U.S. patent application number 13/008679 was filed with the patent office on 2011-05-12 for method and material for enhanced tissue-biomaterial integration.
Invention is credited to Jennifer H. Elisseeff, Anthony Ratcliffe, Dong-an Wang.
Application Number | 20110111034 13/008679 |
Document ID | / |
Family ID | 34435375 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110111034 |
Kind Code |
A1 |
Wang; Dong-an ; et
al. |
May 12, 2011 |
METHOD AND MATERIAL FOR ENHANCED TISSUE-BIOMATERIAL INTEGRATION
Abstract
The present invention relates to the covalent binding of a
hydrogel to an extracellular matrix (ECM). The integration of the
hydrogel with the tissue is superior to that in previous
techniques. Moreover, unlike previous techniques, the present
invention does not require a photoinitiator. Potential therapeutic
applications include tissue repair and delivery of drugs or cells.
The ECM is first exposed, then treated with a priming agent. Then a
polymerizable agent is added and crosslinked to the ECM. Two
primary embodiments of methods are disclosed. In the first, the
priming agent is an oxidizer which creates tyrosyl radicals in the
ECM, which are then bound by acrylate groups in the polymerizable
agent. In the second, the priming agent contains aldehydes which
bind amino groups in the ECM.
Inventors: |
Wang; Dong-an; (Baltimore,
MD) ; Ratcliffe; Anthony; (Del Mar, CA) ;
Elisseeff; Jennifer H.; (Baltimore, MD) |
Family ID: |
34435375 |
Appl. No.: |
13/008679 |
Filed: |
January 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11369578 |
Mar 7, 2006 |
7897165 |
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13008679 |
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Current U.S.
Class: |
424/487 ;
424/93.7; 435/180 |
Current CPC
Class: |
A61L 27/3691 20130101;
A61L 27/3616 20130101; A61L 27/52 20130101; A61L 27/3604 20130101;
C12N 5/0062 20130101; A61L 24/0005 20130101; A61L 27/3633 20130101;
A61L 27/38 20130101; A61L 27/50 20130101; A61L 24/0031 20130101;
A61L 27/3687 20130101; C12N 2533/30 20130101; A61K 35/32 20130101;
C12N 5/0655 20130101; C12N 5/0697 20130101; A61L 27/3683 20130101;
A61K 45/06 20130101; A61P 19/00 20180101 |
Class at
Publication: |
424/487 ;
424/93.7; 435/180 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61P 19/00 20060101 A61P019/00; C12N 11/08 20060101
C12N011/08 |
Claims
1. A method of integrating a hydrogel to an extracellular matrix,
comprising the steps of: (a) providing an exposed extracellular
matrix; (b) priming the extracellular matrix by treating with a
priming agent to create a primed extracellular matrix; (c) adding
to the primed extracellular matrix a solution of a polymerizable
agent; and (d) reacting the primed extracellular matrix and
polymerizable agent to create a hydrogel covalently bound to the
extracellular matrix.
2. The method of claim 1, wherein: said extracellular matrix
comprises a plurality of tyrosine residues, said priming step
comprises oxidizing said extracellular matrix, said priming agent
comprises an oxidizing agent, said primed extracellular matrix
comprises a plurality of tyrosyl radicals, said polymerizable agent
comprises an acrylate group reactable with said tyrosyl radicals,
and said reacting step comprises binding said polymerizable agent
to the tyrosyl radicals and crosslinking said polymerizable
agent.
3. The method of claim 2, wherein said extracellular matrix is
disposed in a living body.
4. The method of claim 3, wherein said body is that of a
mammal.
5. The method of claim 4, wherein said body is that of a human.
6. The method of claim 3, wherein said providing step comprises
treating said extracellular matrix with chondroitinase ABC.
7. The method of claim 2, wherein said extracellular matrix
comprises a collagen.
8. The method of claim 7, wherein said collagen is disposed in a
cartilage.
9. The method of claim 2, wherein said oxidizing agent comprises a
hydrogen peroxide solution.
10. The method of claim 2, wherein said priming step further
includes exposing said extracellular matrix and said polymerizable
agent to a source of electromagnetic radiation.
11. The method of claim 2, wherein, said source of electromagnetic
radiation is a source of ultraviolet light.
12. The method of claim 2, wherein said polymerizable agent has at
least one free radical polymerizable group selected from the group
consisting of an of an acrylate, a diacrylate, oligoacrylate,
dimethacrylate, and oligomethacrylate.
13. The method of claim 2, wherein said reacting step comprises
exposing said extracellular matrix and said polymerizable agent to
a source of ultraviolet radiation.
