permits one to modify proteins such as, by way of
example, and not limitation, collagen and elastin which are major
components of some tissue types. In some cases, tissues may possess
high concentration of glycosaminoglycans. An example of such
tissue, without limitation, is cartilage tissue. Glycidyl
methacrylate may be especially useful to modify such tissues rich
with glycosaminoglycans such as, by way of example, and not
limitation, hyaluronic acid. Glycidyl methacrylate may also undergo
transesterification reaction with hydroxyl groups in the tissue.
Many reaction conditions, such as, by way of example, and not
limitation, aqueous or non-aqueous medium, time, temperature,
concentration may, for example, and without limitation, be used to
control degree of substitution. In one preferred embodiment, the
reaction temperature is maintained below 55.degree. C., well below
shrink temperature of the tissue. A base catalyst such as, for
example, without limitation, triethyl amine, trimethyl amine,
pyridine is used to accelerate the reaction. A phase transfer
catalyst such as, for example, without limitation, tetrabutylamine
hydrobromide, tetrapropylamine hydrochloride, may also be employed
to facilitate the substitution. The glycidyl methacrylate or
glycidyl acrylate may, for example, and without limitation, be
used, for example, without limitation, with uncrosslinked tissue or
crosslinked tissue such as, for example, without limitation,
glutaraldehyde fixed tissue. In one preferred embodiment, 5 pieces
of 28 mm pericardial tissues are treated with a 300 ml solution
containing 200 ml PBS, 25 ml glycidyl methacrylate and 25 ml
triethylamine at 50.degree. C. for 4 hours. Alternatively, the
reaction can also be done at room temperature for about 24 hours.
The concentration of glycidyl methacrylate may be varied form 1 ml
to 25 ml to control the amount of unsaturated groups in the
modified tissue.
[0113] In an alternate preferred embodiment, an unsaturated
monoaldehyde such as, for example, without limitation, acrolein or
methacrolein is used to modify the tissue with unsaturated groups.
Acrolein is highly soluble in water and therefore its modification
reaction is very similar to, for example, without limitation,
glutaraldehyde fixation chemistry except that no crosslinking
occurs. FIG. 4 depicts the reaction scheme for tissue crosslinking
using unsaturated aldehydes, in accordance with an embodiment of
the present invention. The acrolein reacts with primary amines
groups on the tissue. Reaction variables such as, by way of
example, and not limitation, time, temperature, concentration,
pressure are controlled in such a way that about 1 to about 100
percent primary amine groups on the tissue are modified. More
preferably, about 10 to about 95 percent amine groups are modified,
even more preferably about 40 to about 95 percent amine groups are
modified. In the present embodiment, an aqueous reaction medium is
preferred for the modification reaction. Non-aqueous or semi
aqueous medium also may, for example, and without limitation, be
used in some embodiments. The concentration of acrolein or
methacrolein may range from about 0.1% to about 20%, more
preferably about 0.1% to about 5%, even more preferably about 0.1%
to about 2%. To prevent unwanted free radical polymerization of
acrolein or methacrolein, solutions may be supplemented with free
radical polymerization inhibitors such as, by way of example, and
not limitation, hydroquinone in the reaction mixture. Like
glutaraldehyde, acrolein or methacrolein also have antimicrobial
properties and therefore may help in reducing bacterial and viral
contamination of the tissue.
[0114] Unsaturated acid derivatives such as, by way of example, and
not limitation, methacrylic anhydride, acrylic anhydride, acryloyl
chloride, methacryloyl chloride may also be used to introduce
unsaturated groups into the tissue. Primary amine residues are
target of such modification chemistries. In one embodiment,
methacrylic anhydride is used to modify bovine pericardial tissue.
The reaction is carried out in basic buffered medium at low
temperature for several days to achieve the desired degree of
modification. In another embodiment, the reaction is carried out in
aprotic solvent such as, by way of example, and not limitation,
dimethylsulfoxide (DMSO) in presence of triethylamine (TEA) as a
base catalyst. Five 1 cm by 1 cm pieces are treated with 10 ml DMSO
containing 0.02 ml methacrylic anhydride and 0.2 ml TEA. The
reaction was carried out at ambient temperature (30).degree. for
24-48 hours
[0115] In many of the modification chemistries discussed above, the
tissue modification may be carried out in aqueous medium or
non-aqueous medium such as, by way of example, and not limitation,
organic solvent. The preferred organic solvents are commonly used
organic solvents dimethyl sulfoxide, n-methylpyrrolidinone, ethanol
and the like. However, other organic solvents may also be employed.
Reaction conditions such as, by way of example, and not limitation,
time, temperature, concentration will depend on the degree of
substitution desired.
[0116] The unsaturated group modification methods, which are taught
by way of example in the present detailed description, may be
modified to obtain a desired degree of substitution. Synthetic
methods as such, and otherwise, will be apparent to those skilled
in the art, in light of the teachings of the present invention. In
most cases, up to about 70 to about 100 percent modification of
primary amine groups is preferred to suppress the immune response
and to be useful in the crosslinking reactions motioned below.
[0117] The unsaturated group incorporation methods for tissues as
discussed above may also be used to modify collagen solid particles
or collagen sponges or fibers in the solid state. For example,
collagen sponges obtained from commercial vendors like Kensey Nash
may be treated with ANHS solution or with methacrylic anhydride in
DMSO to incorporate acrylic/methacrylic groups in the collagen
sponge. The unsaturated group modified collagen sponge is then
crosslinked using methods similar to the tissue crosslinking
methods mentioned in this document. The crosslinked collagen sponge
may have a higher degradation time as compared to uncrosslinked
sponge and may be useful in tissue engineering applications
especially in bone tissue engineering applications. The crosslinked
collagen may be incorporated with BMP to accelerate the bone
formation. Though not required, the BMP may be added prior to
crosslinking and polymerization reaction.
[0118] The first step shown by way of example in the FIG. 1 for the
crosslinking of unsaturated group modified tissue will next be
described in some detail. In the second step, the unsaturated
groups introduced by tissue modification methodologies discussed
above are used in tissue crosslinking. In one approach, the
unsaturated groups are dimerized, polymerized, or copolymerized and
crosslinked with monomers that can undergo free radical
polymerization or cyclic dimerization. In another approach, the
unsaturated groups are reacted with di- or polyfunctional mercapto
compounds to introduce the crosslinking in the tissue.
[0119] Crosslinking of Unsaturated Groups in the Tissue Using Free
Radical Crosslinking
[0120] Another aspect of the present invention is directed to a
method of crosslinking a tissue, including treating the unsaturated
group modified tissue under effective crosslinking conditions with
a free radical crosslinking mechanism. In one embodiment of the
present invention, a method for cross-linking collagen-containing
biological tissue under effective conditions wherein at least a
portion of the tissue functional groups form a covalent bond with a
compound having the formula F--X--U, wherein F, X, and U are as
defined above, then treating the tissue under effective
crosslinking conditions with a compound having at least one free
radical polymerizable group. Accordingly, the tissue used in this
type of crosslinking is first modified using unsaturated groups as
discussed above. More preferably, the tissue modified with
unsaturated groups such as, by way of example, and not limitation,
acrylamide, methacrylamide, acrylate or methacrylate group is used
in the crosslinking reaction. In one embodiment of the present
invention, the method of cross-linking the tissue is achieved by
steps that include, without limitation, treating unsaturated group
modified tissue under effective cross-linking conditions with a
free radical polymerizable monomer.
[0121] In one embodiment, unsaturated group modified tissue is
gradually dehydrated in series of aqueous alcohol solutions. The
dehydration may also be done using other techniques such as, by way
of example, and not limitation, lyophilization or vacuum drying and
the like. The dehydrated tissue is then exposed to or incubated in
a monomer/monomers solution (n-vinyl pyrrolidinone as an example)
containing free radical photoinitiator. The incubation is carried
out until the infusion of monomer/initiator is complete throughout
the tissue matrix. This may require incubation from minutes to few
days. More preferably, tissue is incubated for about 0.1 minute to
about 16 hours. Even more preferably, for about 5 minutes to about
2 hours. The monomer/photoinitiator-soaked tissue is then exposed
to long wavelength ultraviolet light until polymerization and
crosslinking is achieved. The photoinitiator in presence of UV
light initiates polymerization of monomer. The unsaturated groups
on the tissue copolymerize with the monomer and crosslink the
tissue. The overall tissue crosslinking reaction is shown in the
example of FIG. 1. In order to effectively crosslink the tissue,
the growing polymer free radical must copolymerize or react with
unsaturated groups on two collagen chains. This is achieved, for
example, without limitation, by selecting a number of
polymerization variables such as, by way of example, and not
limitation, monomer concentration, percent acrylate modification of
the tissue, initiator concentration, UV light intensity,
temperature, and the like. In one preferred embodiment, acrylic
acid succinimide ester modified or glycidyl methacrylate tissue is
dehydrated in aqueous ethanol solutions. The ethanol-treated tissue
is then transferred to n-vinyl pyrrolidinone (NVP) solution
containing Irgacure 2959
[(4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone)] as a long
wavelength ultraviolet photoinitiator. The NVP-soaked tissue is
irradiated up to 5 minutes using long wavelength UV light
(Black-Ray UV lamp, 360 nm flood light, 10000 mW/cm2 intensity).
The irradiated tissue is washed with PBS solution to remove
unpolymerized monomer and polymerized but uncrosslinked soluble
polyvinyl pyrrolidinone polymer.
[0122] Glycidyl methacrylate tissue incubated in n-vinyl
pyrrolidinone solution but not exposed to light is referred to as
the dark control. Untreated tissue is referred as control tissue.
Table 1 gives pepsin digestion data on pericardial tissue
fixed/crosslinked using glycidyl methacrylate/n-vinyl pyrrolidinone
or glycidyl methacrylate/acrylic acid. The data of Table 1
indicates that the untreated control tissue and dark control tissue
degraded completely in a pepsin solution indicating their
susceptibility to enzymatic degradation. The tissue treated with
glycidyl methacrylate and subsequently treated with n-vinyl
pyrrolidinone or acrylic acid monomer and free radical initiation
showed less than 9 percent weight loss when exposed to pepsin
solution indicating their high stability towards enzymatic
degradation. The source of the tissue such as, by way of example,
and not limitation, bovine pericardium or sheep pericardium did not
show a significant difference in weight loss indicating the
applicability of this treatment to tissues from different animals.
Table 2 lists shrink temperature values for control and treated
tissue. Again, the n-vinyl pyrrolidinone-treated tissue showed a
significantly higher shrink temperature as compared to untreated
control tissue indicating tissue stability and crosslinking.
TABLE-US-00001 TABLE 1 Pepsin digestion data for sheep and bovine
pericardium tissue fixed using glycidyl methacrylate and free
radical polymerizable monomer. Pepsin Digestion Weight Tissue
Treatment Loss (%) Control tissue (untreated tissue) 100* Dark
control (all treatments except exposure to light) 100* Crosslinked
sheep tissue (exposure to UV light and 8.3 photoinitiator, monomer
acrylic acid) Crosslinked sheep tissue (exposure to UV light and
6.5 photoinitiator, n-vinyl pyrrolidinone) Crosslinked bovine
tissue (exposure to UV light and 2.5 photoinitiator and n-vinyl
pyrrolidinone) *The tissue is totally disintegrated and could not
be washed and weighed.
TABLE-US-00002 TABLE 2 Shrink Temperature data for control and
crosslinked tissue Shrink Temperature measured Tissue treatment by
DSC (.degree. C.) Untreated control sheep tissue 60.9 Sheep
pericardium tissue fixed using 82.1 glycidyl methacrylate and
n-vinyl pyrrolidinone
[0123] The crosslinked tissue exhibited a different texture to a
human hand as compared to uncrosslinked tissue indicating
crosslinking. The crosslinked tissue showed substantially higher
shrink temperature as compared to uncrosslinked tissue indicating
crosslinking formation. Unlike glutaraldehyde tissue, the
crosslinked tissue is found to be non-cytotoxic. In another
embodiment of the present invention, a thermal initiator such as,
by way of example, and not limitation, azobisisobutyronitrile is
used to initiate polymerization and crosslinking of tissue. Yet in
another embodiment, radiation or electron beam irradiation or short
UV is used to polymerize and crosslink the tissue in presence NVP
as comonomer. In these systems, no initiator needs to be added. In
some embodiments, a free radical photoinitiator may be chemically
or physically attached to tissue being fixed. One aspect of such
treatment is that the solution can be fixed without external free
radical polymerization initiator.
[0124] Those skilled in the art of free radical polymerization
chemistry will understand that many changes could be made in the
polymerization and crosslinking of tissue, in light of the
teachings of the present invention. The polymerization and
crosslinking could be initiated using a number of monomers. For
example, many monofunctional monomers which form linear soluble
polymers could be used in copolymerization and crosslinking of
tissue and these include, but not limited to, acrylic and
methacrylic esters such as, by way of example, and not limitation,
methyl methacrylate, 1-Adamantyl methacrylate,
1-allyloxy-2,3-propane diol, ethyl methacrylate,
dimethylaminoneopentyl acrylate, propyl methacrylate, isopropyl
methacrylate, n-acryloyl sarcosine methyl ester, dimethylaminoethyl
methacrylate, hexyl methacrylate, butyl methacrylate,
dicyclopentenyl acrylate, 2-ethyl hexyl methacrylate,
2-methacryloyloxyethyl phosphorylcholine, ethoxyethyl methacrylate,
octyl methacrylate, 2-hydroxy-4-acryloylethoxy benzophenone,
stearyl methacrylate, 2-hydroxyethyl methacrylate, 2-aminoethyl
methacrylate, glycidyl methacrylate, isocyanatoethyl methacrylate,
cyclohexyl methacrylate, methyl acrylate, ethyl acrylate, propyl
acrylate, isopropyl acrylate, hexyl acrylate, butyl acrylate,
2-ethyl hexyl acrylate, octyl acrylate, stearyl acrylate,
2-hydroxyethyl acrylate, glycidyl acrylate, isocyanatoethyl
acrylate, ethoxyethyl acrylate, cyclohexyl acrylate,
2,2,3,4,4,4-hexafluorobutyl acrylate; 2,2,3,4,4,4-hexafluorobutyl
methacrylate; 2,2,3,3,3-Pentafluoropropyl acrylate;
2,2,3,3,3-Pentafluoropropyl methacrylate; trifluoroethyl
methacrylate, trifluoroethyl acrylate; tetrafluoroethylene,
fluorinated vinyl ethers, alkyl acrylamides and methacrylamides
such as, by way of example, and not limitation, acrylamide,
methacrylamide, n-propyl acrylamide, butyl acrylamide, isobutyl
acrylamide, ethyl acrylamide, tertiarybutyl acrylamide, n-propyl
methacrylamide, butyl methacrylamide, isobutyl methacrylamide,
ethyl methacrylamide, tertiarybutyl methacrylamide, pentyl
methacrylamide; pentyl acrylamide; styrene and styrene derivatives
such as, by way of example, and not limitation, methyl styrene,
chlorostyrene; vinyl ethers such as, by way of example, and not
limitation, glycidyl vinyl ether; acrylonitrile, acrylic acid,
methacrylic acid, allyl alcohol, allyl amine, polysiloxane or
polyether based macromonomers, polyurethane acrylates and
methacrylate and the like. Many monofunctional monomers which form
linear soluble polymers could be used in copolymerization and
crosslinking of the tissue and these include, but not limited to,
n-vinyl pyrrolidinone, acrylic acid, 2-hydroxyethyl methacrylate,
glyceryl methacrylate, polyethylene glycol acrylate,
2-hydroxypropyl acrylate, monomers used in commercial soft and hard
contact lens manufacturing, 2-hydroxypropyl methacrylate,
acrylamide, vinyl acetate, silicone-based acrylates and
methacrylates, silicone-based monomers used on contact lens
manufacturing, n-isopropyl methacrylate, glycidyl methacrylate,
glycidyl acrylate, methacrylic acid, acrylonitrile, styrene, methyl
methacrylate, methyl acrylate, PEG or PEO acrylates and
methacrylates, monomers obtained from fatty acid and fatty acid
derivatives, monomers obtained from low molecular weight
polyethylene, polypropylene other vinyl polymers and the like.
Monomers which are soluble or substantially soluble in water
(solubility greater than 1 percent in water) are preferred. The
polymerization and crosslinking reaction can be performed in
aqueous, semiaqueous or non-aqueous medium. In general, water or
alcohol water mixtures are preferred. In water, extreme high or low
pH with prolonged exposure times, typically greater than 1 hour are
not preferred at least because such conditions can damage or
denature the tissue. The preferred pH range is between about 10 to
about 2; pH range between about 9 to about 6 is even more
preferred, and physiologic pH range around about 7.2 is most
preferred. Many buffers can be used to control the pH. Buffers such
as, by way of example, and not limitation, phosphate buffer,
triethanol amine buffer, acetate buffer, borate buffer, bicarbonate
buffer, HEPES buffer are preferred. Among non aqueous media,
monomers that can be used as solvents are preferred. For example,
n-vinyl pyrrolidinone is a liquid and can solubilize a variety of
other monomers without significantly affecting the tissue. Other
preferred liquid monomers include liquid PEG-based monomers,
n-vinyl caprolactum, 2-hydroxyethyl methacrylate, glyceryl
methacrylate, and glycidyl methacrylate. organic solvents such as,
by way of example, and not limitation, n-methyl pyrrolidinone,
dimethylsulfoxide, dimethylformamide. dimethylacetamide etc.; lower
ketones (i.e., having from about 3 to 6 carbons) such as, by way of
example, and not limitation, methyl ethyl ketone or cyclohexanone;
and polyhydroxy compounds such as, by way of example, and not
limitation, glycerol, ethylene glycol, or polyethylene glycols
having molecular weights less than about 1000 also could be used
for polymerization and crosslinking. The free radical initiation
necessary for polymerization could also be achieved by exposing the
tissue to gamma radiation, electron beam radiation, UV radiation,
using photoinitiator or thermal initiators. The gamma radiation,
electron beam irradiation or short UV irradiation may not need
external free radical initiator. The photoinitiator could be
selected depending on the wavelength of interest. For long
wavelength UV light, initiator Irgacure 2959, Irgacure 651
(2,2-dimethoxy2-phenyl acetophenone), benzophenone and the like
could be used. For visible light initiation, initiating systems
eosin-triethanol amine (irradiation wavelength 512 nm), methylene
blue-triethanol amine system (irradiation wavelength 632 nm) and
the like could be used. The choice of various photoinitiators and
their corresponding wavelength can be found in photopolymerization
literature art. Preferred photopolymerization conditions,
photoinitiators are described in U.S. Pat. No. 5,410,016 (Hubbell,
et al), which is cited here for reference only as one suitable
technique. There are many free radical polymerization systems that
are known in the dental cement art, contact lens manufacturing art,
or in the radiation-cured coating art which can be used in
polymerization or crosslinking of unsaturated group modified
tissue. However, initiating systems based on long UV or visible
light initiated systems are preferred due to higher penetration of
light inside the tissue. In general, the higher the wavelength
light, the greater penetration in side the tissue. The penetration
of light into the tissue can also be controlled by controlling
photoinitiator concentration or a chromophore that selectively
absorbs light from irradiation sources. The amount of free radical
initiator in the formulation may range from about 0.01% to about 2
percent relative to the weight of monomer. Most preferably, the
preferred initiator amount is about 0.05% to about 1%. Various
light sources such as, by way of example, and not limitation, xenon
lamp, mercury lamp, halogen lamp, argon ion laser, solid sate
semiconductor based lasers may, for example, and without
limitation, be used for photopolymerization and crosslinking of
tissues. The light used in the photopolymerization could be
transported using fiber optic or liquid guide as known in the
photochemistry art or surgical laser instrumentation art. Various
types of thermal initiators could also be used; these include, but
not limited to, azo-based initiators such as, by way of example,
and not limitation, azobisisobutyronitrile, peroxide-based
initiators such as, by way of example, and not limitation, benzoyl
peroxide, inorganic initiators such as, by way of example, and not
limitation, potassium persulfate or ammonium persulfate and the
like. The polymerization medium could be aqueous or non-aqueous. In
one embodiment, the tissue is dehydrated by lyophilization and then
incubated with an aqueous solution of polyethylene glycol acrylate.
The polyethylene glycol acrylate is then photopolymerized and
crosslinked in water in presence of unsaturated group modified
tissue.
[0125] The polymerization and crosslinking may also be conducted in
water under mild conditions. For example, without limitation,
unsaturated group modified tissue is incubated in a 50% aqueous
solution of acrylamide or 2-hydroxyethyl methacrylate containing 2%
Irgacure 2959 for 24 hours. Acrylamide concentration could be
varied from about 10% to about 60% in water with 0.1% Irgacure
2959. The incubated tissue is removed and exposed to long UV light
for 10 minutes to polymerize and crosslink the tissue. The
unreacted monomers are removed by washing. In one embodiment,
glycidyl methacrylate modified bovine pericardial tissue (see
Example 37 described below) was exposed to with 10-50% acrylamide
solution containing 0.1% Irgacure 2959 for 30 minutes and then
exposed to long wavelength UV lamp (360 nm light) for 5 minutes.
The resultant tissue showed significantly higher shrink temperature
as compared to untreated pericardial tissue.
[0126] The degree of polymerization and crosslinking can also be
controlled by adding special additives known in the free radical
polymerization art. For example, a chain transfer agent such as, by
way of example, and not limitation, mercapto ethanol may be added
to control the molecular weight of polymer formed in the tissue.
Other chain transfer agents that can be used include, but are not
limited to, biocompatible thiol, bromine or chlorine containing
organic compounds. These compounds include, but are not limited to
cysteine (amino acid), carbon tetrachloride, chloroform and the
like. Inhibitor may be added to monomers to prevent unwanted
polymerization during storage. The monomer may be purged with inert
gas to remove dissolved oxygen. The molecular weight of polymer
produced may range from 1000 to 5 million g/mole, more preferably
5000 to 150000 g/mole, even more preferably 10000 to 10000 g/mol.
The choice of molecular weight will depend on the intended use.
Typically tissue-polymer composite which require superior
mechanical properties may use high molecular weight polymer
(>10000 g/mole).
[0127] Polymeric crosslinks formed by free radical polymerization
generally results into polymers with verging degree of molecular
weights or chain lengths. The molecular weight distribution of
polymer will depend on experimental conditions used. Typically, the
molecular weight distribution, which is generally defined as ratio
of weight average molecular weight (Mw) to number average molecular
weight (Mn), is 1.1 to 4, more typically 1.3 to 2.5 for most
polymeric systems formed by free radical polymerization. Since
polymer formed during crosslinking reaction as described above have
a range of molecular weight, the polymeric chain length may also
vary. Thus, this invention overcomes the limitation of fixed
molecular length crosslinking methods such as glutaraldehyde
crosslinking and forms polymeric crosslinks with various lengths or
molecular weights.
[0128] Monomers used may be purified before using them in the
polymerization and crosslinking reaction. The purification may be
achieved by techniques such as, by way of example, and not
limitation, vacuum distillation, column chromatography,
recrystallization and the like. Oxygen in the atmosphere may act as
an inhibitor and can alter polymerization kinetics. The effect of
oxygen can be minimized by conducting the polymerization and
crosslinking under inter gas atmosphere. Inert gases such as, by
way of example, and not limitation, nitrogen, carbon dioxide, and
argon are most preferred.
[0129] In one embodiment, the tissue is first crosslinked using a
crosslinker containing two or more tissue reactive groups and a
polymerizable double bond. The double bond in the crosslinker is
then further crosslinked using free radical polymerization. One
aspect of this type of crosslinking is that two methods of
crosslinking are used in preparing the crosslinked tissue. For
example, tissue may be first crosslinked by di- or polyfunctional
unsaturated compounds such as, by way of example, and not
limitation, n,n-methylenebisacrylamide using a Michael addition
reaction known in the synthetic organic chemistry art or a
derivative of unsaturated di- or polyacid. This type of
crosslinking is similar to glutaraldehyde crosslinking known in the
art. The dangling or unreacted double bonds in the crosslinking
reaction and double bonds in the crosslinks may be copolymerized
and crosslinked using free radical photopolymerization as described
earlier. This concept further is further illustrated by way of
example, and not limitation, in FIG. 3 using unsaturated
di-polyacid derivative as an example. The preferred di- or
polyunsaturated compounds that may be used include, but are not
limited to, n,n-methylenebisacrylamide, polyethylene glycol
diacrylate, polyethylene glycol dimethaacrylate, trimethylol
propane triacrylate, trimethylol propane trimethacrylate;
dipentaerythritol methacrylate, and the like. In one exemplary
approach, the tissue is first crosslinked or modified with di- or
polyunsaturated diacids (see FIG. 3). These diacids include, but
are not limited to, fumaric acid, itaconic acid, maleic acid,
castor oil, and the like. Unsaturated acids such as, by way of
example, and not limitation, fumaric acid, maleic acid and itaconic
acid are preferred due to their high reactivity and ability to
copolymerize with variety of monomers. The di- or polyunsaturated
acids could be incorporated into the tissue using several chemical
methods known in the protein modification art. For example,
unsaturated acids like fumaric acid could be incorporated by
incubating the tissue in a 1% solution of fumaric acid in PBS or
MEM buffer at about pH 4.0 to about 8.0 in the presence of
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride (EDC)
as an esterification catalyst (2%) and n-hydroxy succinimide as
co-catalyst (2%). In a preferred method (see Example 32 described
below), an activated derivative of fumaric acid is prepared
separately. The preferred derivative is n-hydroxysuccinimide ester
(NHS) or n-hydroxysulfosuccinimide ester (SNHS) or fumaryl
chloride. The SNHS ester is soluble in water and does not need
organic solvents to disperse it. Tissue such as, by way of example,
and not limitation, pericardial tissue is first reacted with
fumaric acid NHS derivative in PBS. The modified tissue shows
increased shrink temperature as compared to unmodified tissue
indicating crosslinking. The tissue is also less cytotoxic or
non-cytotoxic when compared with tissue fixed using 0.4%
glutaraldehyde. The fumaric acid-modified tissue is further reacted
or crosslinked with monomers capable of undergoing free radical
polymerization as mentioned previously. In one exemplary approach,
fumaric acid-modified tissue is reacted or copolymerized with
n-vinyl pyrrolidinone using UV photopolymerization. The
polymerization reaction variables as well as types of monomers
copolymerized can be changed to obtain a desirable implantable
tissue material. Bovine pericardial tissue modified with fumaryl
chloride and crosslinked with n-hydroxysuccinimide showed higher
shrink temperature as compared to unmodified tissue indicating
crosslinking.
[0130] Yet another embodiment of the present invention provides for
a method for making radio-opaque implantable tissue including the
steps of providing a tissue suitable for human implantation;
exposing the tissue to fluid including a radio-opaque compound
capable of reacting with a tissue; and covalently bonding a
radio-opaque compound to the tissue. Monomers containing
radio-opaque atoms such as, by way of example, and not limitation,
iodine or metal ions are used for polymerization and crosslinking.
The incorporation of radio-opaque polymerizable monomers will
provide radio-opacity to the tissue. The preferred monomers that
may, for example, and without limitation, be used are polymerizable
derivatives or iodinated compounds such as, by way of example, and
not limitation, polymerizable monomer obtained by esterification of
triiodobenzoic acid and 2-hydroxyethyl methacrylate. Other
iodinated derivatives that may be converted into polymerizable
monomers by esterification of hydroxyl groups include, but are not
limited to iohexol, metrizamide, iopamidol, iopentol, iopromide,
erythrosin, and ioversol. The polymerizable derivative of
metrizamide is most preferred. The radio-opacity is controlled by
the changing the amount of radio-opaque monomer incorporated in the
tissue. The iodine content of tissue may vary from about 20 to
about 300 mg iodine per gram of tissue weight. More preferably, the
amount of iodine may be about 40 to about 200 mg/g of tissue.
[0131] Tissue could be chemically modified with unsaturated group
and free radical initiating group such as, by way of example, and
not limitation, eosin or benzophenone. For example, eosin could be
physically adsorbed or chemically bound to the tissue using the
esterification of carboxyl group on the eosin molecule. Such
initiator and unsaturated group modified tissue can be crosslinked
using free radical initialing chemistry such as, by way of example,
and not limitation, photoinitiation chemistry as discussed
previously. In a similar manner, tissue could be chemically
modified with thermal initiator and polymerizable group on the same
protein chain. One aspect of such systems is that no external free
radical initiator is needed to initiate free radical polymerization
and crosslinking.
[0132] In some embodiments, the tissue could be modified with
chemical groups which undergo cyclic dimerization upon exposure to
heat or light energy could be attached to tissue and then
subsequently dimerized to crosslink the tissue. The compounds which
undergo cyclic dimerization include, but are not limited to,
cinnamic acid, coumarin, chalcone and thymine Many functional
groups which undergo cyclic dimerization are known in the
photochemistry art and could be used; however the derivatives of
cinnamic acid are most preferred. Cinnamic acid and its derivatives
are known to undergo photodimerization on irradiation with
ultraviolet light. This reaction is highly specific and does not
require free radical photoinitiator. It can be carried out in
liquid, solid, or solution state. Photocurable polymers which can
be cured or crosslinked by photodimerization reaction of cinnamic
acid have been extensively used in arts related to lithography,
paints and printing. In a preferred embodiment, the tissue is first
modified with cinnamic acid derivative such as, by way of example,
and not limitation, n-hydroxysulfosuccinimide derivative of
cinnamic acid or cinnamoyl chloride. The modified tissue is then
exposed to long wavelength ultraviolet light (360 nm) or visible
light to dimerize and crosslink the unsaturated groups in the
tissue.
[0133] Prosthetic Tissue with Biostable and Biodegradable
Regions
[0134] In some embodiments, photopolymerization and crosslinking is
controlled by the exposure of light to certain parts of the tissue.
This may, for example, and without limitation, be used to crosslink
only selected parts of the tissue. In one exemplary embodiment, the
tissue was exposed to the light through a mask which permits
exposure of light only on a selected predetermined area. The tissue
exposed to the light undergoes polymerization and crosslinking.
Parts of the tissue, which are not exposed to light remain
uncrosslinked and therefore remain susceptible for degradation.
This concept is illustrated, by way of example, and not limitation,
in FIG. 5. In one exemplary embodiment (see Example 40 described
below), a 2 cm diameter tissue treated with glycidyl methacrylate
as shown in Example 37 part 1. The tissue is incubated in, by way
of example, and not limitation, n-vinyl pyrrolidinone solution
containing photoinitiator (Example 37, part 2). The incubated
tissue is kept on a glass plate. Two 4 mm diameter aluminum foil
sections or arrow shape foil sections are cut and placed on the
tissue. The tissue is then exposed to long wavelength UV light for,
by way of example, and not limitation, 5 minutes for polymerization
and crosslinking of the tissue. The same procedure is repeated from
other side of the tissue. The tissue is then exposed to pepsin
solution. The areas of the tissues which are covered by aluminum
foil showed substantial degradation and is completely solubilized
while the light-exposed tissue did not show signs of degradation
and was mechanically intact. Thus a pattern of degradable and
non-degradable regions is created in the same tissue. Many types of
patterns of crosslinked and non-crosslinked tissue may be generated
which include rectangular, circular or complex patterns or shapes
inside the tissue matrix using this approach. The selective
crosslinking and polymerization approach could be, for example,
used to selectively reinforce certain parts of medical devices such
as, by way of example, and not limitation, heart valve. Certain
high-stressed sections of bovine pericardial heart valve tissue are
susceptible to calcification and mechanical failure. Such sections
may be selectively crosslinked by exposing those sections to light.
It can also be used in vivo tissue engineering application.
[0135] In some embodiments, the solvents and other crosslinking
constituents used during the polymerization and crosslinking
process may be subjected to mild or high pressure to obtain a
suitable crosslinking density.