14. The method of claim 2, wherein said reacting step comprises
using a redox initiator.
15. The method of claim 2, wherein said reacting step comprises an
ionic crosslinking reaction.
16. The method of claim 2, wherein said reacting step comprises an
enzymatic crosslinking reaction.
17. The method of claim 2, wherein said method is carried out
transdermally.
18. The method of claim 2, wherein a diffusible drug substance is
added with said polymerizable agent.
19. The method of claim 2, wherein a plurality of living cells are
added with said polymerizable agent.
20. The method of claim 2, whereby a solid repair in a tissue is
created.
21. A hydrogel produced by the method comprising the following
steps: (e) providing an exposed extracellular matrix; (f) priming
the extracellular matrix by treating with a priming agent to create
a primed extracellular matrix; (g) adding to the primed
extracellular matrix a solution of a polymerizable agent; and (h)
reacting the primed extracellular matrix and polymerizable agent to
create a hydrogel covalently bound to the extracellular matrix;
wherein said extracellular matrix comprises a plurality of tyrosine
residues, said priming step comprises oxidizing said extracellular
matrix, said priming agent comprises an oxidizing agent, said
primed extracellular matrix comprises a plurality of tyrosyl
radicals, said polymerizable agent comprises an acrylate group
reactable with said tyrosyl radicals, and said reacting step
comprises binding said polymerizable agent to the tyrosyl radicals
and crosslinking said polymerizable agent.
22. A hydrogel covalently bound to an extracellular matrix at a
plurality of tyrosine residues of said matrix.
23. A hydrogel in accordance with claim 22, wherein said
extracellular matrix is composed of a plurality of collagen fibers
in a cartilage matrix.
24. A method of forming a hydrogel covalently bound to a cartilage
tissue, comprising the steps of: (a) exposing a plurality of
tyrosine residues disposed in a plurality of collagen fibers
further disposed in a cartilage tissue by treating the tissue with
a glycanase to remove a plurality of polysaccharides; (b) oxidizing
the tyrosine residues, by treating with a hydrogen peroxide
solution and a source of ultraviolet light, to produce a plurality
of tyrosyl groups in the collagen; (c) adding a polymerizable agent
comprising one or more acrylate groups; and (d) reacting the
tyrosyl groups and the polymerizable agent to create a hydrogel
covalently bound to the collagen.
25. The method of claim 1, wherein: said extracellular matrix
comprises a plurality of amino groups, said priming agent comprises
a compound with an aldehyde group and a carrier compound with an
amino group, said priming step comprises reacting said priming
agent with said extracellular matrix to covalently bind said
extracellular matrix and said priming agent, said primed
extracellular matrix consists of said extracellular matrix
covalently bound to said priming agent, and said reacting step
consists of covalently binding said polymerizable agent to said
primed extracellular matrix and crosslinking said polymerizable
agent.
26. The method of claim 25, wherein said extracellular matrix is
disposed in a living body.
27. The method of claim 26, wherein said body is that of a
mammal.
28. The method of claim 27, wherein said body is that of a
human.
29. The method of claim 25, wherein said polymerizable agent has at
least one free radical polymerizable group selected from the group
consisting of an of an acrylate, a diacrylate, oligoacrylate,
dimethacrylate, and oligomethacrylate.
30. The method of claim 25, wherein said reacting step comprises
exposing said extracellular matrix and said polymerizable agent to
a source of ultraviolet radiation.
31. The method of claim 25, wherein said reacting step comprises
using a redox initiator.
32. The method of claim 25, wherein said reacting step comprises an
ionic crosslinking reaction.
33. The method of claim 25, wherein said reacting step comprises an
enzymatic crosslinking reaction.
34. The method of claim 25, wherein said method is carried out
transdermally.
35. The method of claim 25, wherein a diffusible drug substance is
added with said polymerizable agent.
36. The method of claim 25, wherein a plurality of living cells are
added with said polymerizable agent.
37. The method of claim 25, whereby a solid repair in a tissue is
created.
38. A hydrogel produced by the method comprising the following
steps: (e) providing an exposed extracellular matrix; (f) priming
the extracellular matrix by treating with a priming agent to create
a primed extracellular matrix; (g) adding to the primed
extracellular matrix a solution of a polymerizable agent; and (h)
reacting the primed extracellular matrix and polymerizable agent to
create a hydrogel covalently bound to the extracellular matrix;
wherein, said extracellular matrix comprises a plurality of amino
groups, said priming agent comprises a compound with an aldehyde
group and a carrier compound with an amino group, said priming step
comprises reacting said priming agent with said extracellular
matrix to covalently bind said extracellular matrix and said
priming agent, said primed extracellular matrix consists of said
extracellular matrix covalently bound to said priming agent, and
said reacting step consists of covalently binding said
polymerizable agent to said primed extracellular matrix and
crosslinking said polymerizable agent.
39. A hydrogel covalently bound via a priming agent to an
extracellular matrix at a plurality of amino groups in said
extracellular matrix.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/369,578, which claims benefit under 35
U.S.C. .sctn.119(e) of U.S. Provisional Application No. 60/416,881,
filed Oct. 9, 2002, the entire disclosures of which are hereby
incorporated by reference.
[0002] This application is related to a utility patent application
claiming priority to U.S. Provisional Application No. 60/413,152
(filed Sep. 25, 2002), entitled "Cross-linked polymer matrices, and
methods of making and using same," and filed on Sep. 25, 2003, the
entire disclosure of which is hereby incorporated by reference.
[0003] This application is also related to a utility patent
application claiming priority to U.S. Provisional Application No.
60/416,882 (filed Oct. 9, 2002), entitled "Multilayered
polymerizing hydrogels for tissue regeneration," and filed on Oct.
9, 2003, the entire disclosure of which is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention pertains to a method of creating a
hydrogel covalently bound to the extracellular matrix. More
specifically, this invention is directed to a polymer hydrogel
covalently bound to an extracellular matrix, and to a method for
creating such a hydrogel, in a tissue-initiated polymerization.
[0006] 2. Background Art
[0007] Integration of biomaterials with the body is a longstanding
problem in medicine. Lack of proper integration with the body
sacrifices implant longevity and function. Hard tissues such as
cartilage and bone present particular challenges to
integration.
[0008] Cartilage lacks the ability to repair itself, and has a
dense extracellular matrix that provides a smooth surface with
weight bearing function, making cartilage extremely challenging to
integrate with other materials. Collagen fibers present in the
cartilage matrix and throughout the body have structural integrity
and are therefore a target for anchoring and biomaterial
integration.
[0009] Earlier work (e.g. Langer et al., U.S. Pat. No. 6,224,893;
Hubbell et al., U.S. Pat. No. 6,258,870; Hubbell et al., U.S. Pat.
No. 6,465,001) on biological applications for polymers relied on
photoinitiators to capture light energy and form free radicals to
initiate polymerization of the polymer precursors. For example, in
U.S. Pat. Nos. 6,258,870 and 6,465,001, the use of ethyl eosin is
recommended. An advantage of certain embodiments of the present
invention, wherein appropriate free radicals are produced following
localized mild oxidation of the tissue, is that no photoinitiator
is required, saving complexity and expense, and alleviating any
issue of toxicity of the photoinitiator.
[0010] Most notably, the polymers of the prior art (e.g. Langer et
al., U.S. Pat. No. 6,224,893; Hubbell et al., U.S. Pat. No.
6,258,870; Hubbell et al., U.S. Pat. No. 6,465,001) do not bind
covalently to the tissue. This profoundly limits the strength and
durability of these inventions, as they do not integrate well as
possible with the tissue.
SUMMARY OF THE INVENTION
[0011] It is a general object of the invention to create a hydrogel
covalently bound to an extracellular matrix.
[0012] In a first preferred embodiment of the invention, the method
comprises the following steps: [0013] (a) providing an .exposed
extracellular matrix; [0014] (b) priming the extracellular matrix
by treating with a priming agent to create a primed extracellular
matrix; [0015] (c) adding to the primed extracellular matrix a
solution of a polymerizable agent; and [0016] (d) reacting the
primed extracellular matrix and polymerizable agent to create a
hydrogel covalently bound to the extracellular matrix.
[0017] As used in this description and in the appended claims,
"solution" means a solution, a suspension, or a colloid.
[0018] In a second preferred embodiment of the invention, the
extracellular matrix comprises a plurality of tyrosine residues,
the priming step comprises oxidizing the extracellular matrix, the
priming agent comprises an oxidizing agent, the primed
extracellular matrix comprises a plurality of tyrosyl radicals, the
polymerizable agent comprises an acrylate group reactable with the
tyrosyl radicals, and the reacting step comprises binding the
polymerizable agent to the tyrosyl radicals and crosslinking the
polymerizable agent.
[0019] In a further preferred embodiment, the extracellular matrix
of the second embodiment is in a living body, preferably that of a
mammal, or most preferably in a human body.
[0020] In yet another preferred embodiment, the extracellular
matrix of the second embodiment is exposed by treating with
chondroitinase ABC.