[0136] Shape Preserving Tissue Fixation
[0137] In some preferred embodiments, the collagen fibers may be
strained or oriented along the direction of pull prior to
polymerization and crosslinking. The tissue orientation may be, for
example, and not by way of limitation, achieved through the use of
mechanical force. For example, unsaturated group modified bovine
pericardial tissue is dried, infused with n-methyl pyrrolidinone
and photoinitiator 2959 and then stretched to about 110 to about
150% of its length to orient the fibers along the direction of
force. The monomer and initiator infusion could also be made after
orienting the tissue. The strained tissue is then exposed to long
UV light for polymerization and crosslinking. The polymerization
and crosslinking will "lock in place" the oriented structure of the
proteins/fibers in the tissue and thus yielding a tissue with
superior mechanical properties, especially along the axis of
orientation. Modification of various variables used in this method
such as, by way of example, and not limitation, direction of force
applied, axis of orientation, % stretching or compression used,
polymerization conditions, monomers used must be optimized for a
given application. It was discovered that some monomers which
produced highly crosslinked and rigid polymers can substantially
preserve the shape of a crosslinked tissue. This concept is
schematically illustrated, by way of example, and not limitation,
in FIG. 6. In one exemplary embodiment (see Example 29 described
below), a 15 cm long and 3 mm wide strip of bovine pericardial
tissue modified with glycidyl methacrylate was infused with
triethyleneglycol dimetharylate or tetraethyleneglycol
dimethacrylate monomer containing 500 ppm Irgacure 651
photoinitiator. The monomer infused strip is then spirally wound,
by way of example, and not limitation, on a 5 mm diameter mandrel
(glass rod) in a helical shape. The tissue was then exposed to long
UV light for 5 minutes (Black-Ray UV lamp, 360 nm light, 10000
mW/cm2 intensity) for polymerization and crosslinking of the
monomer. The glass rod was removed. The tissue shape of helical
coil was substantially preserved after removal of glass rod or
mandrel. When coiled tissue was stretched along the axis of the
helical coil, and the stress is removed, the tissue substantially
returned to the helical coil shape indicating shape memory
property. This ability to remember the shape after fixation on
crosslinking can be beneficial in making novel medical devices. For
example, it can be used to form a tissue-based stent-like devices.
The light exposure and polymerization can be done in situ at a
surgical site. The monomer and photoinitiator infused tissue strip
is immobilized, by way of example, and not limitation, on an
angioplasty catheter. The catheter is capable of emitting 360 nm
light carried using, by way of example, and not limitation, fiber
optic cable on the balloon surface. The tissue is transported at
the angioplasty site using, for example, without limitation,
standard balloon angioplasty techniques, expanded and exposed to
emitting 360 mm light until polymerization is complete (typically
less than 5 minutes). The balloon is withdrawn and coiled tissue is
left at the angioplasty site which provides mechanical support
similar to metal-like stent. In another modification of this
approach, a monomer infused unsaturated group modified pericardial
tissue as mentioned previously was converted or sewn into a 6 mm
diameter tube. The tube was then mounted on a 6.1 mm diameter
stainless steel mandrel and compressed to reduce the length of the
tube (10 to 50 percent reduction in length). The compressed tissue
is then exposed to UV light to crosslink the monomer and fix the
tissue. The shape of the tissue is preserved in a compressed shape.
The mandrel is removed and the compressed tissue tube held the
shape with accordion-like wrinkles in the tube. The tube is
compressible along the axis of the tube. Such a tube may be useful
to make peripheral vascular and coronary graft and other medical
devices. Any desired drug such as, by way of example, and not
limitation, heparin, by way of example and not limitation, may be
incorporated in the tube to influence the desired biological or
therapeutic outcome.
[0138] One embodiment of the present invention provides for a
method for making a tissue capable of remembering a shape.
[0139] In one embodiment, a radiation polymerization or electron
beam-induced polymerization technique is used to form a crosslinked
tissue. Such a reaction can be carried out in the solid state. The
gamma radiation or electron beam irradiation generates several free
radicals in the tissue. The generated free radicals react with
unsaturated groups in the tissue to promote crosslinking. Radiation
polymerization may not require free radical initiators. The dose of
radiation will depend on the amount of polymerization and
crosslinking desired. It is contemplated that such optimization can
be readily undertaken by one skilled in the art, in light of the
teachings of the present invention, without undue experimentation.
Low to moderate radiation doses, typically below about 3-5 MRAD,
are preferred at least because high radiation dose may also degrade
collagen molecular chain.
[0140] One aspect of the present invention is that many monomers
with different properties such as, by way of example, and not
limitation, charge density, hydrophobicity, thermosensitivity could
be used to impart different properties to the crosslinked tissue.
In one embodiment, a functional monomer such as, by way of example,
and not limitation, glycidyl methacrylate which has a glycidyl
group is copolymerized along with vinyl pyrrolidinone. The
polymerization of glycidyl methacrylate provides glycidyl
functional groups on the crosslinked polymer chain which could be
further reacted using standard epoxy chemistry for additional
crosslinking or other types of tissue modifications. Functional
monomers that could be used, but are not limited to, are
2-isocyanate methacrylate, acrylic acid, methacrylic acid,
2-hydroxyethyl methacrylate, glyceryl methacrylate and the like. As
those skilled in the art will appreciate, chemical variables such
as time, temperature, concentrations, pressure, catalysts and the
like may be controlled to promote additional crosslinking.
Moreover, the use of these variables will, at least, depend on a
particular chemical reaction used in crosslinking.
[0141] In another embodiment, monomers containing long alkyl chains
are used in polymerization and crosslinking. The introduction of a
long chain through crosslinking can reduce its calcification
potential and may improve hemocompatibility of the crosslinked
tissue. It is hypothesized that long chain alkyl chains, typically
more than about 8 carbons (C8), more typically with carbon lengths
such as, by way of example, and not limitation, C16-C18,
specifically bind to albumin and thus improve hemocompatibility of
the tissue. However, shorter alkyl chains, typically >C4, may
also be advantageous for some applications.
[0142] In some embodiments, the monomers that give fluorinated
polymers upon polymerization may, for example, and without
limitation, be used to incorporate fluorinated polymers in the
tissue. The monomers including fluorinated monomers such as, by way
of example, and not limitation, acrylates and methacrylates with
fluorinated alkyl chains may, for example, and without limitation,
be used. The fluorinated monomer include, but are not limited to,
2,2,3,4,4,4-hexafluorobutyl acrylate; 2,2,3,4,4,4-hexafluorobutyl
methacrylate; 2,2,3,3,3-pentafluoropropyl acrylate;
2,2,3,3,3-pentafluoropropyl methacrylate; trifluoroethyl
methacrylate, trifluoroethyl acrylate, tetrafluoroethylene and the
like.
[0143] In another embodiment of the present invention, a silicone-
or polydimethylsiloxane-based monomer or macromonomer is used in
polymerization and crosslinking of the tissue. Many silicone- or
polydimethylsiloxane-based monomers could be suitably used and are
known to one skilled in the art. The incorporation of highly
elastomeric silicone-based monomers incorporates silicone rubber in
the tissue matrix which provides elastomeric properties and
improves oxygen permeability of the tissue in many applications.
Polypropylene glycol and some polyurethane acrylates and
methacrylate-based monomers that also form elastomeric polymeric
crosslinks may also be used. Commercially available polydimethyl
siloxane-based macromonomers known in the soft contact lens
manufacturing art can be useful in many applications, at least
because use of such monomers is already commercialized in high
oxygen permeable contact lens applications.
[0144] In another exemplary embodiment (see Example 11 described
below), a monomer which produces a thermosensitive polymer such as,
by way of example, and not limitation, poly(n-isopropyl acrylamide)
is used in polymerization and crosslinking of tissue. The
incorporation of thermosensitive polymer crosslinked network
permits one to rehydrate the collagen chains at low temperature
(<10.degree. C.). At body temperature (37.degree. C.),
poly(n-isopropyl acrylamide) becomes hydrophobic. The hydrophobic
domains thus formed may provide interesting properties to the
tissue. For example, hydrophobic domains may, for example, and
without limitation, be used to lock or dissolve hydrophobic drugs
in the treated tissue. The hydrophobic domains may also provide
additional protection against enzymatic degradation of the tissue.
Many n-alkyl acrylamide-based monomers which upon polymerization
yield thermosensitive polymers could be used, these include, but
are not limited to, n-ethyl acrylamide, n-propyl acrylamide,
n-isopropyl acrylamide, n-butyl acrylamide, n-isobutyl acrylamide,
n-tertiarybutyl acrylamide, n-pentyl acrylamide, and the like. In
one illustrative embodiment, glycidyl methacrylate treated
pericardial tissue (see Example 37, part 1 described below) is
exposed to, for example, without limitation, 50 percent
n-isopropylacrylamide solution in water containing 0.1% Irgacure
2959 for 2 hours. The monomer infused tissue was exposed to long UV
light for 5 minutes (Black-Ray UV lamp, 360 nm light, 10000 mW/cm2
intensity) for polymerization and crosslinking of the monomer. The
tissue was washed with distilled cold water to remove monomer and
polymerized but not covalently bound polymer. The tissue represents
an example of pericardial tissue crosslinked using thermosensitive
polymer. The tissue showed substantially higher shrink temperature
as compared to untreated pericardial tissue indicating crosslinking
using n-isopropylacrylamide. Any number of bioactive compounds such
as, by way of example, and not limitation, paclitaxel, for example,
could be diffused inside the matrix at low temperature (typically
below 15.degree. in water). At body temperature,
n-hydroxysuccinimide becomes hydrophobic trapping the drug in the
hydrophobic matrix and releasing it in a controlled manner.
Hydrophobic drugs such as, by way of example, and not limitation,
paclitaxel, rapamycin, chlorhexidene gluconate are preferred. Other
hydrophobic drugs may also be used in alternate embodiments of the
present invention. Certain polymeric monomers or macromonomers,
such as, by way of example, and not limitation, monomer made from
block copolymers of PEG and polypropylene oxide or polypropylene
oxide, for example PEG-PPG-PEG acrylates and methacrylates which
are soluble in water at low temperature may also be used.
Alternatively, thermosensitive polymer-based crosslinkers can also
be used to crosslink the tissue. The crosslinking may be carried
out when the thermosensitive polymer crosslinker is soluble in
water.
[0145] In another embodiment, the monomers containing charged
groups such as, by way of example, and not limitation, sulfonic
acid salts or carboxylic acid salts are used for polymerization and
crosslinking. It is believed that incorporation of charged groups
in the crosslinked tissue may reduce the propensity for
calcification and improve hemocompatibility. The monomers
containing charged groups that may be used, but are not limited to,
are 2-acrylamido-2-methylpropane sulfonic acid, vinylsulfonic acid,
styrene sulfonic acid, sulfoethylmethacrylate,
sulfopropylmethacrylate, or other vinyl sulfonic acids; acrylic
acid, methacrylic acid, fumaric acid, maleic anhydride, maleic
acid, fumaric acid, itaconic acid, and the like. Monomers
containing sulfonic acid salts are most preferred. The
polymerization of such charged groups creates localized acidic or
basic environment which may reduce calcification upon implantation.
In one exemplary embodiment, crosslinking of glycidyl methacrylate
modified tissue using 2-acrylamido-2-methylpropane sulfonic acid
showed substantially higher shrink temperature as compared to
untreated tissue indicating crosslinking.
[0146] In another embodiment, the monomers containing free amine
groups are used for polymerization and crosslinking of the tissue.
The amine groups in the monomers can be used to introduce
additional crosslinking in the tissue. The amine-containing
monomers that may be used, but are not limited to, are 2-aminoethyl
methacrylate, allyl amine, 3-aminostyrene, and the like. After
polymerization, amine groups in the polymeric crosslinks may be
further crosslinked using difunctional amine reactive crosslinkers
such as, by way of example, and not limitation, glutaraldehyde,
EDC, di- or polyepoxides based crosslinkers. For example, without
limitation, tissue crosslinked using allyl amine is further treated
with 0.2% glutaraldehyde solution for 24 hours. The glutaraldehyde
treatment creates additional crosslinking tissue proteins and amine
groups of polymerized allyl amine.
[0147] In some embodiments, monomers containing phosphorylcholine
moiety may be used to crosslink the tissue. Monomers such as
2-methacryloyloxyethyl phosphorylcholine (MPC) could be infused
into the tissue and homopolymerized or copolymerized to promote
crosslinking and/or grafting. The MPC polymers and copolymers are
well-known for their excellent biocompatibility and blood
compatibility presumably due to cell membrane like properties of
MPC copolymers
[0148] In one embodiment, polyvinyl alcohol is incorporated in the
tissue. This is achieved by polymerizing vinyl acetate monomer in
the glycidyl methacrylate modified tissue. The polyvinyl acetate
incorporated in the tissue is converted into polyvinyl alcohol by
exposing the tissue to mild acid hydrolysis conditions. The
polyvinyl alcohol incorporated in the tissue could be further
crosslinked by methods known in the art such as, by way of example,
and not limitation, glutaraldehyde crosslinking, metal ion
crosslinking or freezing and thawing. Using a similar approach,
semicrystalline polymer polyacrylonitrile can be incorporated in
the tissue and is then partially hydrolyzed to produce polyacrylic
acid. The partially hydrolyzed polyacrylonitrile produces strong
mechanical hydrogels.
[0149] In another embodiment of the present invention, a monomer
which forms a polymer with ability to form complexes with ions,
drugs, small molecules and polymers is used in crosslinking and
polymerization. In one exemplary embodiment, vinyl pyrrolidinone
monomer is used to polymerize and crosslink the modified tissue as
(see Example 37 described below). The polymerized polyvinyl
pyrrolidinone (PVP) can form complexes with various bioactive
compounds such as, by way of example, and not limitation, free
iodine, iodide ions and antibiotics. Using a similar scheme,
polyacrylic acid introduced by crosslinking with acrylic acid
monomer is complexed with polyethylene glycol. It is understood
that many other polymer-polymer complexes or polymer-small compound
complexes could be used. A monomer-containing cyclodextran may also
be used. The cyclodextran moiety is useful for complexing variety
of bioactive compounds. The selection of such complexes will depend
on the properties desired and can readily be practiced by one
skilled in the art in light of the teachings of the present
invention. For example, an iodine-polyvinyl pyrrolidinone complex
may, for example, and without limitation, be used to impart
antibacterial properties to the tissue. Polyacrylic acid or acid
groups present in the tissue may be complexed or ionized with
silver salts to produce an antimicrobial effect due to release of
silver ions. Many silver salts can be used to form a silver salts;
these include, but are not limited to, silver acetate, silver
benzoate, silver chloride, silver carbonate, silver iodide, silver
iodate, silver nitrate, silver laureate, silver sulfadiazine,
silver palpitate, silver aspartate, silver succinate, and mixtures
thereof. Silver phosphate, silver sulfadiazine, silver citrate,
silver lactate, and mixtures thereof are preferred. In one
embodiment, the ionized polyacrylic acid modified tissue
material/collagen implant is incubated in a concentrated solution
of sodium lactate (for example, without limitation, 0.1M to 1.0M)
for about 10 to about 60 minutes, most preferably about 30 minutes.
The materials are then transferred to a selected silver salt
solution (e.g., silver lactate) for about 5 to about 120 seconds,
preferably about 60 seconds, in order to produce an anti-microbial
surface that retains silver ions and slowly releases them over an
extended period.
[0150] In another embodiment of the present invention, a di- or
polyfunctional water soluble macromonomers such as, by way of
example, and not limitation, polyethylene glycol diacrylate or
polyethylene glycol dimethaacrylate is used to polymerize and
crosslink the animal tissue. In one embodiment, a 5 to 35%, more
preferably 10-25%, even more preferably 23% solution of
polyethylene glycol, molecular weight 10000 moles/g (PEG10 KDA) is
used to crosslink the tissue. Briefly, unsaturated groups modified
tissue is soaked in the PEG10DA solution containing Irgacure 2959
as photoinitiator and heparin as a bioactive compound. The PEG10DA
in the tissue is then polymerized and crosslinked. The tissue is
washed with water to remove unreacted PEG10DA. The heparin
entrapped in the crosslinked tissue-PEG10DA matrix is slowly
released. Other monomers such as, by way of example, and not
limitation, polyethylene glycol dimethaacrylate, polypropylene
glycol diacrylate or methacrylate, to name a few, may also be used.
Polyethylene glycol diacrylate with molecular weight from 400 to
100000 Daltons could be used to control the molecular porosity of
the crosslinked polymer network. In some embodiments, the monomers
containing degradable links are used to crosslink the tissue. The
monomers could be small molecule in nature (typically molecular
weight less than 2000) or polymeric macromonomers. In one
embodiment, a polyethylene glycol based degradable macromonomer is
used. Briefly, a biodegradable monomer is synthesized from
polyethylene glycol. The polyethylene glycol chain is extended
using oligomeric polyhydroxyacid or polylactone links and then
terminated with acrylate polymerizable end groups. The
polyhydroxyacid or polylactone links serve as biodegradable sites
in the monomer. These sites undergo hydrolysis or biodegradation
and break the polymeric crosslinks and making the tissue
susceptible for enzymatic degradation. In another embodiment of the
present invention, a liquid hydrophobic biodegradable monomer is
synthesized by polymerization of cyclic lactones and modifying the
terminal group with polymerizable groups such as, by way of
example, and not limitation, acrylic group is used. Briefly,
unsaturated group modified tissue and biodegradable monomers are
copolymerized using a free radical polymerization technique such
as, by way of example, and not limitation, photopolymerization.
Upon crosslinking, the degradable polyhydroxyacid or polylactone
links undergo hydrolysis and the tissue becomes non-crosslinked and
therefore susceptible for enzymatic degradation. Depending on the
type of polyhydroxyacid or polylactone used, the crosslinked tissue
degradation may be controlled. The degradation may range from weeks
to years. In general, oligoglycolate polymer crosslinks will
degrade in weeks, oligolactate crosslinks will degrade in months
and oligocaprolactone crosslinks will degrade in years. In one
embodiment, the hydrophobic liquid biodegradable oligomer is
polymerized in the presence of tissue microparticles obtained by
cryogenic grinding. The polymerization is conducted in a mold and
sodium chloride is used as a porosity-inducing compound. In another
embodiment of the present invention, a degradable monomer
containing hydrolizable bond is obtained from hydroxy alcohols such
as, by way of example, and not limitation, hydroxy amine or ethanol
amine. These hydroxy alcohols are reacted with acryloyl chloride or
methacryloyl chloride to form ester-amide derivative. The ester
bond is susceptible to hydrolysis upon implantation. Biodegradable
monomers containing peptide links that can be broken down by
enzymes such as, by way of example, and not limitation,
collagenease, pepsin, etc. may also be used in crosslinking. The
tissue-synthetic biodegradable crosslinked material composite may,
for example, and without limitation, be used as scaffold for tissue
engineering such as, by way of example, and not limitation,
scaffold for bone tissue engineering. Cells, enzymes or bioactive
compounds may be incorporated during polymerization and
crosslinking process to enhance the therapeutic effect.
[0151] Di- or polyunsaturated monomers containing degradable bonds
have also been disclosed in U.S. Pat. Nos. 6,713,646 (Zhang, et al)
and 5,410,016 (Hubbell, et al), which are cited here for reference
only. Such monomers and their copolymers with other known
biocompatible monomers can also be used to form biodegradable
hydrogels in the tissue matrix. Briefly, monomers described in the
above cited references are reacted with tissue modified with
unsaturated groups and polymerized as described above. The
hydrogels incorporated into the tissue matrix degrade by hydrolysis
making the tissue susceptible to enzymatic degradation.
[0152] In some embodiments, a glutaraldehyde-crosslinked tissue or
EDC crosslinked tissue is first reacted with glycidyl acrylate or
methacrylate to introduce unsaturated groups in the tissue. These
unsaturated groups are then further crosslinked using various
monomers as described above. This approach is especially useful for
heart valve bioprosthesis made using bovine pericardial tissue or
porcine aortic root tissue.
[0153] In some instances, more than one crosslinking method may be
employed to obtain a desirable crosslinked tissue.
[0154] In an alternate embodiment of the present invention, the
method for making the tissue is carried out such that part(s) of
the tissue are biostable and/or biodegradable. Table 3 provides a
summary of experiments performed to stabilize the tissue by various
embodiments discussed above.
TABLE-US-00003 TABLE 3 Summary of experiments related to biostable
tissue stabilized by polymeric crosslinks. The tissue was first
modified with unsaturated groups and then crosslinked by free
radical photopolymerization using various monomers Visual
Observation Unsaturated after 60 day group Monomer used in Shrink
Resistance subcutaneous modifying free radical UV light Temperature
to pepsin implantation Group Type of Tissue reagent polymerization
exposure (.degree. C.) digestion in rat Untreated Bovine No
treatment No Treatment No 60 Digested Substantially Control
pericardium completely degraded No BP No treatment Vinyl
pyrrolidinone, Yes 60 Digested -- Unsaturated 100% (VP) completely
group control Dark Control BP Glycidyl 100% VP NO 60 Digested
Substantially methacrylate completely degraded (GM) VP fixed-1 BP
GM 100% VP Yes >95 Very good No degradation VP fixed-2 BP GM
100% 2- Yes >95 -- -- hydroxyethyle methacrylate VP fixed-3 BP
GM 20% NIPAM Yes >95 -- -- VP fixed-4 BP GM 50% NIPAM + 50% Yes
>95 -- -- VP VP fixed-5 BP GM Tetraethyleneglycol Yes >95 --
-- dimethacrylate VP fixed-6 BP GM 2-acrylamido-2- Yes -- -- --
methylpropane sulfonic acid VP fixed-7 Bovine vein GM 100% VP Yes
>95 Very good No degradation VP fixed-8 Bovine cornea GM 100% VP
Yes -- -- -- VP fixed-9 Bovine GM 100% VP Yes -- -- -- meniscus VP
fixed-10 Sheep skin GM 100% VP Yes >95 Very good -- Acrylic acid
BP GM 100% Acrylic acid Yes >95 Very good fixed Acrylamide BP GM
50% acrylamide in Yes >95 Very good fixed water Acrylamide BP GM
10% acrylamide in Yes >95 Very good fixed water Fumaryl BP
Fumaryl 100% VP Yes >95 Very good chloride chloride Methacrylic
BP MA 100% VP Yes >95 Very good anhydride (MA) Methacrylic BP MA
100% VP No. 73 -- anhydride Used (MA) thermal initiator
[0155] It clear that a variety of tissue types from various animal
sources can be stabilized using the inventive methods described
above. In many cases, the tissue did not show shrinkage in boiling
water indicating high stability and crosslinking of
collagen/protein molecules. The unmodified tissue, dark control
tissue (tissue modified with unsaturated groups and infused with
monomer and photoinitiator but not exposed to light) and
unsaturated group control (tissue not modified with unsaturated
group, but infused with monomer and photoinitiator solution and
exposed to UV light) showed substantial degradation when subjected
to pepsin digestion and low shrink temperature, typically around
about 60.degree. C. Thermally initiated polymerization also showed
higher shrink temperature. Some fixed and unfixed tissues are
subjected to rat subcutaneous implantation for 60 days. The fixed
tissue showed excellent biostability with no sign of degradation.
The untreated tissue and dark control tissue are either completely
digested or substantially digested indicating poor biostability or
biodegradation.
Crosslinking of Tissue with Di- or Polymercapto Compounds
[0156] The tissue modified with polymerizable unsaturated groups
may be crosslinked using di- or polyfunctional mercapto compounds.
The modified tissue is treated under effective cross-linking
conditions with a di- or polymercapto organic compounds to
crosslink the tissue. FIG. 7 is a schematic representation of
exemplary steps involved in tissue crosslinking using mercapto
compounds, in accordance with an embodiment of the present
invention.
[0157] In one embodiment, a method of cross-linking a tissue is
provided that treats the tissue under effective cross-linking
conditions with a di- or polymercapto organic compound.
[0158] By "di or polymercapto" is meant any compound including the
structure:
SH--R--SH
[0159] wherein R is an organic moiety having at least 1 carbon atom
and SH is a thiol or mercapto group capable reacting with
unsaturated carbons such as, by way of example, and not limitation,
acrylamide, methacrylamide, acrylate or methacrylate groups. R may
have one or more mercapto groups.
[0160] Many di- or polymercapto compounds may, for example, and
without limitation, be used in tissue crosslinking. The preferred
compounds include, but are not limited to, 1,3-propane thiol,
1,4-butane thiol, 1,5-hexane thiol, polyethylene glycol terminated
with thiol, trimethylolpropane tris(3-mercaptopropionate), and the
like. In one preferred embodiment, the di- or polymercapto organic
compound is a trimethylolpropane tris(3-mercaptopropionate).
[0161] Preferably, the di- or polymercapto organic compound is a
solute in a fluid including a solvent. The fluid including the di-
or polymercapto organic compound also includes a solvent. The
solvent can be any liquid in which the compound is soluble and in
which the compound does not undergo degradation or side reactions.
If the di- or polymercapto organic compound is not soluble in
water, but is soluble in organic solvents, dimethyl sulfoxide
(DMSO) or acetonitrile, for example, may, for example, and without
limitation, be used as a co-solvent.
[0162] The concentration of the di- or polymercapto organic
compound in the fluid is preferably between about 0.1 mg/mL and
about 100 mg/mL. More preferably, the concentration is between
about 1 mg/mL and about 20 mg/mL.
[0163] The pH of the fluid can be any pH which is not deleterious
to the tissue being treated or the cross-linking reaction. The pH
of the fluid can be adjusted by any appropriate technique.
Typically, the pH of the fluid is between about pH 6 and about pH
10. This pH range allows cross-linking to be relatively rapid and
have a relatively low amount of side-reactions. Preferably, the pH
of the fluid is between about pH 7 and about pH 9. More preferably,
the pH of the fluid is between about pH 8 and about 9.
[0164] The temperature of the fluid may be any temperature at which
the cross-linking reaction is relatively rapid and a relatively low
amount of side reactions occur. Preferably, the temperature of the
fluid is between about 0.degree. C. and about 45.degree. C. More
preferably, the fluid temperature is between about 0.degree. C. and
about 30.degree. C. Conveniently, the reaction may be carried out
at room temperature about 25 to about 30.degree. C.
[0165] One skilled in the art will readily recognize, in light of
the teachings of the present invention, that the duration of
treatment is not critical, so long as the tissue and the di- or
polymercapto organic compound remain in contact long enough for
cross-linking to proceed to the desired extent. The duration of
treatment may vary depending on the tissue being treated or the di-
or polymercapto organic compound being used for cross-linking.
Typically, treatment duration is in the range of from about 1 min
to about 24 hr. Preferably, treatment duration is at least about 30
min, more preferably at least about 6 hr.
Tissue Crosslinking by Hydrogen Abstraction Mechanism
[0166] Aromatic ketones such as, by way of example, and not
limitation, benzophenone are known to undergo hydrogen abstraction
reaction when exposed to long UV light. In one aspect of the
present invention, hydrogen abstraction reaction is exploited for
tissue crosslinking. In one embodiment, a method of crosslinking a
tissue is achieved by steps that include, without limitation,
treating the tissue under effective cross-linking conditions with a
crosslinker capable of undergoing hydrogen abstraction reaction;
and crosslinking the tissue by hydrogen abstraction mechanism.
Surmodics Inc. has commercialized a technology based on hydrogen
abstraction reaction for medical device coatings and surface
modifications. In one exemplary embodiment (see Example 34
described below), a benzophenone derivative of a polymer
(polyethylene glycol terminated with benzophenone) is synthesized.
This polymer is then infused into a tissue and the tissue is
exposed to long UV light (360 nm). The benzophenone undergoes
hydrogen abstraction reaction and crosslinks the tissue. Those
skilled in the art of surface modification will readily recognize,
in light of the teachings of the present invention, that that many
reaction variables can such as, by way of example, and not
limitation, polymer concentration, exposure time, light intensity,
type of chromophore used may be changed to obtain an effectively
crosslinked tissue. Many polymers that can be modified using
benzophenone are useful in crosslinking. These include, but are not
limited to, polyvinyl pyrrolidinone, heparin, polyvinyl alcohol,
polyethylene glycol and their copolymers, and the like. Many
different chemicals that undergo hydrogen abstraction reaction
could be used. Chemicals that undergo hydrogen abstraction reaction
in long UV or visible light range are preferred. One embodiment of
the present invention provides for a method for forming a
crosslinked tissue including the steps of providing a tissue
suitable for human implantation; exposing the tissue to a fluid
including a compound capable of reacting with a tissue and
undergoing hydrogen abstraction reaction when exposed to light;
covalently bonding at least portion of the tissue functional groups
with the a compound capable of undergoing hydrogen abstraction
reaction when exposed to light; and crosslinking the tissue under
effective cross-linking conditions by exposing the tissue to a long
UV or visible light.
[0167] In addition, proteins in the tissue may be chemically
modified using a benzophenone derivative and then crosslinked by
exposing to UV light at 360 nm. Benzophenone undergoes hydrogen
abstraction reaction upon exposure to UV light and undergoes
crosslinking. The crosslinking density can be controlled by amount
of benzophenone substituted on the tissue proteins. In one
embodiment, the present invention provides for an activated
derivative of benzophenone carboxylic acid. For example, carboxyl
groups of 2-carboxyl benzophenone may be activated by forming
n-hydroxysuccinimide ester or -hydroxysulfosuccinimide ester and
then reacted with tissue in water (PBS pH 7.2). The
benzophenone-modified tissue is exposed to high intensity 360 nm UV
light to crosslink the tissue.
[0168] Compositions and methods for making biodegradable tissue or
tissue/synthetic biodegradable polymer composite materials will
next be discussed in some detail.
Tissue Crosslinked Using Degradable Crosslinker
[0169] In another aspect of the present invention, a method of
cross-linking a tissue that is achieved by steps that include,
without limitation, treating the tissue under effective
cross-linking conditions with a crosslinker containing
biodegradable chemical bonds.
[0170] A method is herein provided, by way of example, and not
limitation, for cross-linking biological tissues to be used in the
production of biodegradable animal tissue based medical devices and
controlled drug delivery systems. This is achieved by contacting a
biological tissue of interest with one or more of the disclosed
compounds under conditions effective to cause the desired degree of
tissue cross-linking. The crosslinks in the crosslinked tissue
undergo biodegradation when implanted into human or animal body.
Upon hydrolysis of the crosslinker, the tissue becomes susceptible
to enzymatic degradation and degrades into non-toxic components.
One aspect of the present invention is to provide a straightforward
approach for producing biodegradable animal tissue-based
bioprostheses having desirable mechanical and biocompatibility
features and control over degradation of the tissue.
[0171] FIG. 8 is a schematic representation of an exemplary tissue
crosslinking using biodegradable tissue crosslinker, in accordance
with an embodiment of the present invention. The biodegradable
crosslinkers compounds suitable for use with this invention can be
generally represented by the following structural formula:
F--B--X--F
[0172] wherein F is a functional group reactive with collagen,
elastin or other tissue-based constituents; and X is an organic
molecule or radical at the core of the crosslinker. X may include
Carbon-Carbon, Carbon-Hydrogen, Carbon-Nitrogen, Carbon-Oxygen,
Carbon-Sulfur, Nitrogen-Hydrogen and Oxygen-Hydrogen covalent
bonds.
[0173] F is a functional group that is sufficiently reactive with
the collagen molecules present in the biological tissue to be
treated such that the desired chemical bond is formed. For example,
functional groups reactive with collagen and suitable for use may
include, but are not limited to, anhydride, isocyanate, epoxy,
n-hydroxysuccinimide, aldehyde or other protein reactive
functionalities known in the art. It is desirable to have two or
more functional groups per crosslinker for effective
crosslinking.
[0174] B is a biodegradable link between two F moieties.
"Biodegradable link" denotes a covalent bond or bonds that will
degrade in a biological environment by either a biologically
assisted mechanism, such as, by way of example, and not limitation,
an enzyme catalyzed reaction or by a chemical mechanism which can
occur in a biological medium, such as, by way of example, and not
limitation, hydrolysis.
[0175] The biodegradable linkages B may be chosen such that the
resulting biodegradable biocompatible crosslinked will degrade or
be absorbed in a desired period of time. Preferably, biodegradable
linkages are selected that degrade under physiological conditions
into non-toxic products. The biodegradable linkage may be
chemically or enzymatically hydrolyzable or absorbable.
Illustrative chemically hydrolyzable biodegradable linkages are
ester or amide bonds that undergo cleavage under physiological
conditions such as, by way of example, and not limitation, found in
human body (pH 7.2). Preferred linkages include, but are not
limited to, polymers, copolymers and oligomers of glycolide,
dl-lactide, l-lactide, caprolactone, dioxanone, and trimethylene
carbonate. Illustrative enzymatically hydrolyzable biodegradable
linkages include peptidic linkages cleavable by metalloproteinases
and collagenease enzymes. For example, collagen contains peptide
sequences susceptible to degradation by collagenease. Crosslinker
containing reactive groups and such linkages may be synthesized and
used. Additional illustrative biodegradable linkages include
polymers and copolymers of poly(hydroxy acids,
poly(orthocarbonate)s, poly(anhydride)s, poly(lactone)s,
poly(aminoacid)s, poly(carbonate)s, and poly(phosphonate)s.
Biodegradable linkage may also be non-polymeric; these include, but
are not limited to, ester linkages such as, by way of example, and
not limitation, succinate, glutarate, itaconate, and the like.
[0176] The biodegradable crosslinker preferably contains at least
two F moieties and one B moiety per molecule. The number of F
groups can be greater than two per molecule and number of B groups
can be greater than one per molecule.
[0177] Preferred biodegradable crosslinkers include, but are not
limited to, activated acid esters such as, by way of example, and
not limitation, n-hydroxysuccinimide esters (NHS esters). Some of
the preferred protein crosslinkers that can synthesized and used as
described according to patent WO 9812274, which is cited here for
reference only as one suitable technique. The polyether NHS esters,
more preferably polyethylene glycol glutarate, or succinate esters
are most preferred.
[0178] The cross-linking agents described can be made using any
suitable synthetic methodologies including, but not limited to,
those known to skilled individuals in the art. One preferred
approach involves the use of water soluble polyethylene glycol
based activated acid ester is used. In one exemplary embodiment
(see Example 19 described below), 10 pieces of 1 cm by 1 cm bovine
pericardium tissue are transferred to 50 ml polypropylene
centrifuge tube containing 10 ml PBS. 1.0 g 4
arm-n-hydroxysuccinimide ester of polyethylene glycol
carboxymethylene-butyric acid, average molecular weight 10000
Daltons (obtained from Shearwater Polymers, 4 arm, product
CM-HBA-NS-10K) is added to the tube and the mixture is vortexed for
5 minutes. The tissue is isolated from the tube, washed with
distilled water and lyophilized until further use. The glutarate
ester bond in CM-HBA-NS-10K undergoes hydrolysis when exposed to
physiological conditions such as, by way of example, and not
limitation, PBS pH 7.2. After hydrolysis of the crosslinker, the
tissue degrades by normal enzymatic pathways. Tissue crosslinked
with the 4 arm-n-hydroxysuccinimide ester of polyethylene glycol
carboxymethylene-proprionic acid, average molecular weight 10000
Daltons has a shorter degradation time as compared to glutarate
ester. Thus, by changing the type of ester linkage, degradation of
the tissue can be controlled.
[0179] In another illustrative embodiment (see Example 18 described
below), a non-polymeric biodegradable crosslinker that is
synthesized from hydroxylamine is used. Briefly, the hydroxy amine
is first reacted with succinic anhydride. The acid groups formed
are then activated by forming n-hydroxysuccinimide ester (NHS
ester). The NHS ester is then used to crosslink the tissue. The
hydrolysis of succinate ester makes the tissue degradable.
[0180] The biological tissue of interest is treated with one or
more of the crosslinking agents under conditions effective to cause
the desired degree of tissue cross-linking. The skilled individual
will recognize, in light of the teachings of the present invention,
that the time of treatment is not critical as long as the tissue
and cross-linking agent remains in contact for a time sufficient to
allow the cross-linking to occur. The time of treatment may vary
depending on the type of tissue being treated and/or the particular
cross-linking agent used. Typically, the length of the reaction
will be from about one minute to one day or more. However, the time
of treatment should not be so long as to adversely affect the
cross-linked tissue. Thus, cross-linking times greater than about
one or two days are generally avoided, though such lengthy times
may be appropriate in certain applications. Preferably, the tissue
is treated for a period from about one minute to about six hours,
more preferably for about one hour to four hours. The degree of
cross-linking can to some extent be varied by manipulating the time
of the reaction. A reaction temperature is selected to be effective
in permitting the desired cross-linking reaction to occur while
also being one that does not adversely compromise the integrity of
the tissue being treated. The identification of an optimal
temperature for a particular agent and/or application can be
readily determined by the skilled individual in light of the
teachings of the present invention. The cross-linking reaction can
generally be successfully carried out at an ambient temperature
(20-40.degree. C.), or any other convenient temperature provided it
does not exceed the tissue denaturation temperature of about 60 to
about 65.degree. C. Thus, a suitable reaction temperature for use
in this invention may be from about 0.degree. C. to about
55.degree. C., preferably from about 20.degree. C. to about
50.degree. C., more preferably from about 30.degree. C. to about
40.degree. C. This temperature range is generally applicable to all
the tissues treatments described. The tissue is treated under pH
conditions that are tissue-stabilizing (i.e., which are not
deleterious to the tissue being treated) and which do not adversely
effect the tissue cross-linking reaction. This will typically be in
a range from about pH 6 to about pH 9. More preferably, the pH will
be from about 7.0 and about 8.0. Most preferably, it will be from
about 7.0 to about 7.4. The optimal pH may depend to some extent on
the cross-linking agent employed, the type of crosslinker being
employed, and/or on the tissue being treated, but can be readily
determined, in light of the teachings of the present invention,
without undue experimentation by the skilled individual in this
art. The process of cross-linking biological tissues can be carried
out in any suitable solvent. The choice of solvent is generally not
critical; however, preferred solvents will typically be aqueous
medium with buffering agent. Water-miscible organic solvents that
have minimal toxicity to the tissue and/or the recipient, will be
non-denaturing and will be compatible with the tissue/protein
cross-linking reaction. Some such solvents include, but are not
limited to, linear or branched lower alcohols (i.e., having from
about one to four carbons); aprotic high polarity organic solvents
such as, by way of example, and not limitation,
n-methylpyrrolidinone, dimethylsulfoxide, dimethylformamide.
dimethylacetamide etc.; lower ketones (i.e., having from about 3 to
6 carbons) such as, by way of example, and not limitation, methyl
ethyl ketone or cyclohexanone; and polyhydroxy compounds such as,
by way of example, and not limitation, glycerol, ethylene glycol,
or polyethylene glycols having molecular weights less than about
1000.
[0181] One embodiment of the present invention provides for a
biodegradable absorbable non-crosslinked tissue based implant
material including covalently bonding at least a portion of the
tissue functional groups with a compound having the formula F-X,
wherein F is a functional group reactive with collagen or protein
or components in the tissue; preferably free primary amine groups
in the tissue and X is an organic molecule covalently linking F and
X. X may include Carbon-Carbon, Carbon-Hydrogen, Carbon-Nitrogen,
Carbon-Oxygen, Carbon-sulfur, Nitrogen-Hydrogen and Oxygen-Hydrogen
covalent bonds. X may polymeric or non-polymeric. For example, and
not by way of limitation, the compound F--X may be selected from
polyethylene glycol with tissue reactive groups;
n-hydroxysuccinimide ester of acetic acid, n-hydroxysuccinimide
ester of acrylic acid, polyethylene glycol monomethoxy alcohols
modified with acid group, n-hydroxysuccinimide ester of methacrylic
acid; acrylic anhydride; acetic anhydride; n-hydroxysuccinimide
ester of triiodobenzoic acid; succinic anhydride, glutaric
anhydride, acetyl chloride; acrolein, methacrolein, lactide,
glycolide, methyl isocyanate, phenyl isocyanate, glycidyl
methacrylate, and glycidyl acrylate. FIG. 9 is a schematic
representation of an exemplary biodegradable uncrosslinked tissue
modified using polyethylene glycol, in accordance with an
embodiment of the present invention. The method shown is suitable
for obtaining a biodegradable uncrosslinked tissue modified with
polyethylene glycol.
[0182] Animal Tissue and Synthetic Biodegradable Polymer
Composites
[0183] The present invention provides for novel methods and
compositions for materials that combine the properties of animal
tissue and synthetic or semisynthetic biodegradable polymers,
copolymer and oligomers. In one embodiment, the present invention
provides for a method for forming an animal tissue and synthetic
biodegradable polymer composite implant including the steps of
providing a tissue suitable for human implantation; exposing the
tissue to a fluid including a biodegradable polymer dissolved in
the solvent; and evaporating the solvent. In an alternate
embodiment, the tissue is exposed to a fluid including
biodegradable polymer dissolved in the solvent and a therapeutic or
bioactive substance dispersed in the solvent. In another alternate
embodiment, the present invention provides for a method for forming
an animal tissue and synthetic biodegradable polymer composite
including the steps of providing a tissue suitable for human
implantation; exposing the tissue to a fluid including
biodegradable crosslinkable polymer dispersed in the solvent;
evaporating the solvent; and crosslinking the biodegradable
polymer. In these various embodiments, the tissue may be
dehydrated. Additionally, the biodegradable polymer may be
hydrophilic or hydrophobic.
[0184] Animal tissue such as, by way of example, and not
limitation, bovine pericardium has unique mechanical properties
such as, by way of example, and not limitation, excellent fatigue
resistance and durability. However, animal tissues have found
little utility in delivering bioactive compounds, especially water
soluble compounds, in a controlled manner. A composite material
including synthetic biodegradable polymer and animal tissue is
discussed herein. Methods of such composite preparation as well as
their medical applications are discussed herein. Compositions and
methods that physically or chemically bound synthetic biodegradable
polymers to the tissue are also discussed herein.
[0185] In one illustrative and preferred embodiment (see Example 22
described below), the animal tissue is dehydrated by exposing it to
a series of aqueous ethanol solutions. The dried tissue is then
added to a solution of synthetic biodegradable polymer in an
organic solvent incubated for sufficient amount of time until the
polymer solution penetrates the tissue matrix. The polymer solution
may include biologically active compound. The tissue is removed
from the solution and the polymer solvent is evaporated or removed
by extraction with other solvents. The tissue-biodegradable polymer
composite may, for example, and without limitation, be used in
variety of medical and surgical applications. The tissue may be
mechanically or chemically treated to improve adhesion of
biodegradable polymer to the tissue. Mechanical modification may
involve, for example, creating perforations, micro-porosity to
mechanically interlock the biodegradable polymer in the tissue
matrix. The molecular weight of biodegradable polymer may vary form
1000 to 1 million g/mole, most preferably from 2000 to 500000
g/mole and even more preferably from 5000 to 150000 g/mole. The
amount of polymer incorporated in the tissue may range from 1% to
70%, more preferably 5% to 30%.
[0186] The animal tissue used may be biostable or biodegradable.
The animal tissue used may be crosslinked or non-crosslinked. The
animal tissue used may be crosslinked using conventional
crosslinking technique such as, by way of example, and not
limitation, EDC crosslinking or glutaraldehyde crosslinking methods
known in the heart valve bioprosthesis art. The animal tissue may
be non-crosslinked or chemically modified without crosslinking.
This includes tissue like porcine sub-mucosa tissue or
non-crosslinked but chemically treated tissue. Membrane-like
biodegradable tissue is most preferred for controlled drug delivery
applications. In many medical device applications where
biodegradable tissue is needed, a non crosslinked tissue, EDC
crosslinked tissue or tissue crosslinked using biodegradable
crosslinker as discussed previously may, for example, be used.
Applications were biostable tissue is needed, then glutaraldehyde
or di or polyepoxide crosslinked tissue may, for example, be
used.
[0187] The bioabsorbable or biodegradable polymers used in this
invention include any polymer that degrades into non-toxic products
upon degradation and may include, but are not limited to, polymers,
oligomers or copolymers generally known as polylactones or
polyhydroxy acids; polylactones such as, by way of example, and not
limitation, polylactide poly-L-lactide (PLLA), poly-D-lactide
(PDLA), poly-DL-Lactide (PLA), polyglycolide (PGA),
polylactide-polyglycolide copolymers; polydioxanone,
polycaprolactone (PCL), Polyhydroxyalkanoates are polyesters
produced by microorganisms such as poly(3-hydroxybutyrate),
3-hydroxyvalerate, 4-hydroxybutarate, 3-hydroxyhexanoate,
3-hydroxyoctanoate can also be used. Polycaprolactone-polyglycolide
copolymers, polylactone-polyethylene oxide copolymers are
preferred. Modified cellulose, polylactones collagen,
poly(hydroxybutyrate), polyanhydride, polyphosphoester, poly(amino
acids), poly(alpha-hydroxy acid) or related copolymers materials,
each of which have a characteristic degradation rate in the body,
may also be used. For example, polyglycolide and polydioxanone
copolymers degrade in weeks to months. PLA degrades in months to
few years and polycaprolactone degrades in few years. Their
copolymers have intermediate degradation times depending on the
composition of the copolymer. The biodegradable polymer may be
linear, branched, star type. The biodegradable polymer may be block
or random copolymer or may be blend of two are more polymers. The
block copolymers include, but are not limited to, ABA type, BAB
type or AB type block copolymers. Bioabsorbable polymers such as,
by way of example, and not limitation, PLLA, PDLA, PGA and others
are commercially available from several sources including PURAC
America, Inc.; Aldrich, Polysciences, and Birmingham Polymers. The
biodegradable polymer may be in a variety of forms, for example,
liquid, solid, semi solid, wax type or gel type when incorporated
in the tissue.
[0188] The biodegradable polymer may be covalently linked to the
tissue. Many methods of covalently bonding the polymers may, for
example, and without limitation, be used. One preferred and
illustrative reaction scheme is shown in FIG. 10. In one preferred
and exemplary embodiment (see Example 39 or 40 described below),
the hydroxy groups of proteins in the tissue or collagen matrix
may, for example, and without limitation, be used to initiate
polymerization of cyclic lactones or carbonates. The cyclic
lactones that may, for example, and without limitation, be used
include, but are not limited to, lactide, glycolide, caprolactone,
dioxanone, trimethylene carbonate, and the like. Two or more
lactones may be copolymerized to obtain a desirable degradation
profile of the composite material. The polymerization is usually,
though not necessarily, catalyzed by metal organic catalysts such
as, by way of example, and not limitation, stannous octoate. The
concentration of catalyst added may depend on the catalyst used.
Enzymatic catalysts or biological enzymes that promote
esterification may also be used. Preferred catalyst stannous
octoate is generally added in the range of about 100 ppm to 10000
ppm relative to cyclic lactone concentration. The polymerization
reaction may be carried out in a suitable solvent or under melt
conditions to obtain a desired molecular weight of the polylactone.
Many commonly used organic solvents can be used, but solvents that
do not denature the proteins in the tissue are preferred. Preferred
commonly used organic solvent solvents include, but are not limited
to, acetone, tetrahydrofuran, benzene, toluene, xylene, chloroform,
methylene chloride, dimethyl sulfoxide, dimethylacetamide, and the
like. The preferred temperature is chosen that promotes rapid
polymerization over a period of about 7 days, more typically in
about 2 days even more typically in about a few hours. If using a
tissue, polymerization temperature below shrink temperature is most
preferred. The shrink temperature of uncrosslinked tissue is
generally around about 60.degree. C. and crosslinked tissue is
around about 70-100.degree. C. Polymerization conditions using
temperatures below about 60.degree. C. are most preferred. Lower
temperature usage generally requires longer polymerization times.
The molar ratio of hydroxy groups in the tissue to lactone may, for
example, and without limitation, be used to control the degree of
polymerization of cyclic lactones. For high molecular weight
polymers, a higher ratio of cyclic lactone to hydroxy groups in
tissue may, for example, and without limitation, be used. The molar
ratios (cyclic lactone/hydroxy groups) may vary from about 1 to
about 500 depending on the final molecular weight desired. A molar
ratio of about 1 to 50 is generally preferred. In one embodiment,
lactide polymerization was initiated by tissue or solid collagen
sponge using THF as a solvent and stannous octoate as a catalyst.
The tissue-polylactide composite has a different degradation
profile than this tissue. The pepsin digestion data for dl-lactide
modified tissue is given in Table 4.
TABLE-US-00004 TABLE 4 Pepsin digestion data on dl-lactide modified
tissue Visual Tissue treatment Pepsin digestion Observations Bovine
pericardium untreated Completely Complete control degraded
dissolution in 6 hours. Bovine pericardium, treated with 45%
reduction Partial digestion dl-lactide in presence of stannous in
51 hours after 51 hours octoate Bovine pericardium crosslinked No
degradation No change with glutaraldehyde, treated with in tissue
dl-lactide in presence of stannous appearance. octoate
[0189] The data in Table 4 indicates that the uncrosslinked tissue
treated with dl-lactide showed substantial stability towards
enzymatic degradation as compared to untreated tissue which
degraded in 6 hours. However dl-lactide treated tissue lost 45%
weight in 51 hours indicating its susceptibility to enzymatic
degradation. Thus, it is expected that the dl-lactide tissue can
resist degradation much longer than the untreated tissue and thus
stay much longer than untreated tissue once implanted in the body.
As expected, glutaraldehyde treated tissue shows higher stability
towards enzymatic degradation. Collagen based films, fiber mats,
sponge, porous scaffolds may also be modified using cyclic
lactones.
[0190] In another embodiment, dl-lactide polymerization was
initiated by gelatin or collagen to produce a gelatin-polylactide
graft copolymer. The composite degrades by hydrolysis and an
enzymatic degradation mechanism. The degradation of synthetic
polymer will depend on cyclic lactone used. In general,
polyglycolide degrades in about a few months, polydl-lactide in
about 6 to 18 months, and polycaprolactone in about a few years.
Their copolymers may, for example, and without limitation, be used
to achieve intermediate degradation times. The synthetic polymer
may, for example, and without limitation, be used to entrap a
bioactive compound inside the biodegradable polymers. Techniques
such as, by way of example, and not limitation, solvent diffusion
may, for example, and without limitation, be used to diffuse or
entrap drugs in the degradable polymer matrix in the composite
material.
[0191] The biodegradable polymers may be hydrophobic or
hydrophilic. The biodegradable polymers may be crosslinked or
non-crosslinked. The hydrophobic polymers include, but are not
limited to, polymers, copolymers or oligomers of glycolide,
dl-lactide, d-lactide, 1-lactide, caprolactone, dioxanone and
trimethylene carbonate; polyhydroxyacids, polylactic acid,
polyglycolic acid, polyanhydrides, and polylactones. Hydrophobic
polymers also include polyhydroxyalkanoates which are polyesters
produced by microorganisms such as poly(3-hydroxybutyrate),
3-hydroxyvalerate, 4-hydroxybutarate, 3-hydroxyhexanoate,
3-hydroxyoctanoate. Preferred hydrophilic polymers include, but are
not limited to, polyethylene glycol-polyhydroxy acid or
polyethylene glycol-polylactone copolymers (PEG-PL copolymers),
polyvinyl alcohol co-polylactone copolymers, and derivatives of
cellulose, hyaluronic acid and dextran. The PEG-PL copolymers are
most preferred. PEG-PL copolymers such polyethylene
glycol-polylactone copolymers can have a range of properties from
hydrophobic to hydrophilic depending on the amount of PEG
incorporation in the copolymer and molecular weight of PEG and
polylactone. PEG incorporation that gives up to about 5-70 percent
water absorption is preferred. The PEG-PL copolymers with PEG
molecular weight about 400 to about 30000 Daltons are preferred. In
one exemplary embodiment (see Example 21 described below), a PEG
molecular weight 8000 (Carbowax 8000) is used in the polymerization
of dl-lactide using stannous octoate as a catalyst. The
polymerization reaction is carried out for 16 h at 160.degree. C.
The polymer is isolated and incorporated in the tissue (PEG8K50).
Briefly, a membrane-like tissue such as, by way of example, and not
limitation, pericardial tissue is incubated for 30 minutes each in
20 percent ethanol, 40 percent ethanol, 80 percent ethanol, and
finally in 100 percent ethanol to dehydrate the tissue. The tissue
may also be dehydrated by lyophilization. The dehydrated tissue is
then transferred into polyethylene glycol-lactate copolymer
(PEG8K50) and rifampin solution in chloroform and incubated for 2
hours. The tissue is removed from the solution and the solvent is
evaporated by air drying. The reddish yellow rifampin-PEG-lactate
polymer is clearly visible to the naked eye. The dry tissue is
sterilized using ethylene oxide and used as a biodegradable drug
delivery patch. The polyethylene glycol-lactate polymer in the
tissue acts as a hydrophilic biodegradable polymeric drug delivery
matrix for Rifampin.
[0192] The biodegradable polymer incorporated into the tissue may
be crosslinked in nature. The crosslinked polymer may be
hydrophilic or hydrophobic. It is preferred that the crosslinking
reaction is carried out after infusion of precursor polymers in the
tissue. This permits better distribution of polymer in the tissue
matrix. In one exemplary embodiment (see Example 15 described
below), a hydrophobic free radically polymerizable liquid precursor
is made by initiating the polymerization of lactide using
trimethylol propane triol and then endcapping the terminal hydroxyl
groups with acrylate end groups. This polymerizable precursor is
then infused in the tissue along with photoinitiator and vinyl
pyrrolidinone as comonomer using chloroform as a solvent. After the
solvent removal, the lactide precursor is photopolymerized exposing
the tissue to long wavelength light. In another variation (see
Example 20 described below), a water-soluble precursor based on
polyethylene glycol is incorporated into the tissue first and then
crosslinked. If a bioactive compound such as, but not limited to,
heparin, rifampin, or growth factor is mixed prior to crosslinking,
then the compound remains entrapped in the crosslinked polymer and
is then released slowly upon degradation of biodegradable
crosslinked polymer.
[0193] Radio-opaque implantable tissue Animal tissue such as, by
way of example, and not limitation, porcine pericardium or bovine
pericardium is poorly visible when viewed using standard medical
x-ray imaging techniques. This is probably due to poor absorption
of x-rays by the components of the tissue. In many medical device
applications, especially in surgical procedures which are done
using x-ray fluoroscopic techniques; it is desirable to have an
implantable tissue that can be differentiated from surrounding
tissue when implanted and viewed under standard medical x-ray
imaging technique. A radio-opaque tissue can be easily seen when
viewed using standard medical x-ray imaging technique. This
invention provides compositions and methods for making radio-opaque
implantable tissue. Some of the preferred methods for making
implantable radio-opaque tissue are given below.
[0194] Implantable animal tissue may be chemically modified using a
reagent including at least one functional group capable of reacting
with tissue (F) and at least one radio-opaque chromophore capable
of absorbing x-rays (M). The chemical modification agents used in
accordance with an embodiment of the present invention include an
organic functional molecule with at least one functional group
capable of reacting with tissue and at least one radio-opaque
chromophore. Thus, the compounds suitable for use with this
invention can be generally represented by the following structural
formula:
F-X-M
[0195] wherein F is a functional group reactive with collagen or
protein or components in the tissue; preferably free primary amine
groups in the tissue; X is an organic molecule/radical covalently
linking F and X. X may include Carbon-Carbon, Carbon-Hydrogen,
Carbon-Nitrogen, Carbon-Oxygen, Carbon-Sulfur, Nitrogen-Hydrogen
and Oxygen-Hydrogen covalent bonds. X may polymeric or
non-polymeric.
[0196] M is a chromophore that can absorb x-rays. The preferred
chromophores include, but are not limited to, phenyl ring compounds
such as, by way of example, and not limitation,
2,3,5-triiodobenzoic acid, 3,4,5-triiodophenol, erythrosine, rose
bengal, 3,5-Bis(acetylamino)-2,4,6-triiodobenzoic acid,
3,5-Diacetamido-2,4,6-triiodobenzoic acid, heavy metal ion
complexes, and the like. The iodine in these compound may be
radioactive if desired. A radioactive (I.sup.125 or I.sup.131)
could be potentially used for local radiation therapy.
[0197] F is a functional group that is sufficiently reactive with
the collagen molecules present in the biological tissue to be
treated such that the desired chemical bond is formed. For example,
functional groups reactive with collagen and suitable for use may
include, but are not limited to, anhydride, isocyanate,
n-hydroxysuccinimide, n-hydroxysulfosuccinimide, epoxy, aldehyde or
other collagen reactive functionalities known in the art. It is
desirable to have only one F moiety per molecule to avoid
crosslinking of proteins in the tissue. If crosslinking is desired,
more than 1 F moiety may, for example, and without limitation, be
used.
[0198] Preferred molecules which are capable of modifying the
tissue include, but are not limited to, activated monofunctional
triiodobenzene derivatives such as, by way of example, and not
limitation, n-hydroxysuccinimide esters or
n-hydroxysulfosuccinimide esters of triiodobenzoic acid or
erythrosin.
[0199] The tissue modification using reagents, which are taught by
way of example in the present detailed description, can be made
using any suitable synthetic methodologies, including but not
limited to, those known to skilled individuals in the art. In one
embodiment of the present invention a method of modifying the issue
is achieved by steps that include, without limitation, treating the
tissue under effective modification conditions with a
monofunctional tissue reactive iodinated organic compound. In one
exemplary embodiment (see Example 36 described below), bovine
pericardium tissue is modified with triiodobenzoic acid succinimide
ester (TIBA-NHS). The modification method includes exposing the
tissue to a fluid including reactive iodinated compound that can
react with amino groups in the tissue under mild conditions. As
used herein, the term "activated" as applied to an acid
moiety-containing compound containing an additional moiety such
that the activated acid can react with amino groups under mild
conditions. Preferred activating moieties include, but are not
limited to, disuccinimidyl moieties, n-hydroxy disuccinimidyl
moieties, sulfo-disuccinimidyl moieties, and mixtures thereof.
Briefly, bovine pericardium pieces, cut from a freshly obtained
bovine pericardial sac, are treated with TIBA-NHS dissolved in
dimethyl sulfoxide or PBS buffer. The reaction is carried out in
aqueous medium buffered with PBS (pH 7.2). The modification
reaction is carried out for 6 hours at ambient temperature
(25.degree. C.) and then for 12 hours at 4.degree. C. with gentle
shaking. Some NHS esters such as, by way of example, and not
limitation, acetic acid NHS esters are sparingly soluble in water
therefore a solubility enhancing compound such as, by way of
example, and not limitation, dimethyl sulfoxide may, for example,
and without limitation, be used to facilitate the reaction between
activated acid groups and primary amine groups on the tissue. The
reaction occurs in mild conditions, in water (PBS and at pH 7.2)
and is usually complete in about 24 hours, more preferably within
about six hours. It can also be substantially completed in about 10
minutes and therefore may permit the use of this method in the
operating room during a surgical procedure to modify an autologous
tissue. In another variation of this method, the modification
reaction may be carried out under mild acidic conditions (IVIES
buffer, pH 6.5). In this case, the NHS ester is added every 1.5
hours to replace the hydrolyzed NHS ester. (NHS ester is
deactivated by reaction with water). The NHS groups react with
primary amines groups of the tissue forming stable amide bonds. The
NHS ester reacts with primary amines groups on the tissue. Reaction
variables such as, by way of example, and not limitation, time,
temperature, concentration, pressure may be controlled in such a
way that about 1 to about 100 percent primary amine groups on the
tissue are modified. More preferably, about 10 to about 95 percent
amine groups are modified, even more preferably about 40 to about
95 percent amine groups are modified.
[0200] In another approach, triiodobenzoic acid is reacted with
tissue using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) as a catalyst. EDC promotes the reaction
between carboxylic acid of triiodobenzoic acid and OH and primary
amine groups present in the tissue. EDC treatment also results into
crosslinked non-cytotoxic tissue. Aqueous medium, more specifically
buffered medium, with pH about 5.5 to about 7.5 may, for example,
and without limitation, be used for such reactions. A co-catalyst
such as, by way of example, and not limitation,
n-hydroxysuccinimide or n-hydroxysulfosuccinimide may also be used
to accelerate the coupling reaction. Using a similar approach,
other biocompatible hydrogels such as, by way of example, and not
limitation, hyaluronic acid, chitosan and alginate may also be made
radio-opaque and are considered part of the present invention.
[0201] In another approach, a radio-opaque polymerizable monomer
may, for example, and without limitation, be used in polymerization
and crosslinking as discussed above. For example, a polymerizable
radio-opaque monomer may be obtained by esterification of
triiodobenzoic acid and 2-hydroxyethyl methacrylate. The
radio-opaque ester may be copolymerized and crosslinked with
unsaturated group modified tissue.
[0202] In another approach, heavy metal biocompatible salts such
as, by way of example, and not limitation, silver salts may be
deposited in the tissue to make it radio-opaque. Many methods for
silver salt incorporation can be used. In one exemplary embodiment,
a tissue is first exposed to silver nitrate solution by dipping the
tissue and removing the tissue and then exposing to sodium acetate
or sodium iodide or sodium chloride solution. The exposure to
acetate or chloride ions form insoluble salts inside the
extracellular matrix of the tissue and leach out over a period of
time. The silver salts provide visibility in x-ray based medical
imaging equipment.
[0203] Tissue used in modification may be crosslinked or
non-crosslinked. A non-crosslinked tissue generally yields
biodegradable implantable tissue. A glutaraldehyde crosslinked
tissue gives biostable tissue.
[0204] The tissue also may be coated with polymeric compositions
including radio-opaque compounds. For example, biodegradable
polymers such as, by way of example, and not limitation,
poly(lactic acid) and x-ray contrast agent such as, by way of
example, and not limitation, iohexol are dissolved in common
organic solvent such as, by way of example, and not limitation,
dimethyl sulfoxide or tetrahydrofuran and the solution may be
spray-coated on the tissue. Upon solvent removal, iohexol remains
entrapped in the polymeric coating which makes the tissue
radio-opaque.
[0205] The radio-opaque tissue may, for example, and without
limitation, be used to fabricate many and varied medical devices.
The medical devices may be existing medical devices such as, by way
of example, and not limitation, a heart valve or a vascular graft
or devices that are yet to be developed. More specifically,
radio-opaque tissue may, for example, and without limitation, be
used to fabricate tissue covered coronary or vascular stents or
stent grafts. The visibility of radio-opaque tissue permits
localized drug or device therapy using standard techniques used in
MIS surgery. This radio-opaque tissue may be subjected to
additional treatments such as, by way of example, and not
limitation, anti-calcification treatments known in the prior
art.
[0206] In some applications, modified non-crosslinked radio-opaque
tissue is expected to undergo degradation after in vivo
implantation. The modification compounds and their degradation
products/components must be able to be eliminated from the host
body. The modification compounds may be chosen such that the
resulting biodegradable biocompatible crosslinked tissue will
degrade or be absorbed in a desired period of time. Preferably,
modification reagents are selected in a manner that it will degrade
under physiological conditions into non-toxic products.
[0207] In applications where biodegradable tissue is desired, the
tissue may be heat-treated to make it denatured. Such denaturing is
done to accelerate the degradation of the tissue. The denaturing
may be done by heating a membrane-like tissue such as, by way of
example, and not limitation, porcine pericardium in saline solution
and holding it in the temperature range of about 70 to about
100.degree. C. (above shrink temperature) for about 1 minute to
about 24 hours, more preferably from about 1 minute to about 30
minutes.