[0021] In a still further preferred embodiment, the extracellular
matrix of the second embodiment comprises a collagen. In a yet
further embodiment, the collagen is in a cartilage.
[0022] In yet another preferred embodiment, the oxidizing agent of
the second embodiment comprises a hydrogen peroxide solution.
Alternate oxidizing agents may be used, including vitamin C.
[0023] In a still further preferred embodiment, the priming step of
the second embodiment further includes exposing said extracellular
matrix and said polymerizable agent to a source of electromagnetic
radiation. In an even more preferred embodiment, the source of
electromagnetic radiation is a source of ultraviolet radiation
[0024] In yet another preferred embodiment, the polymerizable agent
of the second embodiment has at least one free radical
polymerizable group selected from the group consisting of an of an
acrylate, a diacrylate, oligoacrylate, dimethacrylate, and
oligomethacrylate.
[0025] In a still further preferred embodiment, the reacting step
of the second embodiment comprises exposing said extracellular
matrix and said polymerizable agent to a source of ultraviolet
radiation.
[0026] In yet another preferred embodiment, the crosslinking of the
second embodiment is by using a redox initiator. In a still further
preferred embodiment, an ionic crosslinking reaction is used. In
still another preferred embodiment, an enzymatic crosslinking
reaction is used.
[0027] In a still further preferred embodiment, the method of the
second embodiment is carried out transdermally.
[0028] In yet another preferred embodiment, a diffusible drug
substance is added with said polymerizable agent of the second
embodiment.
[0029] In a still further preferred embodiment, a plurality of
living cells are added with said polymerizable agent of the second
embodiment.
[0030] In yet another preferred embodiment, a solid repair in a
tissue is created by the second embodiment.
[0031] A third preferred embodiment is the hydrogel produced by the
method comprising the following steps: [0032] (a) providing an
exposed extracellular matrix; [0033] (b) priming the extracellular
matrix by treating with a priming agent to create a primed
extracellular matrix; [0034] (c) adding to the primed extracellular
matrix a solution of a polymerizable agent; and [0035] (d) reacting
the primed extracellular matrix and polymerizable agent to create a
hydrogel covalently bound to the extracellular matrix; wherein said
extracellular matrix comprises a plurality of tyrosine residues,
said priming step comprises oxidizing said extracellular matrix,
said priming agent comprises an oxidizing agent, said primed
extracellular matrix comprises a plurality of tyrosyl radicals,
said polymerizable agent comprises an acrylate group reactable with
said tyrosyl radicals, and said reacting step comprises binding
said polymerizable agent to the tyrosyl radicals and crosslinking
said polymerizable agent.
[0036] A fourth preferred embodiment is a hydrogel covalently bound
to an extracellular matrix at a plurality of tyrosine residues of
said matrix. Yet another preferred embodiment is the hydrogel of
the third embodiment wherein said extracellular matrix is composed
of a plurality of collagen fibers in a cartilage matrix.
[0037] A fifth preferred embodiment is a method of forming a
hydrogel covalently bound to a cartilage tissue, comprising the
steps of: [0038] (a) exposing a plurality of tyrosine residues
disposed in a plurality of collagen fibers further disposed in a
cartilage tissue by treating the tissue with a glycanase to remove
a plurality of polysaccharides; [0039] (b) oxidizing the tyrosine
residues, by treating with a hydrogen peroxide solution and a
source of ultraviolet light, to produce a plurality of tyrosyl
groups in the collagen; [0040] (c) adding a polymerizable agent
comprising one or more acrylate groups; and [0041] (d) reacting the
tyrosyl groups and the polymerizable agent to create a hydrogel
covalently bound to the collagen.
[0042] In a sixth preferred embodiment, the method is that of the
first enumerated embodiment, wherein: the extracellular matrix
comprises a plurality of amino groups, the priming agent comprises
a compound with an aldehyde group and a carrier compound with an
amino group, the priming step comprises reacting the priming agent
with the extracellular matrix to covalently bind the extracellular
matrix and the priming agent, the primed extracellular matrix
consists of the extracellular matrix covalently bound to the
priming agent, and the reacting step consists of covalently binding
the polymerizable agent to the primed extracellular matrix and
crosslinking the polymerizable agent.
[0043] In a further preferred embodiment, the extracellular matrix
of the sixth embodiment is in a living body, preferably that of a
mammal, or most preferably in a human body.
[0044] In yet another preferred embodiment, the polymerizable agent
of the sixth embodiment has at least one free radical polymerizable
group selected from the group consisting of an of an acrylate, a
diacrylate, oligoacrylate, dimethacrylate, and
oligomethacrylate.