[0208] Coated Bioprosthetic Tissue Compositions and Methods
[0209] Properties of biological tissue surface can be changed by
applying different types coatings. For example, the glutaraldehyde
fixed tissue surface is cytotoxic and does not support cell growth.
A surface coating that eliminates the surface toxicity is highly
desirable. Non-crosslinked tissues such as, by way of example, and
not limitation, pericardial tissue supports cell growth and degrade
by enzymatic process when implanted in the human or animal body. In
some applications, such as, by way of example, and not limitation,
prevention of post operative adhesions or hernia repair
application, a barrier surface that is hydrophilic, biocompatible
but does not support cell growth is desirable. A polyethylene
glycol based hydrogel on tissue surface may provide such non-cell
adhesive surface.
[0210] In one aspect of the present invention, various coating
compositions that can be applied on the implantable tissue surface
are provided. The coating may cover all surface areas of the tissue
or may be applied on some parts of the tissue. FIG. 11 is a
schematic representation of an exemplary membrane-like tissue
modifications (A-F), in accordance with an embodiment of the
present invention. Illustrated in the Figure are various
modifications of membrane-like implantable tissue. FIGS. 11A and B
illustrate the coating of a membrane-like tissue such as, by way of
example, and not limitation, bovine pericardium from one and both
sides. The coating may include, for example, cells and/or bioactive
compounds. The coating may also be a biodegradable biocompatible
hydrogel. Multiple layers may also be utilized. Methods for forming
such coated tissue surfaces and their biomedical applications such
as, by way of example, and not limitation, barrier for post
operative adhesion prevention and controlled drug delivery are also
described.
[0211] In some embodiments, the tissue is coated using a
biodegradable polymer. The coated tissue structure is schematically
shown by way of example in FIG. 11. The coating thickness may be
about 0.1 microns to about 5 mm thick. More preferably the coating
may be about 5 micron to about 2 mm thick. The biodegradable
coating may degrade in approximately days to years. The degradation
time will depend on the type of biodegradable polymer used in the
coating. For example, a coating based on polyglycolide polymers and
copolymers may degrade in weeks to months, PLA or PLA-PGA coating
will degrade in months to years, and polycaprolactone based coating
will degrade in years. An elastomeric coating formed from
biodegradable polyurethanes may also be used. The coating may have
biologically active compounds encapsulated in the polymer. The
coatings also may have cells such as, by way of example, and not
limitation, stem cells incorporated in the coating. The hydrophilic
coatings include biodegradable hydrogel coatings formed by
crosslinking and polymerization of polyethylene glycol based water
soluble macromonomers. In one such illustrative embodiment (see
Example 23 described below), a polyethylene glycol based macromer
is first synthesized. An aqueous solution of macromer is then
polymerized on the tissue to form a thin hydrogel coating.
[0212] In another embodiment of the present invention, a synthetic
biodegradable hydrogel is coating is formed by condensation
polymerization (see Example 24 described below). Briefly, the
hydrogel is formed by mixing reactive precursor species including
nucleophilic functional groups and reactive precursor species
including electrophilic functional groups. In one exemplary
embodiment, solutions of polyethylene glycol NHS derivatives and
dilysine are sprayed and polymerized on the 10 cm.times.5 cm bovine
pericardial sac. The solutions of polyethylene glycol NHS
derivatives and dilysine are commercially sold under the trade name
of DuraSeal.TM. by Confluent Surgical, Waltham, Mass. DuraSeal.TM.
sealant can also be used to make a coated tissue. The DuraSeal.TM.
coating may be applied just prior to implantation. Alternatively
tissue such pericardial patch tissue and DuraSeal.TM. product and
spray systems may be supplied as a kit to make a coated tissue
product in the surgical suit. The tissue used may be crosslinked
(biodegradable) or crosslinked (biostable). For example,
AlloDerm.RTM. a processed human skin tissue marketed by LifeCell
corporation may be coated using DuraSeal.TM. and used. In another
embodiment of the present invention, a tissue is first dehydrated
by lyophilization or using other techniques known in the art. The
precursors may be applied as a dry powder without crosslinking.
Upon application on the surgical site, the precursor powder coated
tissue undergoes hydration and crosslinking.
[0213] In another embodiment of the present invention, a tissue is
first dehydrated by lyophilization or using other techniques known
in the art. A biodegradable polymer dissolved in organic solvent is
sprayed on the tissue surface. Alternatively the polymer may be
coated using a dip coating method. The solvent is removed and the
polymer coated tissue is recovered and stored appropriately. The
coating may be applied on one side or on both sides.
[0214] A non-crosslinked tissue such as, by way of example, and not
limitation, bovine pericardial tissue typically degrades in 4
months when implanted subcutaneously in the rat body. Control over
tissue degradation time is highly desirable. A polymeric
biodegradable coating on the tissue may temporarily restrict the
access of degradative enzymes when implanted in the body. After the
coating is resorbed due to biodegradation processes in the body,
the access of enzymatic degradative sites in the tissue is restored
and the tissue is degraded. Thus, coatings may help to lengthen the
degradation time of the non-crosslinked biodegradable tissue. By
choosing a biodegradable polymer that degrades from months to
years, biodegradation of the tissue may be varied from months to
years. The polymer coating may be loaded with drug for a local or
systemic therapeutic effect.
[0215] The coating in the degradable polymer-coated tissue, which
are taught by way of example in the present detailed description,
may be supplemented with collagenease, pepsin or other protease
enzymes which can accelerate tissue degradation. The enzyme is
released from the coating in a controlled manner which then
degrades the tissue. This approach may, for example, and without
limitation, be used to accelerate the tissue degradation.
[0216] The tissue-synthetic biodegradable polymer composite
material or coated product produced according to the present
invention can be modified further, if necessary or desired, by the
addition of any pharmaceutically acceptable compound or agent such
as, but not limited to, antioxidant, plasticizer, coloring agent,
x-ray or MRI imaging agent, filler such as, by way of example, and
not limitation, calcium phosphate salts and the like.
[0217] Another aspect of the present invention is that the tissue
based materials described can be used to deliver biologically
active compounds. The composite/coated material will deliver a
therapeutically effective amount of bioactive substance in a
controlled manner. In one embodiment, rifampin, a commonly used
antibiotic is incorporated in the tissue-biodegradable polymer
composite material. Small organic and inorganic molecules as well
as large molecules such as, by way of example, and not limitation,
proteins, carbohydrates, organic molecules, synthetic peptides,
genes, antibodies, enzymes, etc. can be used. Some examples of
bioactive compounds that can be released inside the human or animal
body include, but are not limited to, antiviral agents;
antiinfectives such as, by way of example, and not limitation,
antibiotics; antipruritics; antipsychotics; cholesterol or lipid
reducing agents, cell cycle inhibitors, anticancer agents,
antiparkinsonism drugs, HMG-CoA inhibitors, antirestenosis agents,
antiinflammatory agents; antiasthmatic agents; antihelmintics;
immunosuppressives; muscle relaxants; antidiuretic agents;
vasodilators such as, by way of example, and not limitation, nitric
oxide or nitric oxide adducts; beta-blockers; hormones;
antidepressants; decongestants; calcium channel blockers; growth
factors such as, by way of example, and not limitation, bone growth
factors, bone marrow proteins, vascular endothelial growth factor,
platelet derived growth factor, acidic growth factors, basic growth
factors, wound healing agents, analgesics and analgesic
combinations; local anesthetics agents, antihistamines; sedatives;
angiogenesis promoting agents; angiogenesis inhibiting agents;
tranquilizers, and the like. In many instances, the duration of the
drug release is affected by the hydrophobicity of the drug and
polymer, drug loading and polymer composition and polymer
degradation characteristics. The amount of bioactive compound
incorporated in the tissue composite materials is in the range of
about 0.1 percent to about 90 percent, more preferably in the range
of about 5 to about 70 percent, and even more preferably in the
range of about 10 to about 50 percent. In some cases, two or more
bioactive compounds may, for example, and without limitation, be
used to achieve a desirable therapeutic effect.
[0218] In a biodegradable drug delivery patch application,
membrane-like tissues such as, by way of example, and not
limitation, porcine pericardial tissue, processed human skin tissue
(AlloDerm.RTM.), bovine pericardial tissue or porcine sub-mucosa
tissue are preferred. The animal tissue used may be crosslinked or
non-crosslinked. A non-crosslinked tissue may, for example, and
without limitation, be used if an absorbable patch material is
desired. A drug delivery patch could be formulated to incorporate a
bioactive compound. The patch will deliver a therapeutically
effective amount of bioactive substance in a controlled manner. The
patch may be coated with biodegradable hydrogels encapsulated with
cells or bioactive growth factors. The patch can be utilized to
deliver a drug or bioactive compound systemically or locally. In
one embodiment, the present invention provides for a method for
localized drug delivery including the steps of providing a sterile
tissue suitable for human implantation; providing a sterile
biodegradable polymer; providing a sterile solvent for
biodegradable polymer; dissolving the biodegradable in the solvent
and optionally adding bioactive compound; exposing the tissue to a
polymer solution; forming a coating of polymer on the tissue; and
implanting the tissue.
[0219] This invention also anticipates the use of non-polymeric
liquid/solids as coating materials for the tissue and as controlled
drug delivery matrices incorporated into the tissue. The
non-polymeric materials used to coat the tissue or to form a
composite material include biocompatible non-polymeric liquids and
solids which can be implanted in the human body. These
non-polymeric materials include those that have a history of human
use. These include, but are not limited to, wax materials such as,
by way of example, and not limitation, bone wax, fatty acids and
their derivatives such as, by way of example, and not limitation,
stearic acid and oleic acid, sugar derivative such as, by way of
example, and not limitation, sucrose acetate, mineral oil, peanut
oil, cotton seed oil, tocopherol, polyethylene glycol, and the
like. PEG-Polylactone based copolymers with melting below
60.degree. C. are most preferred in this application.
[0220] Animal tissue, preferentially a biodegradable tissue, can be
used as carrier for biologically active substances such as, by way
of example, and not limitation, sparingly water-soluble drugs
without the use of carrier. Some examples of such drugs include,
but are not limited to, chlorhexidene acetate, chlorhexidene
gluconate, lovastatin, simvastatin, paclitaxel, silver iodide,
silver acetate, silver lactate, and the like. In one embodiment,
chlorhexidene acetate or chlorhexidene gluconate is incorporated in
the tissue using a solvent method. Briefly dehydrated tissue is
exposed to an organic solvent or semi aqueous solvent such as water
alcohol mixture in which the drug is dissolved. The solvent is
removed and crystals of drug are formed inside the tissue matrix.
These crystals serve as reservoir for the drug to be released. The
drug is released due to slow dissolution of drug crystals. In
another embodiment, poorly water soluble silver salts such as, by
way of example, and not limitation, silver chloride, silver iodide,
silver acetate, silver lactate and the like are precipitated inside
the tissue or extracellular matrix by a reaction of two solutions.
After precipitation, sparingly soluble silver salts release silver
ions from the tissue matrix providing antimicrobial activity to the
tissue. Depending on the solubility, different silver salts such
as, by way of example, and not limitation, silver iodide, silver
acetate, silver lactate and the like may be chosen to have
different silver ion release profiles. Silver salts can be
deposited using many methods such direct salt deposition, ion beam
implantation and the like. In a preferred embodiment, silver salts
are formed in situ inside the tissue. In one preferred embodiment,
tissue is first exposed to silver nitrate solution by incubation in
silver nitrate solution followed by incubation in a solution of
sodium acetate. The silver nitrate in the tissue is converted into
silver acetate in situ which precipitates in the tissue matrix. The
concentration of silver nitrate and silver acetate solution will
very depending on the amount of silver salt is needed in the
tissue. In the preferred embodiment, silver ion leaching of 10-100
microgram of silver per cm square of tissue surface is preferred.
The deposited silver crystals may also improve radio-opacity of the
tissue.
[0221] In many applications, tissue prepared according to an
embodiment of the present invention may be cut into several
geometric shapes. These shapes are then used to construct unique
medical devices such as, by way of example, and not limitation,
heart valve bioprosthesis, vascular grafts, tissue covered stents,
and stent grafts. For example, a bovine pericardial tissue may be
cut and sewn to form a heart valve bioprosthesis. In some
applications, a perforated tissue may, for example, and without
limitation, be used to form a composite material. Also in some
applications, the tissue surface may be textured using different
shapes, holes, grooves to promote healing or to improve adhesion of
biodegradable polymer in the tissue or to deposit controlled drug
delivery compositions. In some applications, multiple coating
layers of tissue may, for example, and without limitation, be used.
For example, a first coating layer may consist of drug and polymer
and a second coating layer on top of first layer may consist of
only polymer without a drug. Such a coating may act as a diffusion
barrier to control the release rate of the drug.
[0222] Embodiments of the present invention also include the use of
grooves or other geometric shapes created on the tissue surface and
their use to fill the biodegradable controlled drug delivery
compositions. FIG. 11 illustrates this concept by way of example,
in particular FIGS. 8C and 8D. Grooves may be of any shape and
pattern. The groove depth and size may depend on the tissue
thickness and intended use. Generally groove depth may range from 1
to 2000 micron most preferably from 20 to 1000 micron. Many groove
shapes may be used these include but not limited to wedged shape,
rectangular shape, cylindrical shape and the like. In a
membrane-like tissue such as, by way of example, and not
limitation, porcine pericardium or bovine pericardium, 10 micron to
2 mm diameter holes (i.e., cylindrical grooves) are created using
any known method in the art such as, by way of example, and not
limitation, laser drilling, mechanical drilling and the like. The
holes or grooves are then filled with the liquid or solid drug
delivery compositions, e.g., controlled release polymer
compositions containing bioactive compounds. For example, the holes
are filled with bioerodable or biodegradable polymer with bioactive
compound such as, by way of example, and not limitation, Lovastatin
or Atrovastatin (HMG-CoA inhibitor). The bioactive compound is then
released from the polymeric matrix. FIGS. 8E and 8F illustrate, by
way of example, and not limitation, the use of perforated tissue.
This tissue may be coated with biodegradable polymer with bioactive
compound. The membrane tissue used may be biostable (crosslinked)
or biodegradable (non-crosslinked).
[0223] A composite tissue-biodegradable composite material or
coated tissue material according to an embodiment of the present
invention can be formulated so that they can be delivered using
minimally invasive surgical (MIS) techniques such as, by way of
example, and not limitation, laparoscopy, thoroscopy and the like
and thus are useful for localized drug delivery inside a living
body. One embodiment of the present invention provides for a method
for the treatment of a medical condition at a localized site,
including the steps providing a implantable membrane like animal
tissue including a bioactive compound; compacting the tissue to a
size suitable for implantation using minimally invasive technique;
transporting the compacted tissue to a localized treatment site;
uncompacting the tissue to its original size; and immobilizing the
tissue on the treatment site wherein the implantable tissue and a
bioactive compound assists in the treatment of the medical
condition. In one exemplary approach, the pericardial
tissue-polymer composite patch loaded with rifampin is rolled into
tube or other compacted shape so that it can be transported to a
localized surgical site using a minimally invasive surgical (MIS)
technique. The drug delivery patch material is then unrolled to
form a flat membrane at the surgical site. The patch is then
applied on the tissue surface and conformed to the localized tissue
or organ geometry. The patch is secured in place by suturing or
stapling to the surrounding tissue. The suture or staple may be
biodegradable or biostable. The patch is then used to releases the
drug in the localized surgical environment
[0224] In some embodiments, a composite of fixed tissue such as, by
way of example, and not limitation, discussed in this invention or
glutaraldehyde fixed tissue is used to incorporate high molecular
weight biocompatible biostable polymers such as, by way of example,
and not limitation, Teflon.RTM. (polytetrafluoroethylene) or
polyethylene vinyl acetate copolymers or their derivatives or
polysulfones may be incorporated using a solvent technique. In one
example, amorphous Teflon.RTM. is dissolved in a proprietary
halogenated solvent obtained from 3M Corporation. A dehydrated
glutaraldehyde fixed bovine pericardial patch tissue is then
exposed to the Teflon.RTM. solution. The solvent is then
evaporated. The Teflon.RTM. polymer-fixed tissue composite is used
as a biostable composite patch for variety of surgical
applications. Alternatively expanded, partially expanded or
unexpanded polytetrafluoroethylene (PTFE) membrane may be used to
make a tissue based composite patch. The PTFE membrane may be sewn
to the using PTFE based sutures. The composite patch may be useful
as hernia surgery application.
[0225] Use of Tissue Engineered Material for Making
Bioprosthesis
[0226] This invention also teaches of compositions and methods
wherein the tissue used in making bioprosthesis is obtained from
non-animal source like cell culturing or tissue engineering
technologies. The use of animal tissue in bioprosthesis manufacture
is known for quite some time. However, animal tissue use has
certain limitations such as size of the maximum single piece that
can be derived from the animal. Also chemical/protein composition
of the animal skin tissue used in conventional animal cannot be
changed and is dependent on the species and type of tissue used.
The tissue derived from animal tissue may also contain pathogenic
viruses and proteins such as Bovine Spongiform Encephalopathy
(BSE). The use of membrane like tissue, made using tissue
engineering technologies, overcomes these limitations. In this
invention, membrane like tissue that may be useful to make
bioprostheses, is derived from modern tissue engineering
technologies which are known in the tissue engineering art or yet
to be developed. In one exemplary embodiment, a sterile porous
scaffold is seeded with smooth muscle cells along with tissue
culture medium that supports the growth of such cells. The
scaffold, cells and cell culture media are incubated at 37.degree.
C. in an atmosphere that supports mammalian cell growth (controlled
carbon dioxide, humidity etc). Care is taken to minimize bacterial
or fungal contamination of the scaffold or culturing cells. The
incubation of cells is continued until cells attach, grow and
create their own extracellular matrix tissue. Additional cell
seeding, cell culture media exchanges may be done to achieve a
desired tissue thickness is reached. The preferred thickness of
engineered tissue may range form 1 micron to 3000 micron, most
preferably 50 microns to 1500 microns. The preferred size of
engineered tissue is >1 square inch, more preferably 1 to 1000
square inch, even preferably 1 to 150 square inch.
[0227] The biodegradable scaffold materials that can be used to
make the engineered tissue include but not limited to are:
degradable polyesters such as polyhydroxy acid, polylactones,
polyhydroxyalkanoates which are the polyesters produced by
microorganisms such as poly(3-hydroxybutyrate), 3-hydroxyvalerate,
4-hydroxybutarate, 3-hydroxyhexanoate, 3-hydroxyoctanoate. Other
degradable polymers include but not limited to are: polyamides,
collagen, gelatin, polyanhydrides, polyglycolate, polyglycolic
acid, polycaprolactone, polycarbonates such as polytrimethylene
carbonate, degradable polyurathanes, fibrinogen, and the like.
Copolymers or blends of such materials may also be used. The
biodegradable scaffold materials may be the form a fiber such as
polyester fiber, collagen fiber, silk fibers, polyamide or nylon
fibers which may be woven to produce a suitable scaffold. The
scaffold used manufacturing of engineered tissue may be porous or
non-porous. Many methods of introducing porosity are known in the
art. These methods include but not limited to: leaching of soluble
salts from the polymer matrix, lyophilization of hydrogel
materials, sublimation of solvents or porosity generating compounds
from the polymers and the like. The porosity is chosen to promote
rapid cell attachment and growth. The porosity of scaffold may
range from 1 microns to 1000 microns, most preferably 10 microns to
500 microns to promote cell infiltration and growth. The scaffold
surface may be chemically or physically modified using bioactive
compounds or peptides that promote cell attachments. Such compounds
include but not limited to peptides such as RGD, RGDS, IGD and the
like. The size, shape and geometry of scaffold may vary depending
on the intended use. A membrane like scaffold with size ranging
from 1 square inch to 1000 square inch may be used for large scale
manufacturing of membrane like tissue. The preferred size be may be
similar to textiles used cloth manufacturing. A continuous or batch
processing methods may be used. The thickness of membrane like
scaffold may vary from 1 microns to 3000 microns, more preferably
from 1 microns to 2 mm. The scaffold could also be circular,
rectangular, triangular, hexagonal or any other 2-dimensional
shape. Square or rectangular shape is preferred due to its
simplicity in large scale manufacturing. Several membrane like
scaffolds may be stacked in a biotechnology tissue engineering
reactor and seeded with mammalian cells to generate large quantity
of engineered tissues suitable for large scale bioprosthesis
manufacturing. The scaffold could also have simple or complex 3
dimensional shapes or geometries. These geometries may include but
not limited are: hollow cylindrical tube, cylinder, sphere or
complex shapes. Membrane shaped tissue engineered tissue may also
be created by using a hollow cylindrical tube like scaffold and
then cutting the tube shaped engineered tissue to make a sheet or
membrane like tissue.
[0228] Chemical compositions of tissue engineered tissue may be
changed by choosing appropriate cell line or combination of cells
in building a desired engineered tissue. The cells could be
obtained from many animals. Cells from mammalian source are most
preferred. The cells may be obtained form animals source which
include animals but not limited to: human, cow, pig, sheep, deer,
ostrich, crocodile, rabbit, rat, snake and the like. Human, pig,
sheep or bovine cells are most preferred. Many types of cells could
be used to produce a tissue engineered material. The preferred
cells chosen which produce large amount of extracellular matrix
proteins such as collagen, elastin and keratin and the like. The
cell types that can be used but not limited to include: fibroblasts
cells, endothelial cells, smooth muscle cells, muscle cells, cells
found in heart tissue or pericardial membrane, cells found in human
or animal skins, sub-mucosa cells, epidermal cells, epithelial
cells, and the like. The cell may be may be purchased from
commercial sources such as ATCC along with their cell culture media
or may be obtained from animals using surgical biopsy technique,
separated and cultured using known tissue culture techniques. For
example, if a collagen rich tissue is desired, mammalian cells that
produce pericardial type tissue may be used. For skin tissue, cells
that generate a combination of collagen and elastin may be used.
Stem cells which can be converted into any organ may also be used.
Two or more cell types may also be used to create a desired tissue.
For example animal cells such as bovine or porcine cells may be
used to create a structural part of the tissue and the outer layer
may be derived from human cells to give feel, texture and
biological properties of a human tissue. Genetically modified cells
may also be used to influence the properties of the engineered
tissue.
[0229] Cell culture medium that is used to grow cells may include
several chemical substances that support the growth of mammalian
cells. Cell culture medium may be obtained from commercial sources
such as Sigma-Aldrich. These substances include chemicals that have
been developed or yet to be developed but not limited to are:
water, pH controlling buffers such as phosphate buffer, HEPES
buffer; amino acids; sugars; antibiotics; pH indicator dyes;
proteins such as albumin; growth factors such as fibroblast growth
factors; blood products such as serum and the like Cell lines used
to grow the tissue will dictate the choice of cell culture medium
and incubation period. Cell culture medium that does not have serum
components (serum free medium) is most preferred for large scale
manufacturing operations. The cell culture medium composition may
be used to influence the texture and other properties of the
engineered tissue. For example, non-natural aminoacid may be used
to influence the chemical composition of the extracellular matrix
produced or cells may be genetically modified to produce a given
tissue type. The engineered tissue may be decellularized prior to
medical device use. This step is particularly useful if non-human
derived cells are used to make the engineered tissue.
[0230] The tissue generated by tissue engineering methods described
above may be decellularized to generate the extracellular matrix
suitable for implantation. Many methods of decellualrization are
known in the art and could be used without limitation. The
preferred methods of tissue decellualrization are described in the
experimental section. Tissue may also be crosslinked or chemically
modified to improve tissue properties. Some preferred crosslinking
methods are described in this invention. Other conventional
crosslinking methods such as glutaraldehyde crosslinking, EDC
crosslinking, dye mediated photooxidation, hexamethylene
diisocyanate crosslinking, crosslinking and the like may also be
used. The tissue generated by tissue engineering methods may be
rendered biostable or biodegradable my many methods known in the
art or methods described in this invention. For example, tissue
treated with glutaraldehyde, hexamethylene diisocyanate,
photooxidation may render the tissue substantially biostable. In
applications where biodegradable tissue is needed, uncrosslinked
tissue or crosslinking catalyzed by -ethyl-3-(3-dimethylaminopropyl
carbodiimide) hydrochloride (EDC) is most preferred The EDC
crosslinking is generally known as "zero length cross-linking" in
the protein modification chemistry art. EDC is a class of compounds
generally known as carbodiimides. Carbodiimides generally promote
reaction between carboxylic acid-amine and carboxylic acid-hydroxyl
groups to form amide or ester bond respectively. Water soluble
carbodiimides are most preferred crosslinkers. Water soluble
carbodiimide such as EDC is most preferred. Other carbodiimides
that can be used include but not limited to:
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide;
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate;
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and the like. EDC can
catalyze a reaction in water over a wide pH range. The pH range may
vary from 1 to 9. Most preferred pH range is 5 to 7. The desired pH
may be achieved by using a biocompatible buffering agents. The
preferred buffers that can be used include, but not limited to,
phosphate buffered saline (PBS) (pH 7 to 7.5), morpholino
ethanesulfonic acid (MES) (pH 5.5 to 6.5) and triethanol amine
buffer (pH 7 to 7.5), sodium acetate buffer and the like. A buffer
concentration in the range of 10 mM to 100 mM is preferred. Among
the buffers, PBS or MES buffers are most preferred. A co-catalyst
that may accelerate the crosslinking reaction is added. Examples of
such co-catalyst include, but not limited to, n-hydroxysuccinimide
or n-hydroxysulfosuccinimide. The molar concentration of
co-catalyst is in the same range as that of EDC. The cross-linking
reaction is generally completed with in 1 to 600 minutes, more
preferably between 1 to 30 minutes. The preferred reaction
temperature is below the shrink temperature of tissue, typically 0
to 45.degree. C. Crosslinking reaction at 4 to 37.degree. C.
temperature range is even more preferred.
[0231] The crosslinked tissue is then used to manufacture
bioprostheses such as heart valve, stent grafts, vascular grafts,
hernia surgical repair, rotating cuff repair and the like. If
desired, smaller engineered tissue sizes may be stitched together
to make a larger size tissue or thinner tissue may be combined or
stacked and glued to make a thicker tissue for a particular medical
device application. The engineered tissue may be crosslinked using
several methods known in the bioprosthesis processing art or
methods yet to be developed. If desired, smaller engineered tissue
sizes may be stitched together to make a larger size tissue or
thinner tissue may be combined or stacked and glued to make a
thicker tissue for a particular medical device application. The
engineered tissue may be crosslinked using several methods known in
the bioprosthesis processing art or methods yet to be developed
[0232] Medical Applications of Tissue Materials
[0233] Tissue prepared according to an embodiment of the present
invention may, for example, and without limitation, be used for
variety of medical and surgical applications, e.g., implantable
tissue or for manufacture of bioprosthesis as defined earlier.
These applications include, but are not limited to, applications
which are already known such as, by way of example, and not
limitation, vascular patch, heart valve, venous valve
bioprosthesis, vascular grafts, surgical patch, or applications
which are yet to be developed. Complex medical devices such as, by
way of example, and not limitation, heart valve, stent grafts and
sewing cuffs are also included. Many variations of valve designs
may, for example, and without limitation, be used. The tissue used
may be subjected to additional anticalcification treatments such
as, by way of example, and not limitation, treatment with ethanol
solutions (>50% buffered ethanol solution) or surfactants or
chemical modifications with long alkyl chains. Medical devices such
as, by way of example, and not limitation, coronary stents or
peripheral stents may be partially or fully covered with the tissue
such as, by way of example, and not limitation, bovine pericardial
or porcine pericardial tissue or porcine sub-mucosa tissue and
used. In one embodiment, the present invention provides for a
method for making a tissue/stent graft including the steps of
providing an expandable metal or polymeric stent capable of
supporting mammalian cell growth on its surface; exposing the stent
to a mammalian cell culture medium including live mammalian cells
capable of attaching and growing on the stent; growing the cells on
the stent surface until the stent surface is partially or
completely covered by the cells and extra cellular matrix; and
exposing the stent to a tissue fixative solution. Coated tissue may
also be used make stent grafts used in the vascular surgery. The
present invention also encompasses an expandable stent wherein the
stent is fully or partially wrapped or covered with a non-cytotoxic
membrane-like tissue. Bovine ureter, bovine thoracic artery or
other arterial tissue may, for example, and without limitation, be
used as small or medium bore vascular grafts. The tissue used may
be loaded with biological active compounds by methods known in the
art or methods described herein. Hybrid devices such as, by way of
example, and not limitation, graft tissue coated with polymeric
elastomers may, for example, and without limitation, be used to
make improved vascular grafts. In one embodiment, a polymeric
vascular graft made from polyurethane elastomer may be completely
wrapped in a membrane like tissue. Such wrapping provides animal
tissue as blood contacting surface for the graft. The polyurethane
provides a self sealing property to the graft. In another
embodiment, crosslinked bovine thoracic artery is dehydrated using
a series of alcohol solutions and finally using dimethyl acetamide
(DMAC) as a solvent. A 10% solution of Biomer.RTM. polyurethane in
DMAC is sprayed on the outer surface of the graft. The solvent is
removed by air drying and finally by vacuum drying. The
polyurethane-tissue composite graft may offer improved mechanical
properties.
[0234] The animal tissue, especially a membrane-like tissue such
as, by way of example, and not limitation, porcine pericardial
tissue, bovine pericardial tissue or porcine intestinal tissue may,
for example, and without limitation, be used in wound management
applications. The wound management applications include, but are
not limited to, venous ulcers, drainage wounds, diabetic ulcers,
chronic vascular ulcers, pressure ulcers, trauma wounds, surgical
wounds, burn wounds, partial and full thickness wounds and the
like. Membrane-type tissue coated with biodegradable polymer
capable of releasing bioactive substances is preferred. The
preferred bioactive substances for wound management include, but
are not limited to, growth factors derived from blood such as, by
way of example, and not limitation, PGDF, anti-infective compounds
like rifampin, and chlorhexidene acetate.
[0235] The tissue-based patch may also be used in various surgical
applications. Depending on the application, a biostable or
biodegradable tissue may, for example, and without limitation, be
used. In one embodiment, the present invention provides for a
method sealing a leaking tissue during a surgical procedure
including the steps of providing a membrane like animal tissue
suitable for human implantation; providing a surgical adhesive
capable of adhering with the tissue; and curing a surgical adhesive
between a leaking site and membrane like tissue. The applications
include, but are not limited to providing pubourethral support for
the treatment of urinary incontinence; abdominal wall repair;
vascular surgery applications like intracardiac defect repair,
septal defect repair, great vessel repair and anulus repair;
suture-line buttressing; hernia repair; perforated tissue repair;
general tissue repair such as, by way of example, and not
limitation, prolapsed tissue repair, thoracic wall repair, carotid
patch, pelvic floor repair, bladder repair; dural substitute for
closure of dura during neurosurgery;
[0236] Membrane-like tissue (biostable or biodegradable) such as,
by way of example, and not limitation, bovine pericardium, porcine
pericardium, porcine submucosa may be cut into strips, strands or
fibers. Many known methods to cut the tissue into small fiber-like
strands may, for example, and without limitation, be used. The
methods reported in the prior art may, for example, and without
limitation, be used. These methods include, but are not limited to,
methods such as, by way of example, and not limitation, mechanical
shredders, ultraviolet, visible and infrared laser cutting-based
methods, water jet cutting methods, ultrasonic wave-based cutting
methods and the like. The shredded tissue or fiber may be braided
or woven to generate cloth like structures or rope like structures.
These braided or woven structures may, for example, and without
limitation, be used to fabricate many devices such as, by way of
example, and not limitation, vascular patch, vascular grafts, stent
grafts etc. Synthetic fibers such as, by way of example, and not
limitation, polyester fiber, Teflon.RTM. fiber, polyethylene fiber,
poly propylene fiber, silk fiber, poly(hydroxy acid) or polylactide
or carbon fiber may be braided or woven along with braided tissue
to form composite structures. Such composite may provide additional
properties to the woven or braided structures. Such braided
structures may, for example, and without limitation, be used as
substitutes for ligaments. The synthetic fibers used may be
biodegradable.
[0237] Tubular tissue such as, by way of example, and not
limitation, arterial tissues may, for example, and without
limitation, be used to make catheters. For example, catheters that
require a longer implantation time may be made using crosslinked
animal tissue. The outer layer of nerve tissue may, for example,
and without limitation, be used to make nerve guide repair
devices.
[0238] Transparent tissues such as, by way of example, and not
limitation, porcine cornea may, for example, and without
limitation, be used to obtain a transparent material If unsaturated
monomers are used in such tissue fixation, then monomers which give
transparent hydrogel may, for example, and without limitation, be
used. For example, tissue may be fixed with 2-hydroxyethyl
methacrylate, vinyl pyrrolidinone monomers which give optically
transparent hydrogels. In general, monomers which are used in
commercial contact lens manufacturing are preferred. Such
transparent materials may, for example, and without limitation, be
used in various ophthalmic applications such as, by way of example,
and not limitation, corneal transplants, implantable contact lens
or intraocular lens, etc. Tissue fixed with a degradable
crosslinkers may, for example, and without limitation, be used as a
scaffold for tissue engineering.
[0239] In many medical applications, implantable tissue may be
subjected to additional crosslinking or anticalcification
treatments which are known in the art. Such alternations,
combinations or modifications are encompassed by the present
invention even if specific examples or embodiments are not provided
in this manuscript.
[0240] The membrane-like tissue is especially useful as a barrier
for prevention of postoperative adhesions. Membrane tissue coated
with non-cell adhesive hydrogels or polymars such as, by way of
example, and not limitation, polyethylene glycol based hydrogel or
PTFE based membranes is especially useful. A localized release of
cell cycle inhibitor such as, by way of example, and not
limitation, Rapamycin, paclitaxel, Actinomycin, Lovastatin through
coated or composite tissue may also assist in reducing
postoperative adhesions.
[0241] Hernia repair is among the most common general surgery
procedures. Complications cited in the medical literature include
intestinal fistulas and surgical adhesions, which may cause
intestinal obstruction. The coated tissue compositions or
tissue-PTFE composite membrane like materials described in this
invention are especially useful for hernia repair applications. A
membrane like tissue (biodegradable or biostable) described in this
invention or commercially available tissue patch such as
AlloDerm.RTM. marketed by LifeCell Corporation is coated with
polyethylene glycol coating on one side is especially useful for
Hernia repair application. The PEG coating acts as a non-cell
adhesive layer which reduces postoperative adhesions. The other
non-coated side promotes cell adhesion and integrates into the
native tissue. The PEG based coating operation may be carried out
prior to implantation. For example, AlloDerm.RTM. patch may be
coated with DuraSeal.TM. sealant from Confluent Surgical Inc.,
Waltham, Mass. just prior to implantation and may be used for
hernia repair applications. The PEG coating may be replaced by
non-cell adhesive membrane like PTFE membrane such as Gore-Tex
surgical patch which is also known to reduce post-operative
adhesions. The ePTFE membrane-tissue or AlloDerm.RTM. composite may
be prepared by suturing the two membranes using ePTFE based
sutures.
[0242] Membrane-like tissue such as, by way of example, and not
limitation, bovine pericardium or porcine pericardium may, for
example, and without limitation, be used as structural backing
material for tissue adhesives. Many types of surgical adhesives
could be used including those that are commercially available or
yet to be developed. In one approach, structural adhesive material
or their precursors may be coated on one side of pericardial
tissue. The tissue provides the mechanical support for the adhesive
material. Upon application, adhesive materials develop strong
adhesive bond between surgical site and tissue backing material
used. The surgical adhesive and pericardial tissue could be
biodegradable or biostable. In one exemplary approach, a 2 cm by 5
cm non-crosslinked pericardial tissue or sub-mucosa tissue is
coated with a liquid surgical adhesive such as, by way of example,
and not limitation, a photopolymerizable liquid diacrylate along
with a visible light photoinitiator (see Example 35 described
below) and cured between a lung tissue and pericardial tissue to
seal air leaks. In another approach, cynaocaylic acid ester is used
as a adhesive. Many cynoacrylate adhesives could be used these
include ester already known in the art or ester yet to be develop
for medical device applications. The preferred cynoacrylate esters
include but not limited to: methyl, ethyl, butyl or octyl ester and
is cured between a lung tissue and uncrosslinked pericardial
tissue. Butyl cynoacrylate may also be substituted with
FocalSeal.RTM. surgical adhesive available from Genzyme Biosurgery.
Because uncrosslinked pericardial tissue and polybutyl cynoacrylate
are degradable, the patch and adhesive combination serves as a
degradable adhesive patch system. In another exemplary approach,
polyethylene glycol n-hydroxysuccinimide based crosslinker (PEGNHS)
and amine based crosslinking agent are mixed and coated on the
tissue in dry state. Upon hydration, the PEHNHS and amine
crosslinker react to form a crosslinked polymer and form a strong
adhesive bond. Other surgical adhesives or glues such as, by way of
example, and not limitation, albumin glue may also be used to make
an adhesive patch.
[0243] Porcine/bovine meniscus tissue modified according to an
embodiment of the present invention may also be used in meniscus
repair procedures.
[0244] Catheters or catheters parts may be made from implantable
animal tissue. Catheters designed to stay with in the body for 3
days or more may be made using compositions according to an
embodiment of the present invention.
[0245] Membrane-like tissue (biodegradable or biostable) may, for
example, and without limitation, be used to fabricate medical
devices such as, by way of example, and not limitation, tissue
coated stents or stent-grafts. The tissue covering on the stent may
be done from outside, inside or form both sides. The stent may be a
vascular stent such as, by way of example, and not limitation,
coronary stent or peripheral stent or non-vascular stent. Stent
used may be biodegradable (made form biodegradable polymers such
as, by way of example, and not limitation, cyclic polylactones and
their copolymers) or biostable such as, by way of example, and not
limitation, metallic stent (made from stainless steel or Nitinol).
The tissue used on the stent may be loaded with drugs such as, by
way of example, and not limitation, cell cycle inhibitors which are
known to inhibit restenosis.
[0246] In minimally invasive surgical applications thin but strong
membrane tissue is desired. Membrane-like tissue such as, by way of
example, and not limitation, bovine pericardium may be too thick in
MIS surgical applications. In stent graft application, a strong
membrane-like tissue which is about 10 to about 1000 micron thick
is highly desirable. Such thin tissue reduces the profile for a
stent graft delivery system therefore permits its use in making
small diameter (low profile) devices. The thickness of the tissue
can be reduced by many methods known in the art such as, by way of
example, and not limitation, cutting, polishing and the like. The
most preferred method for controlling the thickness is laser-based
ablation technique used in vision correction surgery such as, by
way of example, and not limitation, photorefractive keratectomy or
IntraLase.RTM. surgical technique. Laser-based reshaping of tissue
is routinely used in Lasik surgery, short for
laser-assisted-in-situ keratomileusis, normally used in vision
correction surgery. The membrane-type tissue such as, by way of
example, and not limitation, porcine submucosa or porcine
pericardium or bovine pericardium tissue can be treated using
Lasik-like surgery to reduce the desired tissue thickness.
Lasik-like technique may, for example, and without limitation, be
used to reduce the tissue thickness to about 5 to about 500
microns, most preferably about 50 to about 500 micron thick tissue
without significantly compromising other properties of the
tissue.
[0247] The coated tissue described in this invention may be useful
to form strip-like structures that can be used in bone repair. For
example, biodegradable tissue like porcine or bovine pericardium
may be loaded with bone growth factors such as, by way of example,
and not limitation, BMP-2 or BMP-7. The BMP loaded tissue may, for
example, and without limitation, be used to heal bone surface
defects. Multiple layers of such structures may, for example, and
without limitation, be used in bone fracture repair. The
membrane-like porcine pericardium tissue may be coated with
dematerialized bone matrix. The membrane tissue provides structural
strength while demineralized bone matrix provides growth factors
and matrix that promotes bone growth. Such composite materials may,
for example, and without limitation, be used as a "bonewrap" in
bone fracture repair. In another approach, the pericardial tissue
may be coated with demineralized bone matrix particles and the
composite may, for example, and without limitation, be used as a
bone wrap.
[0248] Tissue hydroxyapatite composite matrix may be used in bone
repair applications. The hydroxyapatite or other bone promoting
calcium salts is added to the tissue matrix using many methods
known in the orthopaedic biomaterials art. In one exemplary
embodiment, hydroxy apatite is synthesized inside the tissue matrix
by combining calcium ion and phosphate ion solution in presence of
tissue. The calcium nitrate [Ca(NO3)2, 4H2O, Aldrich], may be used
as calcium source and ammonium hydrogen phosphate
[(NH.sub.3).sub.2HPO.sub.4, Aldrich] may be used as phosphorus
source. In presence of tissue, the calcium nitrate solution is
added to ammonium hydrogen phosphate solution at 40.degree. C.
while maintaining the pH using ammonium hydroxide to 10 and 10.6 to
form hydroxyapatite crystals. The reaction mixture is stirred for
24 hours to stabilize the crystals. The ratio of The Ca ion and P
ion precursors is maintained 1.6 to make hydroxyapatite crystals.
This reaction is carried in presence of tissue such as pericardial
tissue so that hydroxyapatite is generated in side the tissue. It
is contemplated that the tissue-hydroxyapatite can potentially be
used as a matrix for bone formation. If needed bone promoting
bioactive compounds such as bone morphogenic proteins (BMP) may be
added in the composite matrix to accelerate the bone formation.
[0249] In a biodegradable drug delivery patch application,
membranes like tissue such as, by way of example, and not
limitation, porcine pericardial tissue, bovine pericardial tissue
or porcine sub-mucosa, porcine bladder tissue are preferred. The
animal tissue used in the patch application may be crosslinked or
non-crosslinked. A non-crosslinked tissue is used if an absorbable
patch material desired. Drug delivery patch could be formulated to
incorporate bioactive compounds. The patch will deliver a
therapeutically effective amount of bioactive substance in a
controlled manner. The patch may be coated with biodegradable
hydrogels encapsulated with cells or bioactive growth factors.
[0250] In one potential application, an animal tissue-based patch
is used to induce angiogenesis in the heart. Briefly, an animal
tissue-based patch is formulated to deliver vascular endothelial
growth factor (VEGF) in a controlled manner. The patch is then
transported to a surgical site in the body such as, by way of
example, and not limitation, the surface of a beating heart and
immobilized on the heart surface. The localized release of VGEF
induces angiogenesis in the heart. In another application, a patch
is formulated to release cell cycle inhibitor such as, by way of
example, and not limitation, Rapamycin, Paclitaxel, Actinomycin,
Lovastatin, and the like. The patch is then transported to the site
where vascular stent or graft is implanted to a site that is
susceptible for restenosis. The patch is wrapped around the vessel
where restenosis is likely to occur and cell cycle inhibitor is
released locally. The local release of cell cycle inhibitor reduces
or eliminates restenosis. In some applications, a patch may be
coated with hydrogel coatings containing living species such as, by
way of example, and not limitation, mammalian cells, stem cells,
and endothelial cells. After implantation, the cells may perform a
therapeutic function. Cell encapsulation methods such as, by way of
example, and not limitation, those described in U.S. Pat. No.
5,529,914, which cited here for reference only as one suitable
technique, may be preferentially used in cell encapsulation.
[0251] Some of the tissue modifications or crosslinking methods may
be performed on autologous tissue obtained during a surgical
procedure. Chemicals and instrumentation, and tools required for
such procedures may be provided as a kit in the operating room.
Autologous or single tissue may, for example, and without
limitation, be used as a carrier for biologically active compounds.
The drug-tissue based combination may be prepared in the operating
room during a surgical procedure. A kit including biodegradable
polymer such as, by way of example, and not limitation, polylactic
acid/polyglycolic acid copolymer, a non-toxic biocompatible
preferably water soluble solvent capable of dissolving
biodegradable polymer is provided according to an embodiment of the
present invention. The preferred solvents that can be used include,
but are not limited to, n-methylpyrrolidinone, dimethyl sulfoxide,
acetone, polyethylene glycol, and the like. The biodegradable
polymers include, but are not limited to, polymers, copolymers or
oligomers of glycolide, dl-lacide, d-lactide, l-lactide,
caprolactone, dioxanone and trimethylene carbonate;
polyhydroxyacids, polylactic acid, polyglycolic acid,
polyanhydrides, polylactones, polyhydroxyalkanoates which are
polyesters produced by microorganisms such as
poly(3-hydroxybutyrate), 3-hydroxyvalerate, 4-hydroxybutarate,
3-hydroxyhexanoate, 3-hydroxyoctanoate. Preferred hydrophilic
polymers include, but are not limited to, polyethylene
glycol-polyhydroxy acid or polyethylene glycol-polylactone
copolymers (PEG-PL copolymers), polyvinyl alcohol co-polylactone
copolymers, and derivatives of cellulose, hyaluronic acid and
dextran. The PEG-PL copolymers are most preferred. PEG-PL
copolymers such polyethylene glycol-polylactate copolymers can have
a range of properties from hydrophobic to hydrophilic depending on
the amount of PEG incorporation in the copolymer and molecular
weight of PEG and polylactone. The biodegradable polymer solution
may be made in the operating room from the sterile components. The
biologically active compound is dissolved, suspended or dispersed
in the polymer solution. An autologous tissue piece is cut using a
surgical incision and then incubated in the polymer solution. The
tissue is then removed from the solution and washed with saline and
implanted. The polymeric coating holds and releases the bioactive
compounds in a controlled manner. For example, rifampin, an
antibiotic may be released locally to control infection. The
autologous tissue serves as a matrix that can be sutured to
immobilize it at a localized disease site. The low toxicity of
solvents and biodegradable nature of polymer used in this system
provide a simple method for preparing a tissue-based controlled
drug delivery device.
[0252] The present invention also provides for a method for
localized cell therapy including the steps of providing or
obtaining a sterile tissue suitable for human implantation;
providing a sterile crosslinkable polymer; providing a sterile
aqueous based solvent for the crosslinkable polymer; providing or
obtaining mammalian cells for therapeutic use; dissolving the
polymer in the solvent and dispersing cells in the polymer
solution; forming a coating of polymer on the tissue and
crosslinking the polymer without damaging the cells; and implanting
the tissue.
[0253] It is contemplated that the shape memory capable tissue may
be used to make many novel medical devices. For example, tissue
based vascular grafts may be compressed and the compressed shape
may be preserved to improve kink resistance of the graft. The
compressed graft become longitudinally compliant as a result of
compression and may have better patency rates. A heart valve
leaflet shape may be preserved to reduce stresses during heart
valve device use. A tissue based breast implant with appropriate
shape may be preserved using methods described in this
invention.
[0254] The selective modification of tissue to create biodegradable
and biostable regions may be used to create new medical
devices.
[0255] In many medical device applications, bioprostheses prepared
may be sterilized by many methods known in the art. These methods
include but not limited are: gamma radiation sterilization,
ethylene oxide or propylene oxide based sterilization methods,
electron beam sterilization, iodine-based sterilization methods,
and the like. The preferred method is ethylene oxide gas
sterilization or gamma radiation sterilization. The sterilization
process parameters will depend on the tissue based medical device
to be sterilized.
[0256] In some applications proteins such as, by way of example,
and not limitation, collagen or albumin are treated with silver
salt solution to form silver salts/complexes of amino acids present
in the proteins. For example, aspartic acid which produces free
carboxyl acid groups in the proteins may be treated with silver to
form a silver salt. The resultant silver salt then releases silver
ions upon implantation producing an antimicrobial effect due to
silver ions.
[0257] The tissue-based medical devices described herein may be
packaged using variety of packaging methods including packaging in
a liquid solution such as, by way of example, and not limitation,
glutaraldehyde solution. In preferred packing method, the tissue is
packaged in sealed plastic bag. If desired, the tissue may be
packaged and stored under inert gas atmosphere such as, by way of
example, and not limitation, nitrogen, carbon dioxide or argon gas.
The gas used may be kept under pressure.
[0258] The following non-limiting examples are intended to
illustrate the inventive concepts disclosed in this document. Those
skilled in the art will appreciate that, in light of the teachings
of the present invention, modifications can be made to these
examples, drawings, illustrations and claims which are contemplated
to fall within the scope the present invention.
[0259] Materials and Methods
[0260] Tissue-like bovine pericardium, porcine pericardium, porcine
sub-mucosa, porcine aortic root, porcine meniscus tissue and bovine
thoracic arterial tissue are acquired from local abbotair or
slaughter house. The animals from abbotair or the tissue obtained
form abbotair may be screened or tested for infectious viruses,
bacteria or proteins such as, by way of example, and not
limitation, BSE which cause disease such as, by way of example, and
not limitation, Bovine Spongiform Encephalopathy. Tissue infected
with harmful virus or proteins may be discarded. Preferably, the
tissues are collected from slaughter house with in 30 minutes after
the kill. Within 36 hours, the tissue collected is cleaned to
remove excess fat, connective tissue, blood and other cellular
components and rinsed with cold phosphate buffered saline and
stored in hyperosmotic storage solution (ESWHS solution) until
fixation or other modifications. In many instances, the tissue may
be treated with detergents, organic solvents such as, by way of
example, and not limitation, aqueous ethanol or long chain alcohols
such as, by way of example, and not limitation, octanol or
1,2-octane diol other tissue cleaning solutions that remove
cells/cell debris from the tissue and leaves behind only
extracellular matrix. The a cellular extracellular matrix is
preferred in many medical device applications. Acrylic acid
n-hydroxysuccinimide ester, acetic acid n-hydroxysuccinimide,
polyethylene glycol n-hydroxysuccinimide esters, acrolein are
purchased from Sigma-Aldrich or Pierce or Sherewater. Polyethylene
glycol can be purchased form various sources such as, by way of
example, and not limitation, Nektar Therapeutics (formerly
Shearwater Polymers), Dow Chemicals (Union Carbide), Fluka and
Polysciences. Multifunctional hydroxyl and amine terminated
polyethylene glycol are purchased from Nektar Therapeutics, Dow
Chemicals and Texaco Amine-terminated polyethylene glycols also can
be synthesized using methods known in the prior art. Other
specialized polyethylene glycol derivatives may also be purchased
or custom synthesized from Nektar Therapeutics. DL-lactide,
glycolide, caprolactone and trimethylene carbonate can be obtained
from commercial sources like Purac, DuPont, Polysciences, Aldrich,
Fluka, Medisorb, Wako and Boehringer Ingelheim.
N-hydroxysulfosuccinimide can be purchased from Pierce. All other
reagents, solvents are of reagent grade and can be purchased from
commercial sources such as, by way of example, and not limitation,
Polysciences, Fluka, ICN, Aldrich and Sigma. Most of the
reagents/solvents are purified/dried using standard laboratory
procedures such as, by way of example, and not limitation,
described by Penin et al. Monomers may be purified to remove
inhibitors just prior to use. For example, liquid monomers such as,
by way of example, and not limitation, 2-hydroxyethyl methacrylate
may be vacuum-distilled to remove the inhibitor. Most commercially
available monomers are purchased from Polysciences, Fluka, ICN,
Aldrich and Sigma. Photoinitiator
2-Hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone
(Irgacure 2959) and 2,2-dimethoxy-2-phenylacetophenon (Irgacure
651) are purchased from Ciba Specialty Chemicals or Sigma-Aldrich.
Small laboratory equipment and medical supplies can be purchased
from Fisher or Cole-Parmer. Cell culture experiments are performed
using a standard mammalian tissue culture laboratory or
microbiology laboratory capable of handling and growing mammalian
and human cell cultures.
[0261] Removal of Cellular Components from the Tissue (Tissue
Decellualrization)
[0262] Tissue suitable for implantation may be treated to remove
cells and cellular components or fragments, DNA fragments and the
like. The removal of cellular components is believed to reduce
immunogenicity of the implanted tissue. Upon removal of cellular
components, only extracellular matrix components such as, by way of
example, and not limitation, collagen, elastin, laminin, keratin,
glycosaminoglycans are retained in the tissue. The extracellular
matrix is believed to be non-immunogenic. Many protocols for
removal of cellular components are reported in the prior art, one
of the preferred protocols is provided below. Ten 1 cm by 1 cm
bovine pericardium pieces, cut from a freshly obtained bovine
pericardial sac, are transferred to 50 ml conical flask containing
10 ml phosphate buffered saline (PBS, pH 7.2). The tissue is then
incubated in 100 ml distilled water at 4.degree. C. for 48 h,
followed by incubation in 30% sodium chloride and/or saturated
sodium chloride solution for 48 hours followed by washing with 100
ml PBS solution three times. The tissue is then treated with 40 ml
of 0.25% trypsin-EDTA, for 30 minutes at 37.degree. C.; followed by
washing with warm (37.degree. C.) 100 ml PBS solution three times.
It is then treated with 40 ml of micrococcal nuclease, 1 unit/ml,
for 20 minutes at 37.degree. C.; followed by washing with warm
(37.degree. C.) 100 ml PBS solution three times. It is then further
treated with 40 ml of Triton X-100, 10%, for 10 minutes at
37.degree. C.; followed by washing with warm (37.degree. C.) 100 ml
PBS solution three times. It is then treated with 40 ml of 0.1 M
sodium hydroxide at 37.degree. C. for 30 minutes; followed by
washing with warm (37.degree. C.) 100 ml distilled water five
times. It is then treated with 40 ml of 0.1M hydrochloric acid at
37.degree. C. for 30 minutes; followed by washing with warm
(37.degree. C.) 100 ml distilled water five times. Finally the
tissue is washed with 70% isopropanol three times and stored in 70%
isopropanol at -20.degree. C. until further use.
[0263] In a simplified cell removal protocol (tissue
decellualrization protocol)), tissue is first treated with a 0.25%
trypsin-EDTA solution in PBS for 30 minutes at 37.degree. C.
followed by a treatment with 10% surfactant solution (Triton X-100)
for 2 hours to remove cellular debris or soluble proteins.
Shrink Temperature by Differential Scanning Calorimetry
[0264] Shrink temperature is evaluated by differential scanning
calorimetry. Briefly, 3-10 mg of tissue sample is heated in a
sealed aluminum sample pan and heated at 10.degree. C. per minute
up to 200.degree. C. under nitrogen atmosphere. The endotherm
around 55.degree. C. to 110.degree. C. is attributed as shrink
temperature.
[0265] Shrink Temperature by Visual Method
[0266] A 3 cm.times.1 cm tissue strip is cut and the rectangular
shape of the tissue is traced on a paper to record its size. In a
500 ml beaker, 300 ml saline solution is transferred. The tissue is
then suspended in the saline solution using a cotton or metal
thread and is completely immersed in the saline solution. The
solution is slowly heated on a hot plate until boiling. The tissue
is visually observed until signs for shrinkage. The temperature at
which tissue significantly shrinks is recorded. The tissue is
removed from the solution, washed with water. The size of the
tissue again traced on a paper and compared with the original size.
The shrunken tissue shows significant reduction in size (typically
>20% shrinkage). Untreated tissue and treated tissue are usually
compared in the same experiment.
[0267] Viability and Spreading of Bovine Endothelial Cells on
Crosslinked Bovine Pericardial Tissue
[0268] Tissue samples fixed using crosslinking agents such as, by
way of example, and not limitation, photopolymerization, are used
in this experiment. Untreated bovine pericardium tissue is used as
control. 1 cm.times.1 cm size tissue samples are soaked in 70%
ethanol (overnight), washed with sterile PBS (15 minutes, 3 times)
and soaked in Minimum Essential Medium (MEM) supplemented with
aminoacids, antibiotics and 30% fetal bovine serum (FBS) for 1.5 h.
The tissue samples are then transferred to a 24 well sterile tissue
culture plate. Bovine aortic endothelial cells passaged in MEM/10%
FBS are resuspended in MEM/10% FBS and approximately 200000 cells
are carefully placed on the tissue samples. The cells are allowed
to adhere for 30 minutes before adding 0.75 ml of the same medium
to each sample. After 24 hours of incubation in a 5% carbon dioxide
atmosphere at 98% humidity and 37.degree. C., the samples are
transferred to new wells and fresh medium is added to these wells.
The samples are incubated for a further period of 24 to 72 hours.
At the end of this incubation period, the tissue samples/cells are
washed with PBS (3 times, 5 minutes per dish at room temperature)
and fixed using 4% formaldehyde for 10 minutes. After rinsing the
samples in PBS as described above, they are treated with 0.1%
Triton X100 in PBS for 3 minutes. The cells are then stained with
phalloidin/rhodamine (diluted 1:40 in PBS) in dark for 45 minutes,
rinsed 3 times in PBS and viewed immediately under a fluorescence
microscope. The morphology and coverage of endothelial cells on
tissue samples are recorded. Some representative snapshots are
photographed for each sample. Glutaraldehyde-treated tissue is used
a negative control and untreated tissue is a positive control. The
untreated and photopolymerization treated tissue (vinyl
pyrrolidinone treated) showed substantially higher cell growth and
attachment as compared to glutaraldehyde fixed tissue. It is
generally known that glutaraldehyde fixed tissue does not support
tissue growth.
[0269] Subcutaneous Implantation of Tissues
[0270] 1 cm2 rectangular tissue pieces are implanted using a
standard surgical protocol. Prior to implantation, the tissues are
rinsed for 3 minutes in each of three basins containing 500 ml of
sterile PBS, accompanied by gentle shaking. Specimens are implanted
subcutaneously 1 cm lateral from the abdominal midline in 3 week
old Sprague-Dawley rats and retrieved after 60 days. Unimplanted
samples (two per crosslinking treatment) are used as unimplanted
control. Untreated tissue is used as positive control.
Glutaraldehyde-fixed tissue is used as a negative control. Tissue
modified with unsaturated group and infused with monomer and
photoinitiator but not exposed to light is treated as dark
control.
[0271] X-Ray Imaging and Histology
[0272] The subcutaneously implanted samples are recovered after 30,
60 or 90 days. The explanted samples are subjected to standard
medical x-ray imaging for calcification/mineralization as a result
of implantation. The unimplanted samples and samples fixed with
glutaraldehyde are also imaged and used as controls. The calcified
deposits can be seen in samples treated with glutaraldehyde. An
untreated unimplanted sample does not show calcific deposits when
viewed using medical x-ray imaging technique or when stained using
Van Kossa stain. After visual analysis, the parts of the samples
are sectioned, stained with H&E, Masson's trichromeand and Van
Kossa stain and then evaluated histologically for inflammation,
vascularization, calcification and collagen organization.
Inflammation consists of aggregates of lymphocytes and plasma
cells, and the presence of mast cells in the implants and is graded
from 0=None, to 5=Severe (most of the section with large lymphoid
aggregates). Calcification is also graded from 0 to 5 based on Van
Kossa stain.
[0273] Calcium Assay
[0274] The calcium content of the subcutaneously implanted samples
is determined by the wet-washing technique. Briefly, up to 0.5 g of
explanted tissues are dried and weighed and by heated up to
250.degree. C. with 4 ml of concentrated sulfuric acid followed by
heating up to 300.degree. C. with addition of concentrated nitric
acid. The resulting digested sample is brought to 40 ml with
addition of high purity water. Calcium concentration in the
solution is determined by standard ICP methodology.
[0275] Pepsin Digestion Assay-Analysis of Tissue Fragments by Gel
Electrophoresis
[0276] Pepsin digestion is done to evaluate the ability of tissue
to resist enzymatic degradation. Briefly, the fixed and control
tissue samples are exposed to a pepsin solution and size and weight
of the tissue is monitored. Untreated and uncrosslinked tissue
sample is used as a control. In another method, tissue samples are
clipped into small fragments and digested for 4 h at 37.degree. C.
in 10 mM HCl solution containing 4 mg/ml pepsin. Sham digestions
are also prepared in HCl without the enzyme. Following the
incubation, the samples are centrifuged at 4.degree. C. for 1 hour
at 14,000 rpm. The supernatants are analyzed by gel electrophoresis
using 10-20% acrylamide:bisacrylamide (37.5:1) gradient gel.
Control samples with the same quantity of enzyme are also treated
as above, but in the absence of tissue. The fixed tissue shows
significantly lower proteins on gel as compared to unfixed tissue.
The fixed tissue also shows lower weight loss when compared to
unfixed tissue indicating stability of the tissue.
[0277] Pepsin Digestion Assay-Gravimetric Analysis
[0278] The tissue to be treated is washed with distilled water and
incubated in distilled water for 10 minutes. The same is then dried
in air and then kept in vacuum oven for drying until constant
weight is observed. The dried tissue is then incubated in 10 mM HCl
containing 40 mg of pepsin for 12 hours. The tissue is removed,
washed with distilled water and again dried till constant weight.
The difference in the tissue weights before and after the test is
noted. The loss in weight shows the amount of protein digested by
pepsin. The unfixed or uncrosslinked tissue generally shows
substantial loss as compared to fixed or crosslinked tissue.
[0279] Collagenease Digestion Assay
[0280] The resistance to enzymatic digestion also can be found by
exposing the tissue to collagenease solution. Briefly, a tissue is
exposed to a 0.01 mg/ml collagenease enzyme solution in 10 mM TES
buffer (N-tris[hydroxymethyl]methyl-2 aminoethane sulfonic acid)
and 25 mM calcium chloride solution at 37.degree. C. for 24 to 96
hours and the weight loss of the tissue is monitored and compared
with the control untreated tissue.
[0281] Free Amine Content of Tissue
[0282] The free amine content of the treated tissue is determined
by the ninhydrin assay. Briefly, tissue is exposed to ninhydrin
solution and the absorbance of ninhydrin solution is measured at
570 nm and is compared with control tissue (untreated uncrosslinked
tissue). The absorbance is normalized by dividing by weight of
tissue before comparison.
[0283] Biodegradation and Biocompatibility of Tissue and
Tissue-Biodegradable Polymer Composites
[0284] In vitro degradation of the polymers is followed
gravimetrically at 37.degree. C., in aqueous buffered medium such
as, by way of example, and not limitation, phosphate buffered
saline (pH 7.2). In vivo biocompatibility and degradation life
times are assessed after subcutaneous implantation of tissue
samples. The implant is surgically implanted in the animal body.
The degradation of the implant over time is monitored
gravimetrically or by chemical analysis. The biocompatibility of
the implant is assessed by standard histological techniques.
[0285] General Analysis
[0286] Chemical analysis such as, by way of example, and not
limitation, structural determination is done using nuclear magnetic
resonance (proton and carbon-13) and infrared spectroscopy. High
pressure liquid chromatography or UV-visible spectrophotometry is
used to determine drug elution profiles. Gel permeation
chromatography is used for molecular weight determination. Thermal
characterization such as, by way of example, and not limitation,
melting point and glass transition temperature is done by
differential scanning calorimetric analysis. The aqueous solution
properties such as, by way of example, and not limitation, self
assembly, micelle formation, and gel formation are determined by
fluorescence spectroscopy, UV-visible spectroscopy and laser light
scattering instruments.
Example 1
[0287] Modification of tissue using unsaturated succinimide ester
(acrylic acid succinimide ester)
[0288] Modification of Bovine Pericardium Tissue.
[0289] Ten 1 cm by 1 cm bovine pericardium pieces, cut from a
freshly obtained bovine pericardial sac, are transferred to 50 ml
conical flask containing 10 ml phosphate buffered saline (PBS, pH
7.2). 250 mg acrylic acid succinimide ester (ANHS) (Sigma-Aldrich
Product Number: A8060) dissolved in 0.5 ml dimethyl sulfoxide is
added to the conical flask and the solution is vortexed for 15
minutes. 0.1 g of ANHS in 0.1 ml DMSO is added to the fixation
solution every 2 hours up to six hours. The modification reaction
is carried for 6 hours at ambient temperature (25.degree. C.) and
then for 12 hours at 4.degree. C. with gentle shaking. The reaction
is terminated by washing the tissue with 20 ml PBS 3 times.
Finally, the tissue is stored in 30 ml 38% isopropanol and 2%
benzyl alcohol solution at 4.degree. C. until further use.
[0290] In another variation of this method, porcine pericardial sac
is obtained from a local abbotair and is cleaned to remove blood
and fatty tissue contaminants from the tissue surface. Ten 1 cm by
1 cm pieces are cut from the cleaned pericardial sac and
transferred to 10 ml 0.20 M 2-(N-morpholino)ethanesulfonic acid
(MES) buffered solution (pH 6.5). 0.5 g acrylic acid n-hydroxy
succinimide ester (ANHS) is added to the buffered solution and the
reaction is vortexed for 15 minutes and continued for 6 hours at
ambient temperature. An additional 0.1 g ANHS ester is added to the
mixture at every 1.5 hours. After the reaction, the tissue is
isolated and washed several times with PBS and finally stored in
40% isopropanol solution.
[0291] Modification of Arterial and Porcine Heart Valve Tissue.
[0292] One 1.5 cm piece of bovine thoracic artery and one third
section of porcine aortic root containing one valve cusp and one
attachment zone and wall is transferred to 50 ml polypropylene
centrifuge tube containing 10 ml PBS. 250 mg of acrylic acid
succinimide ester dissolved in 0.5 ml dimethyl sulfoxide is added
to the tube and the solution is vortexed for 15 minutes. The
modification reaction is carried for 6 hours at ambient temperature
(25.degree. C.) and then for 12 hours at 4.degree. C. with gentle
shaking. The reaction is terminated by washing the tissue with 20
ml PBS 3 times. Finally, the tissue is stored in 30 ml 40% HEPES
buffered isopropanol solution.
[0293] Modification of Ligament and Meniscus Tissue.
[0294] Using a similar procedure described above 1 cm ligament
tissue and 1 cm by 1 cm porcine meniscus tissue is modified by 250
mg of acrylic acid succinimide ester dissolved in 0.5 ml dimethyl
sulfoxide.
[0295] Modification of Corneal Tissue.
[0296] Porcine cornea is obtained from a local abbotair and is
washed with PBS (pH 7.2) to remove blood and other biological
contaminants. The cornea is placed in 100 ml beaker containing 50
ml PBS solution. To this solution, 0.2 g acrylic acid succinimide
ester dissolved in 2.0 ml dimethyl sulfoxide is added. The reaction
continued for 12 h at ambient temperature. The tissue is removed,
washed several times with PBS and stored until use. The reaction
conditions are chosen to maintain the transparency of corneal
tissue (minimum change in refractive index).
Example 2
Modification of Tissue Using Glycidyl Methacrylate
[0297] 10 pieces of 1 cm by 1 cm bovine pericardium tissue are
transferred to 100 ml conical flask containing ten ml PBS and a
magnetic stir bar. To this solution, 2 ml glycidyl methacrylate, 2
ml triethyl amine and 0.5 g tetrabutylamine hydrobromide are added.
The reaction is carried for 16 hours at ambient temperature
(25.degree. C.) and then 50.degree. C. for 4 hours with gentle
stirring. The solution is cooled and the tissue is isolated from
the reaction mixture. The isolated tissue is washed with PBS and
40% ethanol to remove traces of glycidyl methacrylate and other
catalysts.
Example 3
Modification Tissue Using Acrolein
[0298] Ten 1 cm by 1 cm bovine pericardium pieces are transferred
to 50 ml conical flask containing 10 ml PBS and 0.60 ml acrolein.
The tissue is incubated for 6 hours at ambient temperature
(25.degree. C.). An additional 0.1 g acrolein is added to the
mixture at every 1.5 hours up to six hours. Finally, the reaction
is continued for 4.degree. C. for 48-120 hours with gentle
stirring. The tissue is isolated from the reaction mixture and is
washed with PBS and 40% ethanol to remove unreacted acrolein.
Example 4
Modification Tissue Using Methacrylic Anhydride or Methacryloyl
Chloride
[0299] Treatment in Aqueous Medium
[0300] Ten 1 cm by 1 cm bovine pericardium pieces are transferred
to 100 ml round bottom flask containing 10 ml 100 mM sodium borate
buffer, pH 9.5 and a magnetic stir bar. To this solution, 2 ml
methacrylic anhydride and 2 ml triethylamine are added. The
reaction is carried out for 6 hours at ambient temperature
(25.degree. C.) and then at 0.degree. C. for 72 hours with gentle
stirring. The solution is cooled and the tissue is isolated from
the reaction mixture. The isolated tissue is washed with PBS and
40% ethanol to remove unreacted methacrylic anhydride.
[0301] Treatment in Organic Solvent (Dimethyl Sulfoxide)
[0302] Ten 1 cm by 1 cm bovine pericardium pieces are transferred
to 100 ml round bottom flask containing 10 ml dimethyl sulfoxide
and a magnetic stir bar. To this solution, 0.1 ml triethylamine and
0.02 ml methacrylic anhydride are added. The reaction is carried
for 24-48 hours at ambient temperature (30.degree. C.) and then the
tissue is isolated from the reaction mixture. The isolated tissue
is washed with PBS and 40% ethanol to remove unreacted methacrylic
anhydride and stored at 4.degree. C. in refrigerator until further
use. In some cases the tissue is used immediately and without
ethanol treatment in polymerization and crosslinking step.
Example 5
Crosslinking of Unsaturated Group Modified Tissue Using Free
Radical Photopolymerization
[0303] Two 1 cm by 1 cm pieces of unsaturated group modified bovine
pericardium tissue prepared per Examples 1-4 are incubated for 30
minutes each in 20% ethanol, 50% ethanol and 70% ethanol. 50 mg of
Irgacure 2959 (free radical photoinitiator) is dissolved in 1 ml
n-vinyl pyrrolidinone to prepare a monomer solution. The 70%
ethanol incubated tissue is transferred to Irgacure solution and
incubated for 4 h at 25.degree. C. and then for 12 h at 0.degree.
C. The tissue is removed from solution is placed on a glass plate
and exposed to a Black-Ray UV lamp (360 nm light, 10000 mW/cm2
intensity) for 5 minutes for each side. The crosslinked tissue is
removed and washed with 10 ml PBS to remove unreacted monomer,
initiator fragments and water soluble non-crosslinked polymer. The
crosslinked tissue shows higher shrink temperature as compared to
uncrosslinked tissue indicating crosslinking of the tissue.
Presence of polyvinyl pyrrolidinone in the tissue is conformed by
IR spectrophotometer. The crosslinked tissue also showed a
different texture as compared to uncrosslinked tissue when touched
and felt by a human hand. The crosslinked tissue also shows
resistance to pepsin digestion.
[0304] In some embodiments, the tissue is infused with monomer
without dehydrating using alcohol solutions and the subsequent
photopolymerization treatment showed higher shrink temperature
indicating crosslinking.
Example 6
Crosslinking of Unsaturated Group Modified Tissue Using Thermally
Induced Radical Polymerization
[0305] Two 1 cm by 1 cm pieces of unsaturated group modified bovine
pericardium tissue prepared per Examples 1-4 are incubated for 30
minutes each in 20% ethanol, 50% ethanol and 70% ethanol. 10 mg of
azobisisobutyronitrile (thermal free radical photoinitiator) is
dissolved in 1 ml vinyl pyrrolidinone. The 70% ethanol incubated
tissue is transferred to the vinyl pyrrolidinone solution and
incubated for 4 h at 25.degree. C. and then for 12 h at 0.degree.
C. The tissue is removed, placed between two glass slides. The
tissue is then heated in an oven maintained at 55.degree. C. (below
the shrink temperature of uncrosslinked tissue) for 4 hours. The
tissue is washed with 20 ml acetone and 20 ml PBS to remove
water/acetone soluble unwanted products.
Example 7
Crosslinking of Unsaturated Group Modified Tissue Using Radiation
or Electron Beam Induced Polymerization
[0306] Two 1 cm by 1 cm pieces of unsaturated group modified bovine
pericardium tissue prepared per Example 1-4 are incubated for 30
minutes each in 20% ethanol, 50% ethanol, 70% ethanol and finally
in n-vinyl pyrrolidinone. The tissue is removed from vinyl
pyrrolidinone and exposed to gamma radiation (total dose 3 Mrad).
The exposed tissue is washed with PBS and examined for
crosslinking. No photoinitiator is used to initiate the
polymerization and crosslinking of tissues.
Example 8
Crosslinking of Unsaturated Group Modified Tissue Using Mixture of
Hydrophilic and Hydrophobic Monomers
[0307] Two 1 cm by 1 cm pieces of unsaturated group modified bovine
pericardium tissue prepared per example 1 are incubated for 30
minutes each in 20% ethanol, 50% ethanol and 70% ethanol. 50 mg of
Irgacure 2959 (free radical photoinitiator) is dissolved in 0.8 ml
vinyl pyrrolidinone (hydrophilic monomer) and 0.2 ml octyl
methacrylate (hydrophobic monomer). The 70% ethanol incubated
tissue is transferred to Irgacure solution and incubated for 4 h at
25.degree. C. and then for 12 h at 0.degree. C. The tissue is
removed from solution and then is placed on a glass plate and
exposed to Black-Ray UV lamp (360 nm light, 10000 mW/cm2
intensity). The crosslinked tissue is washed with large amount of
water and 100 percent ethanol to remove organic and water soluble
impurities. The ratio of hydrophilic to hydrophobic monomer may be
changed to control the water content of the crosslinked tissue.
Many monomers known in the free radical polymerization chemistry
art that provide hydrophobic and hydrophilic polymers can be used
to crosslink the tissue.
Example 9
Crosslinking of Unsaturated Group Modified Tissue Using Functional
Monomer
[0308] Two 1 cm by 1 cm pieces of unsaturated group modified bovine
pericardium tissue prepared per example 1 are incubated for 30
minutes each in 20% ethanol, 50% ethanol and 70% ethanol. 50 mg of
2,2-demethoxy-2-phenyl acetophenone (free radical photoinitiator)
is dissolved in 0.5 ml glycidyl methacrylate (monomer containing
epoxy group) and 0.5 ml n-vinyl pyrrolidone. The 70% ethanol
incubated tissue is transferred to glycidyl methacrylate solution
and incubated for 4 h at 25.degree. C. and then for 12 h at
0.degree. C. The tissue is removed from solution is placed on a
glass plate and exposed to Black-Ray UV lamp (360 nm light, 10000
mW/cm2 intensity) for 15 minutes each side. The crosslinked tissue
is washed with water and 100 percent methanol to remove water and
organic soluble impurities. The epoxy functionality of glycidyl may
be further used to introduce other chemical moieties such as, by
way of example, and not limitation, bioactive compounds or it may,
for example, and without limitation, be used to further crosslink
the tissue using di- or polyfunctional amines such as, by way of
example, and not limitation, hexamethylene diamine.
Example 10
Crosslinking of Unsaturated Group Modified Tissue Using Monomers
that Produce Elastomeric Polymers
[0309] Crosslinking Using Poly(Dimethyl Siloxane) Based Monomer
[0310] 50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radical
photoinitiator) is dissolved in 0.2 Poly(dimethylsiloxane), vinyl
terminated (Sigma-Aldrich Product Number: 43, 301-2) and 0.8 ml
n-vinyl pyrrolidone. Two 1 cm by 1 cm pieces of unsaturated group
modified bovine pericardium tissue prepared per example 1 are
incubated for 30 minutes each in 20% ethanol, 50% ethanol and 70%
ethanol and finally in Poly(dimethylsiloxane) solution prepared as
indicated earlier. The tissue is removed from solution and is
placed on a glass plate and exposed to Black-Ray UV lamp (360 nm
light, 10000 mW/cm2 intensity) from both sides. The crosslinked
tissue is washed with water and 100 percent methanol to remove
water and organic soluble impurities. Incorporation of synthetic
silicone based rubber can assist to improve mechanical properties
of the tissue and improve oxygen permeability.
Example 11
Tissue Modified with Thermosensitive Polymer
[0311] Crosslinking of Acrylic Modified Tissue Using Monomer that
Produces Thermosensitive Polymer/Hydrogel
[0312] 50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radical
photoinitiator) is dissolved in 0.4 ml n-vinyl pyrrolidone and 0.6
g n-isopropylacrylamide. Two 1 cm by 1 cm pieces of acrylic acid
succinimide ester modified or glycidyl methacrylate modified bovine
pericardium tissue prepared per example 1 or 37 is incubated for 30
minutes each in 20% ethanol, 50% ethanol and 70% ethanol and
finally n-vinyl pyrrolidinone solution. The tissue is then
transferred to n-isopropylacrylamide solution in n-vinyl
pyrrolidinone (40%) containing 1000 ppm Irgacure 2959 and incubated
for 4 h. The tissue is removed from solution is placed on a glass
plate and exposed to Black-Ray UV lamp (360 nm light, 10000 mW/cm2
intensity) for 5 minutes each side. The exposed tissue is washed
with 100% alcohol to remove unreacted monomers and initiator
fragments and then incubated in cold PBS solution (4.degree. C.)
for 24 hours. The polymerized n-isopropylacrylamide absorbs water
at low temperature (4.degree. C.) and also permits collagen chains
to re-hydrate. The fully absorbed tissue is incubated in PBS
maintained at 37.degree. C. At 37.degree. C., n-isopropylacrylamide
turns hydrophobic but collagen chains remain hydrated.
Alternatively, a 20-50 percent n-isopropylacrylamide solution in
water may also be used in place of ml n-vinyl pyrrolidone and
n-isopropylacrylamide mixture for polymerization and crosslinking
reaction using 500 ppm Irgacure 2959 as photoinitiator.
Example 12
Bioprosthetic Tissue and Polyvinyl Alcohol Composite
[0313] Crosslinking of unsaturated group modified tissue using
vinyl acetate which is converted into polyvinyl alcohol
[0314] Vinyl acetate is vacuum distilled prior to use to remove
polymerization inhibitor. 50 mg of 2,2-demethoxy-2-phenyl
acetophenone (free radical photoinitiator) is dissolved in 1 ml
inhibitor free vinyl acetate. Two 1 cm by 1 cm pieces of acrylic
acid succinimide ester modified bovine pericardium tissue prepared
per example 1 are incubated for 30 minutes each in 20% ethanol, 50%
ethanol and 70% ethanol and n-methylpyrrolidinone. The tissue is
then transferred to vinyl acetate solution and incubated for 4 h.
The tissue is removed from solution and then placed on a glass
plate and exposed to Black-Ray UV lamp (360 nm light, 10000 mW/cm2
intensity) for 15 minutes each side. The exposed tissue is washed
with 100% alcohol to remove unreacted monomers and initiator
fragments. The tissue is then incubated in 1% p-toluene sulfonic
acid solution in water to hydrolyze polyvinyl acetate. Briefly, the
treated tissue is incubated in 10 ml distilled water containing 100
mg p-toluene sulfonic acid at 50.degree. C. for 3 days.
[0315] Using a similar procedure, acrylonitrile monomer can be can
be used in place of vinyl acetate in tissue crosslinking. The
nitrile groups in polymerized polyacrylonitrile can be partially or
completely hydrolyzed under mild acidic/basic conditions to produce
a mechanically strong polyacrylonitrile-co-polyacrylic acid
hydrogel in the tissue matrix.
Example 13
Crosslinked Tissue Complexed with Biologically Active Compounds or
Synthetic Polymers
[0316] Iodine solution is prepared by dissolving elemental iodine
(0.1%) and potassium iodide (0.4%) in 10% ethanol. Vinyl
pyrrolidinone crosslinked tissue (prepared per Example 5 or 37) is
incubated with iodine solution for 3 hours at 25.degree. C.
temperature and is then removed from the solution and washed with
PBS solution. The iodine complexes with polyvinylpyrrolidinone in
the treated tissue. The brown color of iodine-PVP complex is
noticeable on the tissue surface. The iodine complexed tissue may
be applied on a wound and the iodine released in the wound area is
used to control infection or bacterial growth. Other compounds that
form complex with polyvinyl pyrrolidinone such as, by way of
example, and not limitation, antibiotics may also be used.
Example 14
Tissue and Biodegradable Polymer Composite
[0317] Tissue and Biodegradable Polymer Composite Wherein the
Biodegradable Polymer is crosslinked
[0318] Crosslinking of Acrylic Modified Tissue Using Biodegradable
Multifunctional Macromonomer (Water-Soluble)
[0319] Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer
(10KL5)
[0320] 30.0 g of PEG 10000, 4.3 g of dl-lactide and 30 mg of
stannous octoate are charged into 100 ml Pyrex pressure sealing
tube. The tube is then connected to an argon gas line and sealed
under argon. The tube is then immersed in oil bath maintained at
140.degree. C. The reaction is carried out for 16 h at 140.degree.
C. The polymer from the tube is recovered by breaking the Pyrex
tube. The polymer is then dissolved in 70 ml toluene and
precipitated in 2000 ml cold hexane. The precipitated polymer is
recovered by filtration and dried under vacuum for 1 day at
60.degree. C. It is then immediately used in the next reaction.
[0321] Part 2: End-Capping of 10KL5 with Polymerizable or
Crosslinkable Group (10KL5A2)
[0322] 30 g of 10KL5 is dissolved in 450 ml dry toluene. About 50
ml of toluene is distilled out to remove traces of water from the
reaction mixture. The warm solution is cooled to 65.degree. C. To
this warm solution, 1.6 g of triethyl amine and 1.5 g acryloyl
chloride are added. The reaction mixture is then stirred for 30
minutes at 50-60.degree. C. and filtered. The reactive precursor is
precipitated by adding the filtrate to 2000 ml cold hexane. The
precipitated polymer is recovered by filtration. It is then dried
under vacuum for 12 h at 50.degree. C.
[0323] Part 3: Crosslinking of Acrylic Modified Tissue Using
10KL5A2
[0324] 50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radical
photoinitiator) is dissolved in 0.9 ml n-vinyl pyrrolidone (NVP)
and 0.1 g 10KL5A2. Two 1 cm by 1 cm pieces of acrylic acid
succinimide ester modified bovine pericardium tissue prepared per
examples 1 to 4 is incubated for 30 minutes each in 20% ethanol,
50% ethanol and 70% ethanol and n-vinyl pyrrolidinone. The tissue
is then transferred to 10KL5A2 solution and incubated for 4 h. The
tissue is removed from solution is placed on a glass plate and
exposed to Black-Ray UV lamp (360 nm light, 10000 mW/cm2 intensity)
for 15 minutes each side. The exposed tissue is washed with 100%
alcohol to remove unreacted monomers and initiator fragments and
then incubated in cold PBS solution (4.degree. C.) for 24 hours.
The lactate ester bonds in the polymerized 10KL5A2 hydrolyze in
presence of water and make the tissue susceptible for enzymatic
degradation. The ratio of NVP to 10KL5A2 could be changed to
control biodegradation, swelling and other properties of the
tissue/polymer composite matrix.
[0325] In another variation of this procedure, a bioactive compound
such as, by way of example, and not limitation, heparin may be
added in the monomer mixture prior to photopolymerization. The
bioactive compound is released as 10KL5A2 undergoes hydrolysis. In
another variation of this method, 5 ml of 20% solution of 10KL5A2
in PBS containing 400 ppm Irgacure 2959 is used to suspend 1
million human fibroblast cells. The unsaturated group modified
tissue is incubated for 30 minutes in the polymer solution. The
solution is taken out and about 0.2 ml solution is sprayed on the
tissue surface. The solution on the surface and tissue are exposed
to 2 minutes with Black-Ray UV lamp (360 nm light, 10000 mW/cm2
intensity). The polymerized 10KL5A2 with encapsulated cells is
clearly seen on the tissue surface. Most of the cells remained
viable and tolerate the encapsulation process.
Example 15
Tissue and Biodegradable Polymer Composite
[0326] Tissue and Biodegradable Polymer Composite Wherein the
Biodegradable Polymer is Hydrophobic
[0327] Crosslinking of Acrylic Modified Tissue Using Biodegradable
Multifunctional Macromonomer (Water-Insoluble or Substantially
Water Insoluble)
[0328] Part 1: Preparation of Trifunctional Lactate Liquid
Copolymer (TMPTL).
[0329] Trimethylol propane triol (TMPT) is dried under vacuum at
60.degree. C. for 16 hours. 2 g of dry TMPT, 17.5 g of dl-lactide,
and 20 mg of stannous octoate are charged into a 3 necked flask
equipped with Teflon coated magnetic stirring needle and nitrogen
inlet. The flask is then immersed in a silicone oil bath maintained
at 160.degree. C. The reaction is carried out for 5 h under
nitrogen atmosphere. The reaction mixture is then cooled to room
temperature. The mixture is then dissolved in 10 ml toluene. The
hydroxyl-terminated liquid lactate polymer is isolated by pouring
the toluene solution in large excess cold hexane. It is further
purified by repeated dissolution-precipitation process from
toluene-hexane solvent-nonsolvent system and dried under vacuum at
60.degree. C. It is then immediately used for acrylate end capping
reaction described below.
[0330] Part 2: End Capping of Trifunctional Polylactide Polymer
with Acrylate Group (TMPTLA).
[0331] 10 g of TMPT initiated lactate synthesized previously is
dissolved in 150 ml dry benzene. From this solution 20 ml benzene
is distilled out to remove unwanted moisture from the reaction
mixture. The solution is cooled to 0.degree. C. in ice bath. 2.7 ml
of triethyl amine and 1.8 ml acryloyl chloride are added dropwise
to the cold lactate solution. The mixture is refluxed under
nitrogen atmosphere for 3 h. The solution is filtered to remove
triethylamine hydrochloride. The acrylate ester is then isolated by
pouring the filtered solution in large excess cold hexane. It is
further purified by repeated (3 times) precipitation from
toluene-cold hexane system. The liquid polymer is dried under
vacuum at 40.degree. C. It is stored in an amber-colored bottle
under nitrogen atmosphere.
[0332] Part 3: Crosslinking of Acrylic Modified Tissue Using
TMPTLA.
[0333] 50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radical
photoinitiator) is dissolved in 0.9 ml n-vinyl pyrrolidone and 0.1
g TMPTLA. Two 1 cm by 1 cm pieces of acrylic acid succinimide ester
modified bovine pericardium tissue prepared per example 1 are
incubated for 30 minutes each in 20% ethanol, 50% ethanol and 70%
ethanol and n-vinyl pyrrolidinone. The tissue is then transferred
to TMPTALA solution and incubated for 4 h. The tissue is removed
from the solution is placed on a glass plate and exposed to
Black-Ray UV lamp (360 nm light, 10000 mW/cm2 intensity) for 5
minutes each side. The exposed tissue is washed with 100% alcohol
to remove unreacted monomers and initiator fragments and then
incubated in cold PBS solution (4.degree. C.) for 24 hours. The
lactate ester bonds in the polymerized TMPTALA hydrolyze in
presence of water and make the tissue susceptible for enzymatic
degradation.
Example 16
Tissue-Synthetic Biodegradable Crosslinkable Polymer Composite
[0334] Five 1 cm by 1 cm pieces of acrylic acid succinimide ester
modified bovine pericardium tissue are cryogenically ground (at
liquid nitrogen temperature) to make a tissue powder. 50 mg of
2,2-demethoxy-2-phenyl acetophenone (free radical photoinitiator)
is dissolved 0.1 ml n-vinyl pyrrolidone and 0.9 g TMPTLA. 1 g of
tissue powder is mixed with 200 mg TMPTLA monomer and 1 g sodium
chloride (to induce porosity) and the mixture poured into a mold
cavity. The shape of the cavity is similar to shape of human
meniscus tissue. The mixture in the cavity is exposed to Black-Ray
UV lamp (360 nm light, 10000 mW/cm2 intensity) for 15 minutes to
cure the TMPTLA polymer. The cured product is washed with 100%
ethanol to remove unreacted monomer and then with water to remove
sodium chloride (to induce porosity). The cured tissue/synthetic
polymer can be used as a biodegradable meniscus implant or a
scaffold for tissue engineering.
[0335] Collagen protein powder modified with unsaturated groups per
Examples 1-4 may also be used in place of tissue powder.
Example 17
Crosslinking of Unsaturated Group Modified Tissue Using Di or
Polyfunctional Mercapto Compounds
[0336] In this crosslinking method, mercapto group from the
crosslinker react with unsaturated group in the modified tissue
(Examples 1-4) and produce a crosslinked tissue.
[0337] In a 50 ml polypropylene tube, 7.5 ml 50 mM sodium
bicarbonate buffer (pH 9-12) and 2.5 ml acetonitrile are mixed.
Five 1 cm by 1 cm pieces of acrylic acid succinimide ester modified
bovine pericardium tissue (Examples 1-3) are suspended in the
acetonitrile solution. 0.2 ml trimethylolpropane
tris(3-mercaptopropionate) (Sigma-Aldrich Product Number: 38,
148-9) is added to the tube and the mixture is vortexed for 60
minutes. The reaction is continued for 6 h at room temperature and
12 hours at 4.degree. C. The tissue is separated from the mixture,
washed with PBS and 100% ethanol and then finally stored in 100%
ethanol until use.
Example 18
Crosslinking of Tissue with Biodegradable Crosslinker
[0338] Crosslinking of Tissue Using Non-Polymeric Degradable
Crosslinker
[0339] Synthesis of a Biodegradable Crosslinker; Hydroxylamine
Succinate NHS Ester
[0340] Part 1: Hydroxylamine Succinate
[0341] To a solution of 2 g hydroxy amine in 100 ml dry benzene and
100 ml pyridine, 9.4 g succinic anhydride are added and the
reaction mixture stirred at room temperature for 2 h and then
refluxed for 2 h. The solvent is evaporated under vacuum and the
residue is purified by flash chromatography using silica gel.
[0342] Part 2: Hydroxylamine Succinate NHS Ester.
[0343] Part 2: To a cold (4.degree. C.) solution of 5 g
Hydroxylamine Succinate and 5.1 g n-hydroxysuccinimide in 120 DMF,
11.8 g 1,3-dicyclohexyl carbodiimide in 30 ml of DMF and is added
under nitrogen atmosphere. The reaction is continued at room
temperature for 8 h and urea precipitate is filtered. The filtrate
is evaporated and the crude compound is recovered. The compound is
further purified by flash chromatography.
[0344] Part 3: Crosslinking of Pericardial Tissue with
Hydroxylamine Succinate NHS Ester.
[0345] 10 pieces of 1 cm by 1 cm bovine pericardium pierces are
transferred to 50 ml polypropylene centrifuge tube containing 10 ml
PBS. 250 mg Hydroxylamine Succinate NHS ester in 0.5 ml dimethyl
sulfoxide is added to the tube and the solution is vortexed for 15
minutes. The modification reaction is carried for 6 hours at
ambient temperature (25.degree. C.) and then for 12 hours at
4.degree. C. with gentle shaking. The reaction is terminated by
washing the tissue with 20 ml distilled water 3 times. Finally, the
tissue is lyophilized and stored at -20.degree. C. until use.
[0346] The ester linkage hydroxylamine succinate in the crosslinker
is hydrolyzed when exposed to physiological conditions such PBS, pH
7.2. The hydrolyzed tissue degrades by normal enzymatic
degradation.
Example 19
Crosslinking Using Polymeric Degradable Crosslinker
[0347] 10 pieces of 1 cm by 1 cm bovine pericardium tissue are
transferred to 50 ml polypropylene centrifuge tube containing 10 ml
PBS. 1.0 g 4 arm-n-hydroxysuccinimide ester of polyethylene glycol
carboxymethylene-butyric acid, average molecular weight 10000
Daltons (obtained from Shearwater Polymers, 4 arm, product
CM-HBA-NS-10K) is added to the tube and the mixture is vortexed for
5 minutes. The tissue is isolated from the tube, washed with
distilled water and used as a biodegradable matrix for various
medical and tissue engineering applications. Upon implantation, the
glutarate ester bond in CM-HBA-NS-10K undergoes hydrolysis. After
hydrolysis of the crosslinker, tissue degrades by a normal
enzymatic pathway. Additional crosslinking agents such as
crosslinking with EDC may also be used to control tissue properties
or degradation time.
Example 20
Bioprosthetic Tissue Incorporated with Crosslinked Biodegradable
Hydrogel
[0348] Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer
(PEG20KL5).
[0349] 50 g PEG molecular weight 20000 is dried at 120.degree. C.
under vacuum for 10 hours. 10.0 g dry PEG molecular weight 20000,
2.2 g of dl-lactide and 100 mg of stannous octoate are charged into
100 ml flame dried round bottom flask. The flask is then connected
to an argon gas line and then immersed in an oil bath maintained at
160.degree. C. The polymerization reaction is carried out for 16 h
at 160.degree. C. The polymer is then dissolved in 100 ml toluene
and precipitated in 2000 ml cold hexane. The precipitated polymer
is recovered by filtration and dried under vacuum for 1 day at
60.degree. C. It then immediately used in next step.
[0350] Part 2: End-Capping of PEG20KL5 with Polymerizable or
Crosslinkable Group (PEG20KL5A).
[0351] 30 g of PEG20KL5 is dissolved in 450 ml dry toluene. About
50 ml of toluene is distilled out to remove traces of water from
the reaction mixture. The solution is cooled to 65.degree. C. To
this warm solution, 0.6 g of triethyl amine and 0.5 g acryloyl
chloride are added. The reaction mixture is then stirred for 30
minutes at 50-60.degree. C. and filtered. The reactive
crosslinkable precursor is precipitated by adding the filtrate to
2000 ml cold hexane. The precipitated polymer is recovered by
filtration. It is then dried under vacuum for 12 h at 50.degree.
C.
[0352] Part 3: Incorporation of Hydrophilic Crosslinkable
Biodegradable Polymer in the Pericardial Tissue.
[0353] A 3 cm.times.3 cm piece is cut from a cleaned uncrosslinked
bovine pericardial sac. The tissue is then incubated for 30 minutes
each in 20% ethanol, 40% ethanol, 80% ethanol and finally in 100%
ethanol. In a 500 ml beaker, 10 g PEG20KL5A, 0.5 g Chlorhexidene
Gluconate, 0.050 g Irgacure 2959 are dissolved in 90 g methanol.
The 100% ethanol-treated tissue is transferred into Chlorhexidene
Gluconate solution and is incubated for 2 hours. The tissue is
removed from the solution and exposed to long UV light emitting at
360 nm for 5 minutes (Black-Ray light source, model 3-100A, Flood
365 nm, intensity 30 mW/cm2).
Example 21
Bioprosthetic Tissue-Hydrophilic Non-Crosslinked Biodegradable
Polymer Composite
[0354] Biodegradable Tissue Based Patch for Controlled Drug
Delivery
[0355] Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer
(PEG8K50)
[0356] 50 g PEG molecular weight 8000 (Carbowax 8000) is dried at
120.degree. C. under vacuum for 10 hours. 10.0 g dry PEG molecular
weight 8000, 18.0 g of dl-lactide and 100 mg of stannous octoate
are charged into 100 ml flame dried round bottom flask. The flask
is then connected to an argon gas line and then immersed in an oil
bath maintained at 160.degree. C. The polymerization reaction is
carried out for 16 h at 160.degree. C. The polymer is then
dissolved in 100 ml toluene and precipitated in 2000 ml cold
hexane. The precipitated polymer is recovered by filtration and
dried under vacuum for 1 day at 60.degree. C.
[0357] Part 2: Incorporation of Hydrophilic Non-Crosslinked
Biodegradable Polymer PEG8K50) in the Pericardial Tissue.
[0358] A 3 cm.times.3 cm piece is cut from a cleaned uncrosslinked
bovine pericardial sac. The tissue is incubated for 30 minutes each
in 20% ethanol, 40% ethanol, 80% ethanol and finally in 100%
ethanol. In a 500 ml beaker, 10 g polyethylene glycol-lactate
copolymer (PEG8K50), and 1 g rifampin are dissolved in 90 g
chloroform. The 100% ethanol-treated tissue is transferred into
rifampin solution and is incubated for 2 hours. The tissue is
removed from the solution and the solvent is evaporated by air
drying. The reddish yellow rifampin-PEG-lactate coating is clearly
visible to the naked eye. The dry tissue is sterilized using
ethylene oxide and used as a biodegradable drug delivery patch. The
polyethylene glycol-lactate polymer acts as a hydrophilic
non-crosslinked biodegradable polymeric drug delivery matrix for
Rifampin.
[0359] The tissue may be mechanically resurfaced or perforated to
improve the adhesion of polymer to the tissue.
[0360] The same procedure may also be used to incorporate
biodegradable polymer in glutaraldehyde crosslinked tissue or EDC
crosslinked tissue.
Example 22
Incorporation of Hydrophobic Non-Crosslinked Synthetic
Biodegradable Polymer in the Pericardial Tissue
[0361] Biodegradable Tissue Based Patch for Controlled Drug
Delivery
[0362] Seven 1 cm.times.1 cm pieces are cut from a cleaned
uncrosslinked bovine pericardial sac. The tissue is then incubated
for 30 minutes each in 20% ethanol, 40% ethanol, 80% ethanol and
finally in 100% ethanol and dried.
[0363] In a 50 ml beaker, 200 mg polylactide-co-polyglycolide
(50:50), molecular weight 50000 (Sigma-Aldrich Catalog number 43,
044-7) is dissolved in 3 ml chloroform. 20 mg rifampin is added to
the polymer solution. The 100% ethanol treated tissue is
transferred into rifampin solution and is incubated for 2 hours.
The tissue is removed from the solution and the chloroform is
evaporated by air drying. The reddish yellow rifampin-polymer
coating is clearly visible to the naked eye. If necessary, vacuum
drying may, for example, and without limitation, be used to
completely remove the chloroform. Finally the tissue is sterilized
using ethylene oxide.
[0364] Two pieces of rifampin-treated tissue are incubated in 2 ml
PBS at 37.degree. C. and the release of rifampin is monitored over
a period of 72 hours. The PBS is exchanged at 10 minutes, 6 hours,
24 hours, 48 hours and 72 hours time intervals. All incubated
solutions had light yellow color visible to the naked eye
indicating the presence of rifampin in the solution. The
concentration of rifampin in the eluted solution is analyzed by
UV-VIS spectrophotometry.
[0365] Using a similar method, a high molecular weight
polydl-lactide, molecular weight 100000 (Sigma-Aldrich Catalog
Number 53, 116-2) is used in place of polylactide-co-polyglycolide
(50:50) to form a tissue-biodegradable polymer composite. This
polymer takes a much longer time to degrade and shows a different
rifampin release profile.
[0366] The biodegradable polymer used could be liquid or low
melting solid. Using similar methods to those described above, a
non-polymeric drug carrier such as, by way of example, and not
limitation, sucrose acetate or vitamin E or vitamin E acetate may
be also used as a carrier and incorporated in the tissue. If a
biostable tissue is matrix is desired, then a crosslinked tissue
such as, by way of example, and not limitation, glutaraldehyde
crosslinked tissue may, for example, and without limitation, be
used in place of non-crosslinked tissue.
Example 23
Porcine Pericardial Patch Coated with Biodegradable Polymer
Synthetic Biodegradable Hydrogel
[0367] From a freshly obtained piece of porcine pericardial sac, A
3 cm.times.3 cm piece of tissue is cut. This tissue is incubated in
1% Eosin Y solution in PBS for 10 minutes. The tissue is rinsed
with fresh PBS to remove excess of eosin solution from the tissue
and used in the coating process.
[0368] 20 g 10KL5A2 (Example 14), 80 g saline solution buffered
with 1000 mM triethanol amine buffer (pH 7.4) and 1 ml vinyl
pyrrolidinone are mixed in 1000 ml beaker. Eosin stained
pericardial tissue is placed inside the solution. Care is taken to
completely cover the tissue surface with 10KL5A2 solution. The
tissue is then exposed to 514 nm light either from argon laser
(American Argon ion laser, Model 905 emitting at 532 nm, 100
mW/cm2) or xenon lamp (intensity 0.5 W/cm2). The
eosin-triethanolamine-10KL5A2-light initiates a photopolymerization
reaction at the tissue interface forming a thin hydrogel film on
the tissue surface. The thickness of the film is dependent on the
exposure time, monomer concentration, eosin concentration and light
intensity. A to 2000 micron coating is obtained. The biodegradable
hydrogel coated tissue can be used as a coated tissue patch. If
necessary, multiple eosin and monomer solution treatments may be
performed to achieve a desirable thickness.
[0369] Using a similar approach to that described above, an albumin
modified with polymerizable groups may, for example, and without
limitation, be used to coat tissue surface. The albumin may be
bovine albumin or human albumin. The human albumin may be obtained
by recombinant technology.
[0370] A crosslinked tissue such as, by way of example, and not
limitation, glutaraldehyde crosslinked tissue may, for example, and
without limitation, be used in place of uncrosslinked tissue if a
biostable material is desired.
Example 24
Porcine Pericardial Patch Coated with Synthetic Biodegradable
Polymer Hydrogel Formed by Condensation Polymerization
[0371] An ethylene oxide sterilized air assisted sprayer is used in
conjunction with aqueous solutions of polymerizable monomers.
Solution consisted of a 14.4% solution of 4
arm-n-hydroxysuccinimide ester of polyethylene glycol
carboxymethylene-butyric acid, average molecular weight 10000
Daltons (Shearwater 4 arm CM-HBA-NS-10K) is dissolved in 0.01M
phosphate buffer at pH 4.0 and is sterile-filtered (Pall Gelman
syringe filter, p/n 4905) and drawn up in a sterile 5 cc syringe.
Solution 2 consisted of a 1.2% solution of a dilysine (purchased
from Sigma Chemicals) dissolved in 0.1M borate buffer at pH 11 with
0.5 mg/mL methylene blue for visualization and is also
sterile-filtered and drawn up in a sterile 5 cc syringe. These
solutions, when combined 1:1 on a volumetric basis, result in a 1:1
ratio of NHS ester to amine end group. The final % solids after
combination is 7.5%. The two syringes are individually loaded in
the two separate receptacles through a luer-lok type of linkage.
Airflow from a regulated source of compressed air (an air
compressor such as, by way of example, and not limitation, those
commercially available for airbrushes) is connected to the device
using a piece of Tygon tube. On compressing the syringe plungers a
steady spray of the two liquid components is observed. A 10
cm.times.5 cm piece is cut from a cleaned uncrosslinked bovine
pericardial sac or AlloDerm.RTM. tissue marketed by LifeCell
Corporation, N.J. The spray from the syringe is directed to the
piece of pericardial tissue, and a hydrogel coating is formed on
the surface of the tissue. Alternatively, the coating compositions
and spray system can be purchased as DuraSeal from Confluent
Surgical Inc., MA and used. Within a short period of time (less
than a minute) an area of 10 cm.times.5 cm could be coated with
ease. The coating can be applied on both the sides of the tissue.
The polyethylene glycol coating makes the tissue surface non-cell
adhesive. The hydrogel coating is rinsed with saline (the hydrogel
coating is resistant to rinsing) and is observed to be well
adherent to the tissue surface. The hydrogel coating can be used to
modify biological properties of the tissue. If the coating is
applied on one side only, then one surface (coated surface) becomes
non-cell adhesive while the uncoated surface remains as a cell
adhesive surface. The coating can also be used to deliver drugs in
a controlled manner. This is achieved by adding drug or drug
encapsulated microspheres in a coating formulation before coating
the tissue or by incubating the coated patch in drug solution in
organic solvent or water and then removing the solvent. The coated
tissue can be used as a barrier for reducing surgical adhesions.
Such a non-cell adhesive tissue patch can be used to reduce
post-operative adhesions. The coated tissue loaded with BMP
proteins could be used to generate a bone tissue.
[0372] A crosslinked tissue such as, by way of example, and not
limitation, glutaraldehyde crosslinked tissue may, for example, and
without limitation, be used on place of uncrosslinked tissue if a
biostable material is desired.
Example 25
Prevention of Post-Operative Adhesions Using Coated Tissue Patch or
Composite Tissue Patch
[0373] Rat Cecum Model
[0374] Fourteen Sprague Dawley rats with average weight around 260
g are divided into 2 groups, 7 animals in each group. After
following the standard procedures for administrating anesthesia, a
midline incision is made on the abdomen and a cecum is located in
the abdominal cavity. Using a standard surgical cotton gauze pad,
an injury is made to the Cecum surface by abrading the surface of
cecum. The approximate area of injury is maintained around 1-2 sq.
cm. The injury elicits some bleeding from the injured surface. A
porcine pericardial tissue-coated with PEG based hydrogel or
DuraSeal based hydrogel is wrapped around the injured surface
(Examples 20-24, preferably example 24). Composite patch made using
polytetrafluoroethylene (PTFE) membrane and membrane tissue wherein
PTFE membrane serves as non-cell adhesive layer and tissue serve as
cell adhesive layer may also be used. The coated tissue is wrapped
around the injured area such that non-adhesive surface is facing to
the injured surface and immobilized by suturing it either to itself
or by suturing it to abdominal wall. Care is taken to ensure that a
hydrogel surface or PTFE in place of a composite is completely
covering the injured cecum surface. 7 animals are treated with
coated tissue and 7 animals are used as controls which did not
receive any tissue composite. The animals are closed after the
treatment using sutures and staples, topical antibiotic is applied
and the animals are subjected to standard diet and care as
recommended by National Institute of Health. After 14 days the rats
are subjected to CO2 asphyxiation.
[0375] The incisions are reopened and the cecum is observed for the
adhesions in the area of injury. The adhesions are scored as the
percent injured area involved in adhesion formation with the
surrounding organs and peritoneal wall. The results of treated and
control animals are analyzed using standard statistical methods
(t-test).
[0376] Coated or composite tissue patch prepared as above may be
used in hernia surgery to reduce complications due to surgical
adhesions.
Example 26
Chemical Modification of Tissue without Crosslinking
[0377] Modification of Uncrosslinked Tissue Using Monomethoxy
Polyethylene Glycol Derivatives
[0378] Part 1: Synthesis of Monomethoxy Polyethylene Glycol
Succinic Ester. Conversion of PEG Hydroxyl Groups into Carboxylic
Groups.
[0379] 10 g monomethoxy polyethylene glycol, molecular weight 400
(PEG-400M) is dried at 60.degree. C. overnight under vacuum prior
to use. 10 g PEG-400M is dissolved in 35 ml dry pyridine and 35 ml
benzene. 3.5 g succinic anhydride or 4.0 g of glutaric anhydride is
added to it and the solution is refluxed for 2 h under nitrogen
atmosphere. Most of the pyridine is distilled out and the polymer
is isolated by pouring the cold concentrated pyridine solution to
cold 4000 ml hexane and dried under vacuum at 60.degree. C. and
used immediately in a subsequent carboxyl group activation
reaction.
[0380] Part 2: Activation of Acid Group Using N-Hydroxysuccinimide
(PEG-400MNHS).
[0381] To a solution of 10 g of PEG-400M succinate or glutarate in
100 ml dry methylene chloride are added 2.5 g n-hydroxysuccinimide
and 5.9 g 1,3-dicyclohexyl carbodiimide. The reaction mixture is
cooled to 0.degree. C. using ice bath and stirred overnight under
nitrogen atmosphere. Dicyclohexylurea is removed by filtration. The
filtrate is evaporated and the residue obtained is redissolved in
10 ml toluene. The toluene solution is precipitated in 2000 ml cold
hexane.
[0382] Alternatively, monomethoxy PEG NHS derivates may be
purchased from Sigma-Aldrich or Sherewater Inc.
[0383] Part 3: Modification of Tissue Using Monomethoxy
Polyethylene Glycol Succinic Ester (PEG-400MNHS)
[0384] Porcine pericardial sac is obtained from a local abbotair
and is cleaned to remove blood and fatty tissue from the surface.
Ten 1 cm by 1 cm pieces are cut from the cleaned pericardial sac
and transferred to 10 ml 20 mM phosphate buffer solution (PBS, pH
7.2).
[0385] 200 mg monomethoxy polyethylene glycol succinic ester or
glutaric ester (PEG-400MNHS, part 2) is added to the tissue/PBS
mixture. The solution is vortexed for 5 minutes and tissue is
incubated at room temperature for 5 minutes to 12 hours, preferably
6 hours. The tissue is separated from the crosslinker mixture and
washed with 20 ml PBS solution 3 times to remove unreacted
chemicals. The PEG-modified tissue shows does not show significant
increase in shrink temperature and the modified tissue degrades
when exposed to collagenease or pepsin solution for prolonged
periods of time indicating its susceptibility to enzymatic
degradation.
[0386] The tissue treatment can be done just prior to surgical
implantation if the PEG derivative, tissue and PBS is provided as a
kit.
Example 27
Chemical Modification of Tissue without Crosslinking
[0387] Modification of Uncrosslinked Tissue Using Anhydrides
[0388] Porcine pericardial sac is obtained from a local abbotair
and is cleaned to remove blood and fatty tissue from the surface.
Ten 1 cm by 1 cm pieces are cut from the cleaned pericardial sac
and transferred to 10 ml 20 mM sodium borate buffer (pH 9.5). 0.5 g
succinic anhydride is added to the tube. The solution is vortexed
for 5 minutes and tissue is incubated at room temperature for 12
hours. The tissue is separated, washed and stored until use.
[0389] Using a similar reaction scheme, the tissue is modified
using 0.5 g acetic anhydride or 0.5 g glutaric anhydride. Briefly,
two 1 cm by 1 cm pieces of bovine pericardium tissue are incubated
for 30 minutes each in 20% ethanol, 50% ethanol and 70% ethanol and
n-methyl pyrrolidinone. The tissue is then transferred to a 50 ml
flask containing 5 ml n-methyl pyrrolidinone, 0.5 g acetic
anhydride and 0.5 g triethyl amine. The tissue is incubated for 6 h
at 40.degree. C. and then removed. It is washed with 20 ml 100%
ethanol and 20 ml PBS.
Example 28
Chemical Modification of Tissue without Crosslinking
[0390] Modification of Uncrosslinked Porcine Pericardial Tissue
Using Activated Acid (Acetic Acid N-Hydroxy Succinimide Ester)
[0391] Porcine pericardial sac is obtained from a local abbotair
and is cleaned to remove blood and fatty tissue contaminants from
the tissue surface. Ten 1 cm by 1 cm pieces are cut from the
cleaned pericardial sac and transferred to 10 ml 0.20 M
2-(N-morpholino)ethanesulfonic acid (MES) buffered solution (pH
6.5). 0.5 g Acetic acid n-hydroxy succinimide ester) AANHS is added
to the buffered solution and the reaction is vortexed for 15
minutes and continued for 6 hours at ambient temperature. An
additional 0.1 g AANHS ester is added to the mixture at every 1.5
hours. After the reaction, the tissue is isolated and washed
several times with PBS and finally stored in 50% ethanol solution.
The treated tissue is sterilized using ethylene oxide.
[0392] The modified tissue is degradable upon implantation. The
tissue becomes less inflammatory due to chemical modification.
[0393] The tissue treatment can be done just prior to surgical
implantation if the acetic acid n-hydroxy succinimide ester, tissue
and PBS is provided as a kit.
Example 29
Shape Preserving Tissue Fixation Methods and Devices
[0394] a) Preparation of Helical Coil Shaped Tissue (Tissue
Stent)
[0395] Untreated long bovine pericardium (BP) tissue strips
(approximately 10 to 20 cm long and 2-4 mm wide) are transferred to
a 500 ml flask containing 100 ml PBS, 12.5 ml triethylamine (TEA)
and 12.5 ml glycidyl methacrylate (GM). The solution was heated to
50.degree. C. with constant stirring using magnetic stir bar and
held there for 8 hours. The tissue strips are removed from the
solution and washed with PBS, distilled water, & incubated in
70% isopropanol solution in water for 30 minutes. Finally, strips
are then transferred to a tetraethyleneglycol dimethacrylate
(TEGDM) monomer containing 500 ppm 2,2-dimethoxy 2-phenyl
acetophenone (photoinitiator) and incubated at room temperature
(30.degree. C.) for one hour. One TEGDM infused strip is helically
or spirally wound on a 6 mm diameter glass rod. Both ends of the
strip are tied with a thread to prevent unwinding. The helically
wrapped monomer infused tissue strip was then exposed to 360 nm
light (Black-Ray UV lamp, 360 nm flood light, 10000 mW/cm2
intensity at a distance of 10 cm) for 10 minutes while gently
rotating to expose the tissue from all sides. The coiled tissue was
removed from the glass rod and was washed with PBS and 70%
isopropanol to remove unreacted monomer and initiator fragments.
The tissue preserved the helical shape after removal of glass rod
mandrel support.
[0396] b) Preparation of Compressible or Wrinkled Tube from
Membrane Like Tissue.
[0397] Preparation of compressible vascular graft from flat tissue
such as, by way of example, and not limitation, pericardial tissue
or submucosa tissue
[0398] Untreated long bovine pericardium (BP) tissue strips
(approximately 18 to 20 cm long and 15-18 mm wide) are transferred
to a 500 ml flask containing 250 ml PBS, 32 ml triethylamine (TEA)
and 32 ml glycidyl methacrylate (GM). The solution was heated to
50.degree. C. with constant stirring using magnetic stir bar and
held there for 8 hours. The tissue strips are removed from the
solution and washed with PBS, distilled water, & incubated in
70% isopropanol solution in water for 30 minutes. Finally, strips
are then transferred to a 50% n-vinyl pyrrolidinone (NVP) solution
in water containing 500 ppm 2,2-dimethoxy 2-phenyl acetophenone
(photoinitiator) and incubated at room temperature (30.degree. C.)
for 1 hour. The NVP infused tissue strip is wrapped on a 6 mm
diameter stainless steep screw having threads. The tissue was
compressed in such a manner that it took shape of a threaded region
of the screw. Both ends of the strip are tied with thread to
prevent unwinding. The helically-wrapped monomer-infused tissue
strip was then exposed to 360 nm light (Black-Ray UV lamp, 360 nm
flood light, 10000 mW/cm2 intensity at a distance of 10 cm) for 10
minutes while gently rotating to expose the tissue from all sides.
The tissue was removed from the rod and was washed with PBS and 70%
isopropanol to remove unreacted monomer and initiator fragments.
The tissue duplicated the shape of the threaded screw. This tissue
was sewn to make 5 mm diameter tube. The tubular tissue was
compressible similar to commercially available polyester based
vascular grafts.
[0399] In another approach, a pericardial tissue patch modified
with glycidyl methacrylate (see example 37 described below) and
infused with tetraethyleneglycol dimethacrylate (TEGDM) monomer
containing 500 ppm 2,2-dimethoxy 2-phenyl acetophenone
(photoinitiator) was converted into a 6 mm diameter 10 cm long
tube. The tube was mounted on a 6 mm diameter stainless steel
mandrel/rod. The mounted tube was compressed along the axis of the
tube to reduce the its length from 10 cm to 7 cm (30% compression).
The tube has several wrinkles uniformly distributed along the axis
of the tube. The compressed tube was then exposed to UV light
(Black-Ray UV lamp, 360 nm flood light, 10000 mW/cm2 intensity at a
distance of 10 cm) for 10 minutes while rotating the tube. The
crosslinked tube was removed from the mandrel and washed with 70%
ethanol and then PBS. The tube maintained its compressed shape
indicating shape preservation. The compression also made the tissue
more compliant along the axis of the tube. Such compliant
pericardial graft made from flat tissue such as, by way of example,
and not limitation, pericardial tissue may be useful as peripheral,
coronary and AV vascular graft or may be used to make stent graft.
The tissue surface is expected to offer superior hemocompatibility
or patency.
Example 30
Bioprosthetic Tissue Coated with Polyethylene Glycol Crosslinked
with Gamma Radiation
[0400] A 3 cm.times.3 cm piece is cut from a cleaned uncrosslinked
bovine pericardial sac. The tissue is fixed by incubating in 0.2%
glutaraldehyde solution for 7 days. The tissue is washed with PBS
and then incubated for 30 minutes each in 20% ethanol, 40% ethanol,
80% ethanol, and finally in 100% ethanol. In a 500 ml beaker, 1 g
polyethylene oxide molecular weight 100000 Daltons is dissolved in
20 ml chloroform. The ethanol-treated tissue is transferred into
polyethylene oxide solution and incubated for 4 hours. The
chloroform is removed by air drying. The polyethylene oxide-tissue
composite is exposed to gamma radiation (Dose 0.1 to 4 Mrad) to
crosslink polyethylene oxide. The crosslinked polyethylene
glycol-tissue composite is incubated in PBS to rehydrate the
collagen and polyethylene oxide polymer. The crosslinked
polyethylene oxide and tissue composite patch is used as a wound
dressing.
Example 31
Membrane Like Tissue Loaded with Drugs and Used as a Patch for
Local Delivery
[0401] Bioprosthetic Tissue Incorporated with Water Insoluble
Drugs
[0402] Porcine pericardial sac is obtained from a local abbotair
and is cleaned to remove blood and fatty tissue from the surface.
Ten 1 cm by 1 cm pieces are cut from the cleaned pericardial sac
and then incubated for 30 minutes each in 20% ethanol, 50% ethanol
and 70% ethanol, and finally 100 percent ethanol. The
ethanol-treated tissue is then transferred to chlorhexidene acetate
solution (30 percent solution in methanol) and incubated for 5
hours. The methanol is evaporated and the drug crystals are formed
throughout the tissue matrix. When such tissue is implanted in an
animal, it releases the drug in a controlled manner, possibly as a
result of slow dissolution of drug crystals. A Teflon.RTM.-based
release liner is applied on the tissue surfaces to reduce loss of
drug crystals during routine handling, transportation and storage.
The release liner is removed just prior to implantation.
[0403] In another variation of this approach, the tissue is first
exposed to silver nitrate solution followed by exposing to sodium
acetate solution. The exposure of silver nitrate solution inside
the tissue matrix to sodium acetate solution forms silver acetate
`in situ` inside the tissue matrix. The deposited silver salts
release silver ions and provide antimicrobial properties to the
tissue. The silver salt deposition may be carried out in the
operating room prior to implantation if silver salt, implantable
tissue and appropriate salt solution such as, by way of example,
and not limitation, sodium acetate, sodium chloride, sodium lactate
are provided in a sterile manner and as a kit. The tissue used may
be crosslinked (biostable) or uncrosslinked (biodegradable). The
same method also could be used to deposit silver salts in collagen
based products such as, by way of example, and not limitation,
would dressings, collagen sponge products used for implantation
etc. In another example, hydroxyapatite may be deposited or formed
in the tissue matrix to make a hydroxyapatite-tissue composite
matrix. The hydroxyapatite is added to the tissue matrix using
methods known in the orthopaedic biomaterials art. In one
embodiment, hydroxy apatite is synthesized inside the tissue matrix
by combing calcium ion and phosphate ion solution in presence of
tissue. The calcium nitrate [Ca(NO3)2, 4H2O, Aldrich], may be used
as calcium source and ammonium hydrogen phosphate [(NH3)2HPO4,
Aldrich] may be used as phosphorus source. The calcium nitrate
solution is added to ammonium hydrogen phosphate solution at
40.degree. C. while maintaining the pH using ammonium hydroxide to
10 and 10.6 to form hydroxyapatite crystals. The molar ratio of The
Ca and P precursors is maintained 1.6 to make hydroxyapatite
crystals. This reaction is carried in presence of tissue such as
pericardial tissue so that hydroxyapatite is generated in side the
tissue. The tissue-hydroxyapatite can potentially be used as a
matrix for bone formation. If needed bone promoting bioactive
compounds such as BMPs may be added in the composite matrix to
accelerate the bone formation.
Example 32
Crosslinking of Tissues Using Unsaturated Acids
[0404] 32a-1) Synthesis of Activated Unsaturated-Acids
(N-Hydroxysuccinimide Ester)
[0405] Synthesis of Fumaric Acid N-Hydroxysccinimide Ester (FUNHS)
Using Acid Chloride Route.
[0406] 8.2 g N-hydroxy succinimide (NHS) is transferred to 250 ml
flask fitted with magnetic stirrer and nitrogen inlet. 200 ml dry
benzene is transferred to the flask and about 50 ml of benzene is
distilled off. The solution is cooled to 10.degree. C. using an ice
bath. 7.3 g triethylamine and 5.0 g fumaryl chloride are added
dropwise to the NHS solution. The mixture is refluxed for 6 h under
nitrogen atmosphere. At the end of a 6 h period, the solution is
cooled and filtered to remove triethylamine hydrochloride. The
filtrate is concentrated by removing the solvent under vacuum and
the crude product is recovered. The product is further purified by
column chromatography or recrystallization. The final product is
stored under nitrogen atmosphere at -20.degree. C. until further
use.
[0407] 32a-2) Synthesis of Fumaric Acid N-Hydroxysulfosuccinimide
Ester (FUSNHS) Using 1,3-Dicyclohexyl Carbodiimide (DCC).
[0408] 5 g fumaric acid is dissolved in 100 ml dry DMF. The
solution is cooled to 4.degree. C. 25.2 g 1,3-dicyclohexyl
carbodiimide and 20.6 g of N-hydroxysulfosuccinimide are added to
the reaction mixture. The mixture is stirred at 4.degree. C. for 6
h and overnight at room temperature under nitrogen atmosphere.
Dicyclohexylurea is removed by filtration and the fumaric acid NHS
derivative is isolated by removing the DMF under vacuum and further
purified by recrystallization or column chromatography. The product
is stored under nitrogen atmosphere at -20.degree. C.
[0409] 32b) Modification and Crosslinking of Tissue Using Fumaric
Acid N-Hydroxysccinimide Ester (FUNHS).
[0410] Ten 1 cm by 1 cm bovine pericardium pieces, cut from a
freshly obtained bovine pericardial sac, are transferred to 50 ml
polypropylene centrifuge tube containing 10 ml phosphate buffered
saline (PBS, (pH 7.2). 300 mg of fumaric acid succinimide ester
(FUNHS) dissolved in 0.5 ml dimethyl sulfoxide is added to the tube
and the solution is vortexed for 15 minutes. The modification
reaction is carried for 6 hours at ambient temperature (25.degree.
C.) and then for 12 hours at 4.degree. C. with gentle shaking. The
reaction is terminated by washing the tissue with 20 ml PBS 3
times. Finally, the tissue is stored in 30 ml 40% HEPES buffered
isopropanol solution. The tissue shows elevated shrink temperature
as compared to untreated tissue indicating crosslinking of the
tissue, presumably due to crosslinking introduced by a reaction
with a bifunctional crosslinking agent. The crosslinks has
polymerizable unsaturated group. The tissue is further crosslinked
using free radical polymerizable monomer as described below:
[0411] 32c) Additional Crosslinking of Fumaric Acid Modified Tissue
Using Photopolymerization.
[0412] Two 1 cm by 1 cm pieces of fumaric acid crosslinked modified
bovine pericardium tissue are incubated for 30 minutes each in 20%
ethanol, 50% ethanol and 70% ethanol. 50 mg of
2,2-dimethoxy2-phenylacetophenone (Irgacure 651, free radical
photoinitiator) is dissolved in 1 ml n-vinyl pyrrolidinone to
prepare a monomer solution. The 70% ethanol-incubated tissue is
transferred to photoinitiator solution and incubated for 4 h at
25.degree. C. and then for 12 h at 0.degree. C. The tissue is
removed from solution is placed on a glass plate and exposed to
Black-Ray UV lamp (360 nm light, 10000 mW/cm2 intensity) for 5
minutes from each side. The crosslinked tissue is removed and
washed with 10 ml PBS to remove unreacted monomer, initiator
fragments and water soluble non-crosslinked polymer.
Example 33
Implantable Tissue Surface Modification and/or Crosslinking Using
Reagents Capable of Undergoing Hydrogen Abstraction Reaction or
Capable of Initiating Free Radical Polymerization
[0413] a) Synthesis of Benzophenone Modified Polyethylene Glycol
(BPEG)
[0414] A 3 necked 250 ml flask equipped with magnetic stirrer and
nitrogen inlet is charged with 5 g 2-carboxyl benzophenone, 22 g
polyethylene glycol, molecular weight 1000 and 100 ml
dichloromethane (DCM). The solution is cooled to 4.degree. C. and
6.5 g 1,3-dicyclohexyl carbodiimide (DCC) are added under nitrogen
atmosphere. The mixture is stirred at 4.degree. C. for 6 h and
overnight at room temperature under nitrogen atmosphere.
Dicyclohexylurea is removed by filtration and the
benzophenone-terminated PEG ester is isolated by removing the DCM
under vacuum. The PEG-benzophenone ester is further purified
dissolving in toluene and precipitating in cold diethyl ether
hexane.
[0415] b) Crosslinking/Surface Modification of Tissue Using by
Hydrogen Abstraction Mechanism
[0416] Two 1 cm by 1 cm pieces of bovine pericardium tissue samples
are incubated for 30 minutes each in 20% ethanol, 50% ethanol and
70% ethanol and finally in 100% methanol. 200 mg benzophenone
modified polyethylene glycol (crosslinker) is dissolved in 1.8 ml
methanol. The methanol-incubated tissue is transferred to the
crosslinker solution and incubated for 4 h at 25.degree. C. and
then for 12 h at 0.degree. C. The tissue is removed from solution
and the placed on a quartz glass plate and exposed to high
intensity UV lamp (360 nm) for 15 minutes on each side. The
crosslinked/PEG surface modified tissue is removed and washed with
10 ml ethanol to remove unreacted reagent.
Example 34
Chemical Modification of Tissue Using Benzophenone (Hydrogen
Abstraction Moiety) and then Crosslinking Using UV Light
[0417] a) Preparation of Protein Reactive Benzophenone Derivative
(Bezophenone N-Hydroxysulfosuccinimide Ester)
[0418] A 3 necked 250 ml flask equipped with magnetic stirrer and
nitrogen inlet is charged with 5 g 2-carboxyl benzophenone, 5.2 g
n-hydroxysulfosuccinimide (SNHS) and 100 dry dimethylformamide
(DMF) are added. The solution is cooled to 4.degree. C. and 6.5 g
1,3-dicyclohexyl carbodiimide (DCC) is added under nitrogen
atmosphere. The mixture is stirred at 4.degree. C. for 6 h and
overnight at room temperature under nitrogen atmosphere.
Dicyclohexylurea is removed by filtration and bezophenone SNHS
ester is by isolated by removing the DMF under vacuum. The
bezophenone SNHS ester is further purified by flash
chromatography.
[0419] b) Tissue Modification Using Benzophenone SNHS
Derivative.
[0420] Porcine pericardial sac is obtained from a local abbotair
and is cleaned to remove blood and fatty tissue contaminants from
the tissue surface. Ten 1 cm by 1 cm pieces are cut from the
cleaned pericardial sac and are transferred to 10 ml PBS solution
(pH 7.2). 0.5 g bezophenone SNHS is added to the buffered solution
and the reaction is vortexed for 15 minutes and continued for 6
hours at ambient temperature. An additional 0.1 g benzophenone SNHS
ester is added to the mixture at every 1.5 hours. After the
reaction, the tissue is isolated and washed several times with PBS
and ethanol solution finally with 100% methanol. The modified
tissue is then immediately used in tissue modification
reaction.
[0421] c) Crosslinking of Benzophenone Modified Tissue.
[0422] Two 1 cm by 1 cm pieces of benzophenone modified porcine
pericardium tissue samples are incubated for 30 minutes each in 20%
ethanol, 50% ethanol, 70% ethanol, and finally in 100% methanol.
The tissue is removed from solution is placed on a quartz glass
plate and exposed to UV lamp for 5 minutes from each side. The
crosslinked/PEG surface modified tissue is removed and washed with
10 ml ethanol to remove unreacted reagent.
Example 35
Bioprosthetic Tissue Based Adhesive Patch for Surgical
Applications
[0423] Use of Membrane Like Implantable Tissue to Make a Surgical
Adhesive Patch
[0424] Bovine pericardial sac is obtained from a local abbotair and
is cleaned to remove blood and fatty tissue contaminants from the
tissue surface. 2 cm by 5 cm size pieces are cut from the cleaned
pericardial sac. Separately a photopolymerizable adhesive solution
is prepared from polyethylene glycol 400 diacrylate (PEG400DA
monomer). Photoinitiator (Eosin 0.02% and triethanol amine 0.5%) is
dissolved in the PEG400DA monomer liquid and this solution is
applied on the pericardial tissue strip. The monomer
coated/incubated tissue serves as an adhesive patch. The adhesive
coated patch is applied and conformed to the tissue geometry on a
surgical site such as, by way of example, and not limitation,
air-leaking lung tissue. The patch is applied in such a way that
the liquid adhesive layer is between the pericardial tissue and
lung tissue. The liquid adhesive is cured or photopolymerized by
argon ion laser or xenon light emitting at 514 nm light. The green
light passes through the pericardial tissue and initiates the
polymerization of PEGDA. The polymerized PEGDA adheres to the lung
and pericardial tissue. If needed, the patch may also be
immobilized by suturing on the lung tissue. The cured layer of
PEGDA prevents air leak from the air tissue. If an absorbable patch
is desired, then degradable implantable tissue (for example
uncrosslinked bovine pericardial tissue) may, for example, and
without limitation, be used along with absorbable surgical adhesive
such as, by way of example, and not limitation, FocalSeal.RTM.
marketed by Genzyme Biosurgery or DuraSeal.TM. surgical adhesive
marketed by Confluent Surgcial Inc. Other surgical adhesives such
as cynoacrylate based adhesives may also be used.
Example 36
Radio-Opaque Implantable Tissue
[0425] Tissue Modification Using Iodinated Compound
[0426] a) Synthesis of N-Hydroxysuccinimide Ester of Triiodobenzoic
Acid (TIBA-NHS)
[0427] 1.2 g n-hydroxy succinimide and 1.1 g of triethyl amine are
dissolved in 100 ml benzene. The solution is cooled to 0.degree. C.
in ice bath. 1.2 g triiodobenzoyl chloride is added dropwise to the
cold alcohol solution. The mixture is then refluxed under nitrogen
atmosphere for 3 h. The solution is filtered to remove
triethylamine hydrochloride. The ester is then isolated by removing
the solvent. It is further purified by column chromatography.
[0428] b) Chemical Bonding of Triiodobenzoic Acid Derivative to the
Tissue.
[0429] Modification of Bovine Pericardium Tissue.
[0430] Ten 1 cm by 1 cm bovine pericardium pieces, cut from a
freshly obtained bovine pericardial sac, are transferred to 50 ml
conical flask containing 10 ml phosphate buffered saline (PBS, (pH
7.2). 250 mg triiodobenzoic acid succinimide ester, TIBA-NHS ester
dissolved in 0.5 ml dimethyl sulfoxide is added to the tube and the
solution is vortexed for 15 minutes. 0.1 g of TIBA-NHS in 0.1 ml
DMSO is added to the fixation solution every 2 hours up to six
hours. The modification reaction is carried for 6 hours at ambient
temperature (25.degree. C.) and then for 12 hours at 4.degree. C.
with gentle shaking. The reaction is terminated by washing the
tissue with 20 ml PBS 3 times. Finally, the tissue is stored in 30
ml 38% isopropanol and 2% benzyl alcohol solution at 4.degree. C.
until further use. The triiodobenzoic acid moieties incorporated in
the tissue makes the tissue radio-opaque when viewed using medical
x-ray imaging techniques.
[0431] Alternatively radio-opaque compounds such as, by way of
example, and not limitation, iohexol, metrizamide, iopamidol,
iopentol, iopromide, and Ioversol are added in the tissue coating
formulations mentioned above. For example, Metrizamide 0.2 g and
polylactic acid (0.8 g, molecular weight 50000) are dissolved in 10
ml chloroform and the polymer solution is spray-coated or
dip-coated on the tissue surface. The solvent is removed by vacuum
drying. The Metrizamide trapped inside the tissue coating makes the
tissue radio-opaque when viewed using medical x-ray imaging
technique.
[0432] Radio-opaque tissue is also made by treating the tissue with
commercially available x-ray contrast agents such as, by way of
example, and not limitation, Iohexyl or Ioversol in presence of EDC
as a catalyst and n-hydroxysuccinimide as a cocatalyst. The
reaction is performed at pH 6.5 using PBS or other suitable
buffers.
[0433] Tissue may also be crosslinked using a crosslinker
containing radio-opaque groups such as, by way of example, and not
limitation, triiodobenzene moieties.
Example 37
Crosslinking of Tissue Using Free Radical Polymerizable
Monomers
[0434] Tissue Fixation Using Glycidyl Methacrylate and Subsequent
Free Radical Photopolymerization Using Free Radical Polymerizable
Monomers
[0435] Step 1
[0436] Treatment of Bovine or Sheep Pericardial Tissue with
Glycidyl Methacrylate.
[0437] Five pieces of unfixed tissue are attached to circular
immobilization 28 mm diameter ring clamps. The clamped tissues are
suspended in 500 ml beaker containing 200 ml PBS, 25 ml glycidyl
methacrylate and 25 ml triethyl amine. The solution was stirred for
16 hours using magnetic needle stirrer. The beaker was transferred
to a water bath maintained at 50.degree. C. and kept there for 4
hours. The tissue was then removed from the clamps washed with PBS
and used in next step for treatment with free radical polymerizable
monomer and subsequent polymerization and crosslinking.
[0438] Step-2
[0439] Fixation of Glycidyl Methacrylate Treated Tissue Using Free
Radical Polymerization.
[0440] Fixation Using Free Radical Photopolymerization.
[0441] One piece of glycidyl methacrylate treated tissue was
incubated in 15 ml of 70% isopropyl alcohol solution for half an
hour in a glass tube. The tissue was then transferred to 2 ml
n-vinyl pyrrolidinone solution containing 40 mg 2-2 dimethoxy-2
phenyl acetophenone as a free radical long ultraviolet light
photoinitiator and incubated for 1 hour at ambient temperature
(25-30.degree. C.). Care was taken to completely expose all
surfaces of the tissue to monomer solution during incubation. The
tissue was removed from the monomer solution and then placed on
glass plate. The tissue was then exposed to long wavelength
ultraviolet light (Black-Ray UV lamp, 360 nm flood light, 10000
mW/cm2 intensity) at a distance of 10 cm for 5 min from both sides.
The fixed tissue was then washed with PBS and stored in alcohol
solution until further use. Using a similar procedure to that
described above, tissue was treated with acrylic acid monomer
instead of vinyl pyrrolidinone monomer.
Example 38
Modification of Tissue or Collagen by Cyclic Lactone
Polymerization
[0442] Preparation of Tissue-Hydroxyacid or
Collagen-Polyhydroxyacid Copolymer
[0443] a) Modification of Tissue or Collagen Sponge by Cyclic
Lactone (Dl-Lactide)
[0444] Three 2 cm.times.2 cm piece is cut from a cleaned
uncrosslinked bovine pericardial sac. The tissue pieces are
incubated for 30 minutes each in 100% acetone. The dried tissue is
then transferred into 100 ml flask. The tissue is further dried at
50.degree. C. under vacuum for 10 hours and weighed. 10 ml
tetrahydrofuran (THF), 3.0 g of dl-lactide and 100 mg of stannous
octoate are charged into the flask. The solution is refluxed for 24
hours to promote cyclic polymerization of lactide by hydroxyl
groups of tissue proteins (hydroxy proline). At the end of the
reaction, tissue is removed, washed with methylene chloride to
remove lactide monomer and unbound polylactide polymer. The tissue
is dried and weighed. The increase in weight is attributed to
polylactide chemically bound to the tissue matrix. A similar
reaction can be carried out on the lyophilized commercially
available collagen sponge from Knesey Nash or glutaraldehyde fixed
pericardial tissue. Other cyclic lactone monomers such L-lactide,
caprolactone, glycolide, trimethylene carbonate, dioxanone may also
be polymerized or copolymerized in place of lactide using a similar
method mentioned above.
Example 39
Modification of Proteins Using Cyclic Lactones
[0445] A 100 ml round bottom flask is flame-dried under vacuum for
2 hours. 2 g of pharmaceutical grade gelatin is then transferred
the flask. The gelatin is further dried at 50.degree. C. under
vacuum for 10 hours and weighed. 10.0 gram of dl-lactide and 100 mg
of stannous octoate are charged into the flask. The flask is then
heated in an oil bath at 120.degree. C. for 24 hours. The flask is
cooled and 20 ml chloroform is added to the flask. The product is
incubated in chloroform for 3 hours to remove monomer and unbound
dl-lactide from the product. The solvent is removed and the
gelatin-lactide copolymer is dried under vacuum and weighed. Other
proteins or glycosaminoglycans such as, by way of example, and not
limitation, albumin, elastin, fibrinogen, collagen or hyaluronic
acid may also be used in place of gelatin. Other cyclic lactone
monomers such L-lactide, caprolactone, glycolide, trimethylene
carbonate, dioxanone may also be polymerized or copolymerized in
place of lactide using a similar method mentioned above.
[0446] Alternatively, gelatin may also be reacted with hydroxy
acids such a lactic acid in water at 100.degree. C. to form a
copolymer.
Example 40
Preparation of Tissue with Biostable and Biodegradable Regions with
Controlled Geometry
[0447] Bovine pericardium (BP) tissue pieces (approximately 25 mm
dia, 3 pieces) are cut from a fresh pericardium and transferred to
a 100 ml flask containing 10 ml PBS, 1.25 ml triethylamine (TEA)
and 1.25 ml glycidyl methacrylate (GM). The solution was heated to
50.degree. C. with constant stirring using magnetic stir bar and
held there for 8 hours. The tissue strips are removed from the
solution and washed with PBS, distilled water, & incubated in
70% isopropanol solution in water for 30 minutes. Finally, strips
are then transferred to 10 ml vinyl pyrrolidinone monomer solution
containing 500 ppm 2,2-dimethoxy 2-phenyl acetophenone
(photoinitiator) and incubated at room temperature (30.degree. C.)
for one hour. One vinyl pyrrolidinone-infused sample piece was
transferred on a glass plate. An aluminum foil (8 mm diameter, 250
micron thick) was placed at the center of the monomer-infused
tissue. The tissue was then exposed to 360 nm light (Black-Ray UV
lamp, 360 nm flood light, 10000 mW/cm2 intensity at a distance of
10 cm) for 5 minutes to polymerize the vinyl pyrrolidinone monomer
and crosslink the tissue. The same treatment was repeated using
aluminum foil from the other side of the tissue. The exposed tissue
was removed and washed with PBS and 70% isopropanol to remove
unreacted monomer, uncrosslinked polymer and initiator fragments.
The uncrosslinked regions where light could not penetrate due to
aluminum foil and initiate photopolymerization was visually
noticeable and had different "feel" to the human hand when compared
to crosslinked or light exposed tissue. Using a similar method to
that described above, another piece of the vinyl pyrrolidinone
infused tissue was covered with aluminum foil (arrow shape, 8 mm
long and 3 mm thick) and the exposed to polymerization and
crosslinking using 360 nm light for 5 minutes. The tissue under
aluminum foil can be considered as dark control tissue as discussed
previously where no polymerization and crosslinking occurred. The
arrow-imprinted tissue was subjected to pepsin digestion for 48
hours. After 48 hours of treatment with pepsin, the dark control
tissue (in the shape of arrow) was completely digested by the
enzyme while light exposed or crosslinked tissue remained
completely intact or stayed biostable. This observation was
confirmed in vivo by implanting the arrow-shaped imprinted tissue
in a rat subcutaneous cavity for 60 days. After 60 days, the dark
control area of the tissue was (unfixed tissue) substantially or
completely digested by natural protease enzymes present in the
animal body and an arrow-like digested area was clearly visible to
the naked eye on explanted samples. It is understood that a variety
of geometries and shapes may be created inside the tissue by using
various masks to block the light similar to photolithography
technique used in semiconductor processing art. Other techniques
such as, by way of example, and not limitation, electron beam
irradiation or scanning of 360 nm laser light on selected areas may
also be used to create a pattern inside the tissue similar to
principles used in electron beam lithography. In that case, a mask
may not be necessary to create a pattern (i.e., the beam itself can
create a pattern).
Example 41
Membrane Like Thin and/or Large Tissue Obtained Using Tissue
Engineering Art for Bioprosthesis Applications
[0448] a) Engineering a Tissue from as Scaffold
[0449] 5 cm.times.5 cm size porous collagen tissue engineering
scaffold obtained from Knesey Nash is soaked in 70% ethanol
(overnight) to sterilize the scaffold, washed with sterile PBS (15
minutes, 3 times) and soaked in Minimum Essential Medium (MEM)
supplemented with aminoacids, antibiotics and 30% fetal bovine
serum (FBS) for 1.5 h. The scaffold is then transferred to a
sterile tissue culture dish. Human smooth muscle cells or
fibroblast cells passaged in MEM/10% FBS are resuspended in MEM/10%
FBS and approximately 200000 cells per square inch are carefully
placed on the scaffold. The cells are allowed to adhere for 30
minutes before adding 0.75 ml of the same medium to each sample.
After 24 hours of incubation in a 5% carbon dioxide atmosphere at
98% humidity and 37.degree. C., the samples are transferred to new
wells and fresh medium and cells are added to the scaffold. The
addition of cells and media and incubation is continued until a
desired tissue thickness is achieved (1 to 2000 microns). For large
size tissue, a large scaffold is used.
[0450] Porous tissue engineering scaffolds known in the tissue
engineering art based on polyesters such as polylactide or
polyglycolide or their copolymers. Natural scaffolds such as
porcine sub-mucosa tissue may also be used in some cases to obtain
a desired tissue engineered tissue.
[0451] The generated may be decellularized and used in
bioprosthesis manufacturing. The tissue may be crosslinked or
modified using glutaraldehyde, EDC and other methods known in the
art or methods described in this invention prior to using it in
bioprosthesis use.
[0452] In one embodiment of the present invention the composition
of matter produced includes an uncrosslinked biological tissue,
wherein the tissue is chemically modified with unsaturated
polymerizable groups.
[0453] In another embodiment of the present invention the
composition of matter produced includes a biological tissue
modified with unsaturated groups, wherein the unsaturated groups
are used in chemical crosslinking of the tissue.
[0454] In yet another embodiment of the present invention, the
composition of matter produced includes biological tissue modified
with unsaturated groups, wherein a cross-linked biological tissue
is produced by treating the tissue under effective cross-linking
condition comprising a free radical initiator or photoinitiator. In
one embodiment, the crosslinking is done in presence of a mono- or
polyunsaturated compound capable of copolymerizing with the
unsaturated groups in the tissue.
[0455] In an alternate embodiment of the present invention, the
composition of matter produced includes a biological tissue
modified with unsaturated groups, wherein unsaturated groups are
copolymerized with free radical polymerizable comonomers. The
comonomers may include, but are not limited to, functional monomers
with reactive functional groups such as, by way of example, and not
limitation, epoxide or isocyanate; monomers with charged groups;
monomers that undergo crosslinking and biodegradation; monomers
that produce thermosensitive polymers; monomers with long alkyl
chains; monomers that produce crystalline or semicrystalline
polymers; monomers that produce functional polymers upon hydrolysis
such as, by way of example, and not limitation, polyvinyl alcohol;
monomers with radio-opaque moieties; monomers that produce
elastomers, monomers that have phosphorylcholine groups, and
fluorinated monomers
[0456] In yet an alternate embodiment of the present invention, the
composition of matter produced includes a biological tissue
modified and crosslinked with unsaturated groups, wherein modified
unsaturated groups and/or crosslinks with unsaturated groups are
copolymerized with free radical polymerizable comonomers.
[0457] In another alternate embodiment of the present invention,
the composition of matter produced includes a biological tissue
modified with unsaturated groups, wherein unsaturated groups are
copolymerized with free radical polymerizable comonomers having
biodegradable or hydrolizable groups. The biodegradable monomers
may be hydrophilic or hydrophobic.
[0458] In one embodiment of the present invention, a cross-linked
biological tissue is produced by treating the tissue under
effective cross-linking conditions with a biodegradable
crosslinker. In some embodiments, the biodegradable crosslinker may
be a solute in a fluid including a solvent.
[0459] In yet another alternate embodiment of the present
invention, the composition of matter produced includes a biological
tissue modified with unsaturated groups, wherein a cross-linked
biological tissue is produced by treating the unsaturated
group-modified tissue under effective cross-linking conditions with
an organic di- or poly-mercapto compounds. In one embodiment, the
di- or poly-mercapto organic compound may be a solute in a fluid
comprising a solvent.
[0460] In an alternate embodiment of the present invention, the
composition of matter produced includes a membrane-like biological
tissue and a surgical adhesive. The membrane-like tissue and
surgical adhesive are formulated to form a "surgical adhesive
patch". Though not required, the surgical adhesive patch may be
biodegradable in some applications.
[0461] In another embodiment of the present invention, the
composition of matter produced includes a radio-opaque implantable
animal tissue.
[0462] In an embodiment of the present invention, the biological
tissue produced has shape memory properties
[0463] In yet another embodiment of the present invention, certain
parts or regions of the biological tissue are made biostable while
the remaining parts of the tissue are made biodegradable. The
biostable and biodegradable regions within the tissue can be of any
geometry.
[0464] In another embodiment of the present invention, a
non-crosslinked degradable biological tissue is produced by
treating the tissue under effective treatment conditions with a
monofunctional reagent capable of reacting with primary amine
groups on the tissue. In one embodiment, the monofunctional reagent
may be a polyether derivative or an activated acid derivative such
as, by way of example, and not limitation, an n-hydroxysuccinimide
derivative, a cyclic lactone such as, by way of example, and not
limitation, glycolide or lactide, an isocyanate derivative, or an
anhydride derivative.
[0465] In one embodiment of the present invention, a biostable or
biodegradable tissue produced by treating the tissue under
effective treatment conditions with a cyclic lactone to produce a
tissue-polylactone graft copolymer.
[0466] In another embodiment of the present invention a biological
tissue is produced in which the biological tissue/synthetic
biodegradable polymer composite is produced by treating the tissue
with a fluid comprising synthetic biodegradable polymer.
[0467] In another embodiment of the present invention a biological
tissue is produced in which the biological tissue/synthetic
biodegradable polymer composite comprises a synthetic biodegradable
polymer, which is chemically bonded to the biological tissue.
[0468] In one embodiment of the present invention a degradable
biological tissue/synthetic biodegradable polymer composite
produced by treating a non-crosslinked tissue under effective
treatment conditions with a synthetic biodegradable polymer. In one
embodiment, the synthetic biodegradable polymer is polylactone or
polyhydroxyacid derivative in a fluid comprising a solvent. The
synthetic biodegradable polymer can also be a crosslinked
polymer.
[0469] In an alternate embodiment of the present invention the
biological tissue is produced by treating the tissue with a fluid
comprising synthetic biodegradable polymer and a bioactive
compound.
[0470] In an alternate embodiment of the present invention, a
method for cross-linking a tissue, which method may be utilized for
tissue fixation is achieved by steps that include, without
limitation, linking at least a portion of the free radical
polymerizable groups on the tissue with a covalent bond and
crosslinking the free radical polymerizable groups using free
radical mechanism or cyclic dimerization (e.g., treating the tissue
under effective crosslinking conditions with a compound having a
free radical polymerizable group). In another embodiment of the
present invention, a method of modifying a protein or mixture of
proteins in the solid state is achieved by steps that include,
without limitation, covalently bonding at least a portion of the
protein functional groups with a free radical polymerizable group
in a solid state; and treating the protein under effective
cross-linking conditions with a compound having a free radical
polymerizable group.
[0471] In an yet another alternate embodiment of the present
invention, a method for cross-linking a tissue, which method may be
utilized for tissue fixation is achieved by steps that include,
without limitation, covalently linking compounds containing at
least one free radical polymerizable group on the tissue and
crosslinking the free radical polymerizable group(s) using a di- or
poly-mercapto organic compounds. In one embodiment, the method
comprises covalently bonding at least a portion of the tissue
functional groups with unsaturated groups and treating the tissue
under effective cross-linking conditions with a di- or polymercapto
compound.
[0472] In an alternate embodiment of the present invention, a
method for cross-linking a tissue, which method may be utilized for
tissue fixation, is achieved by steps that include, without
limitation, covalently bonding at least a portion of the tissue
functional groups with at least one free radical polymerizable
group and treating the tissue under effective crosslinking
conditions with a compound having a free radical group and a
biodegradable link. In one embodiment, the method comprises
crosslinking the tissue with compounds containing at least one free
radical polymerizable group and further crosslinking the free
radical polymerizable group using free radical chemistry such as,
by way of example, and not limitation, free radical dimerization
and polymerization, free radical crosslinking or free radical
copolymerization with monomer.
[0473] In an alternate embodiment of the present invention, the
method for making a biodegradable biological tissue includes the
steps of without limitation, treating the tissue under effective
cross-linking conditions with a fluid comprising a biodegradable
crosslinker.
[0474] In one embodiment of the present invention, the method for
incorporating a biodegradable polymer in a biological tissue is
achieved by steps that include, without limitation, dehydrating the
biological tissue; treating the dehydrated tissue with a solution
of biodegradable polymer in an organic solvent; and removing the
solvent from the treated tissue.
[0475] In yet another embodiment of the present invention, the
method for incorporating a biodegradable polymer and a bioactive
compound in an implantable biological tissue is achieved by steps
that include, without limitation, dehydrating the biological
tissue; treating dehydrated tissue with a solution of biodegradable
polymer and bioactive compound in an organic solvent; and removing
the solvent from the treated tissue. In an alternate embodiment,
the method comprises the steps of providing a tissue suitable for
human implantation; exposing the tissue to a fluid comprising
sparingly soluble drug dissolved in a solvent; and evaporating the
solvent.
[0476] In an alternate embodiment of the present invention, the
biological tissue based controlled drug delivery patch that
releases at least one bioactive compound.
[0477] In an alternate embodiment of the present invention, the
method for making an implantable degradable drug delivery patch
from the membrane-like tissue is achieved by steps that include,
without limitation, dehydrating the membrane-like biological
tissue; treating dehydrated membrane-like tissue with a solution of
biodegradable polymer and a bioactive compound in an organic
solvent; removing the solvent from the treated tissue; releasing
the compound from the biodegradable polymer. In an alternate
embodiment, the method comprises providing a implantable
membrane-like animal tissue; exposing the tissue to a fluid
comprising controlled release carrier and a bioactive compound
dispersed in the solvent; and evaporating the solvent. The
bioactive compound may be a cell cycle inhibitor including, but not
limited to, Lovastatin (HMG-CoA inhibitor or statin), paclitaxel,
and Rapamycin. The biodegradable polymer may be hydrophobic or
hydrophilic. The biodegradable polymer can be a crosslinked
polymer, though not required.
[0478] In an alternate embodiment of the present invention, the
method for coating the biological tissue with biodegradable polymer
(e.g., forming a coated tissue or tissue implant) is achieved by
steps that include, without limitation, providing a tissue suitable
for human implantation; coating the tissue surface by spraying a
solution comprising a solvent and a polymer dissolved in the
solvent; and evaporating the solvent. In an alternate embodiment,
the method further comprises the steps of providing a tissue
suitable for human implantation; exposing the tissue to a fluid
comprising biodegradable polymer dispersed in the solvent; and
evaporating the solvent. In one embodiment, the method comprises
the steps of providing a tissue suitable for human implantation;
coating the tissue surface by spraying a solution comprising
precursors capable of forming crosslinked polymers; and
crosslinking the precursors. In another alternate embodiment, the
method comprises the steps of providing a tissue suitable for human
implantation; coating the tissue surface by spraying a macromonomer
solution; and crosslinking the macromonomer in the solution. In one
exemplary embodiment, the method comprises dehydrating the
biological tissue; spraying a coating solution comprising
biodegradable polymer in a solvent; and removing the solvent from
the treated tissue. In an alternate embodiment, the method
comprises dehydrating the biological tissue; dipping the dehydrated
tissue in a coating solution comprising biodegradable polymer in a
solvent; removing the solvent from the treated tissue.
[0479] In another embodiment of the present invention, a method for
making a radio-opaque implantable tissue is achieved by steps that
include, without limitation, treating a biological tissue with a
radio-opaque compound under effective treatment conditions to
covalently bond the radio-opaque compound to the tissue. In some
embodiments, the radio-opaque compound is an iodinated organic
compound.
[0480] In another embodiment of the present invention, a method for
making a radio-opaque implantable tissue is achieved by steps that
include, without limitation, treating a biological tissue with a
radio-opaque compound and biodegradable polymer in a solvent and
removing the solvent to produce a tissue coated with biodegradable
compound and radio-opaque compound.
[0481] Another embodiment of the present invention provides for a
method of treating a tissue under effective cross-linking
conditions with a di- or poly-mercapto organic compound.
[0482] In one embodiment of the present invention, a method of
coating a biological implantable tissue with a biodegradable
hydrogel is achieved by steps that include, without limitation,
treating a tissue with a precursor or biodegradable hydrogel
components and crosslinking the precursors to produce a
biodegradable hydrogel coating on the surface of the tissue.
[0483] In another embodiment of the present invention, a method of
coating a biological implantable tissue with biodegradable hydrogel
comprising cells/bioactive compound is achieved by steps that
include, without limitation, treating a tissue with a precursor or
biodegradable hydrogel components comprising cells and/or bioactive
compounds and crosslinking the precursors to produce a
biodegradable hydrogel coating with entrapped cells/drug in the
coating on the surface of the tissue.
[0484] In yet another embodiment of the present invention, a method
of coating a biological tissue with non-crosslinked biodegradable
hydrogel is achieved by steps that include, without limitation,
dehydrating the biological tissue; treating the dehydrated tissue
with a solution of biodegradable polymer in an organic solvent;
removing the solvent from the treated tissue; and exposing the
tissue to a biological environment to hydrate the tissue and
biodegradable polymer.
[0485] In another embodiment of the present invention, a method for
incorporating a biodegradable polymer and bioactive substance in a
biological tissue is achieved by steps that include, without
limitation, forming grooves or holes on tissue surface; filling the
grooves or holes with a biodegradable polymer and bioactive
compound; and releasing the bioactive compound in a controlled
manner.
[0486] In an alternate embodiment of the present invention a
degradable animal tissue is coated with or incorporated with,
Demineralized Bone Matrix (DBM) and/or purified bone morphogenic
proteins (BMP). This mixture provides a matrix that allows the
cellular components of the body to migrate into it and thus produce
osteoinduction where needed. The matrix composition, enzymes (such
as, by way of example, and not limitation, thrombin and plasmin),
BMPs, growth factors and DBM and their concentrations, calcium
salts such as, by way of example, and not limitation, calcium
phosphates may be adequately formulated to optimize the longevity
of this temporal scaffolding structure and the osteoinduction which
needs to occur. All of the animal tissue components are
biodegradable, but during osteogenesis the mixture provides a
non-collapsible scaffold that can determine the shape and location
of the newly formed bone.
[0487] In one embodiment of the present invention the composition
of matter produced comprises a degradable tissue coated with a
biodegradable polymer comprising at least one growth factor and/or
a drug.
[0488] In another embodiment of the present invention, the
composition of matter including a biodegradable implantable animal
tissue coated with biodegradable polymer and an effective
concentration of at least one growth factor, wherein the
concentration of growth factor is effective in promoting wound
healing.
[0489] An alternate embodiment of the present invention provides
for a composition of matter that promotes the growth of cells,
including a degradable animal tissue; a hydrogel coating on the
surface of degradable tissue; and an effective concentration of at
least one growth factor, wherein the concentration of the growth
factor is effective in promoting the directed migration of the
animal cells. In another embodiment, genetically altered cells
and/or other cells may also be included in the tissue-coated
hydrogels of this invention.
[0490] In one embodiment of the present invention, a composition of
matter that promotes the proliferation and/or differentiation of
animal cells is made by including an implantable animal tissue; a
hydrogel; and an effective concentration of at least one growth
factor, wherein the concentration is effective in promoting
proliferation and/or differentiation of animal cells.
[0491] In an alternate embodiment of the present invention, the
composition of matter produced is used to promote the localized
delivery of at least one growth factor. Exemplary, but not
limiting, growth factors are vascular endothelial growth factor
(VEGF) or BMP or mixtures thereof. The use of numerous other growth
factors, both known and yet to be discovered, will be readily
apparent to one skilled in the art, in light of the teachings of
the present invention.
[0492] One embodiment of the present invention provides for a
process for promoting the healing of wounds, which is achieved by
steps that include, without limitation, applying to the wound a
composition that contains a non-crosslinked animal degradable
animal tissue modified with a synthetic polymer and an effective
concentration of at least one growth factor or one small molecule
therapeutic, wherein the concentration is effective to promote
wound healing.
[0493] In one embodiment of the present invention, a degradable
implantable animal tissue-based composition produced is used to
promote the localized delivery of a poorly water-soluble form of a
bioactive compound, such as, but not limited to, chlorhexidene;
chlorhexidene diacetate monohydrate or chlorhexidene
dihydrochloride; chlorhexidene gluconate, silver salts such as, by
way of example, and not limitation, silver chloride, silver iodide,
silver acetate, silver lactate, cell cycle inhibitors such as, by
way of example, and not limitation, paclitaxel, lovastatin,
rapamycin, simvastatin, rifampin; or anti-arrhythmic agents such
as, by way of example, and not limitation, amiodarone.
[0494] In another embodiment of the present invention, the method
for tissue crosslinking or fixation is achieved by steps that
include, without limitation, linking the free radical polymerizable
groups on the tissue with a covalent bond and crosslinking the free
radical polymerizable groups using free radical polymerizable
monomers including a primary amine group. Further crosslinking the
primary amine groups using a di- or polyfunctional crosslinker such
as, by way of example, and not limitation, glutaraldehyde.
[0495] In one embodiment of this of invention a bioprosthesis made
using membrane like tissue is provided wherein the membrane like
tissue is made using tissue engineering methodologies and has a
thickness from 10 micron to 2000 microns and/or has a size >1
square inch.
[0496] In an alternate embodiment of the present invention, the
method for making a degradable tissue matrix is achieved by steps
that include, without limitation, substantially water-insoluble
drug or bioactive compound is provided for in an alternate
embodiment of the present invention. The method includes
dehydrating the membrane-like biological tissue; treating
dehydrated membrane-like tissue with a solution of a substantially
water-insoluble bioactive compound in an organic solvent; and
removing the solvent from the treated tissue.
[0497] Having fully described at least one embodiment of the
present invention, other equivalent or alternative implantable
tissue composition and methods will be apparent to those skilled in
the art. The invention has been described above by way of
illustration, and the specific embodiments disclosed are not
intended to limit the invention to the particular forms disclosed.
The invention is thus to cover all modifications, equivalents, and
alternatives falling with the spirit and scope of the following
claims.
* * * * *