[0045] In a still further preferred embodiment, the reacting step
of the sixth embodiment comprises exposing said extracellular
matrix and said polymerizable agent to a source of ultraviolet
radiation.
[0046] In yet another preferred embodiment, the crosslinking of the
sixth embodiment is by using a redox initiator. In a still further
preferred embodiment, an ionic crosslinking reaction is used. In
still another. preferred embodiment, an enzymatic crosslinking
reaction is used.
[0047] In a still further preferred embodiment, the method of the
sixth embodiment is carried out transdermally.
[0048] In yet another preferred embodiment, a diffusible drug
substance is added with said polymerizable agent of the sixth
embodiment.
[0049] In a still further preferred embodiment, a plurality of
living cells are added with said polymerizable agent of the sixth
embodiment.
[0050] In yet another preferred embodiment, a solid repair in a
tissue is created by the sixth embodiment.
[0051] A seventh preferred embodiment is the hydrogel produced by
the method comprising the steps of [0052] (a) providing an exposed
extracellular matrix; [0053] (b) priming the extracellular matrix
by treating with a priming agent to create a primed extracellular
matrix; [0054] (c) adding to the primed extracellular matrix a
solution of a polymerizable agent; and [0055] (d) reacting the
primed extracellular matrix and polymerizable agent to create a
hydrogel covalently bound to the extracellular matrix; wherein,
said extracellular matrix comprises a plurality of amino groups,
said priming agent comprises a compound with an aldehyde group and
a carrier compound with an amino group, said priming step comprises
reacting said priming agent with said extracellular matrix to
covalently bind said extracellular matrix and said priming agent,
said primed extracellular matrix consists of said extracellular
matrix covalently bound to said priming agent, and said reacting
step consists of covalently binding said polymerizable agent to
said primed extracellular matrix and crosslinking said
polymerizable agent.
[0056] An eighth preferred embodiment is a hydrogel covalently
bound via a priming agent to an extracellular matrix at a plurality
of amino groups in said extracellular matrix.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 is a schematic description of the method as applied
to cartilage using acrylate-tyrosyl chemistry.
[0058] FIG. 2 is a series of spectrograms of treated cartilage at
various stages of the procedure using attenuated total reflectance
Fourier transform infrared spectroscopy (ATR-FTIR).
[0059] FIG. 3 shows the results of tests of the mechanical
functionality of hydrogel alone, and of two hydrogels on a
cartilage surface. FIG. 3A is a schematic of the testing procedure.
FIGS. 3B-D represent the stress-strain-time behavior. FIGS. 3 E-H
represent the rheological behavior. FIG. 31 indicates the applied
strain-time conditions.
[0060] FIG. 4 shows the ATR-FTIR analysis illustrating formation of
a covalent bond between collagen and hydroxyethyl methacrylate.
[0061] FIG 5 shows analysis of the acrylate-tyrosyl reaction
mechanism using electron spin-trapping.
[0062] FIG. 6 illustrates the procedure for measuring
biocompatibility of the process.
[0063] FIG. 7 shows an example of the aldehyde priming embodiment
of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0064] Biomaterials play an important role in medicine today with
applications ranging from medical devices to artificial implants,
drug delivery coatings, and scaffolds for tissue regeneration. The
strategy for biomaterial design has evolved from the philosophy of
creating materials that were "invisible" to the body, to the
development of biomaterials that actively interact with the
surrounding tissue by recruiting cells, stimulating regeneration,
or guiding tissue remodeling. Thus, the body is encouraged to
positively integrate with a biomaterial instead of "walling off"
the implant with a fibrous capsule to silence reactions. The
interface between a biomaterial and surrounding tissue is integral
to its functionality and long-term performance, particularly in
musculoskeletal implants. Integration of biomaterials with hard
tissues such as cartilage and bone is particularly challenging due
to the nature of the dense extracellular matrix and strong
mechanical forces that the tissue must withstand. Furthermore,
cartilage tissue lacks the ability to heal and has difficulty
integrating with natural or engineered cartilage, much less a
biomaterial, and was therefore chosen as our model system.
[0065] Heretofore, true integration of biomaterials by covalent
binding of the materials to an extracellular matrix has not been
achieved.
[0066] The present invention relates to covalent attachment of
biogels to the extracellular matrix. Principles of basic protein
biochemistry are combined with biomaterials to create a general
system for directed integration of biomaterials that may be applied
to multiple tissues types and biomaterial applications.
[0067] The term "hydrogel" as used herein and in the appended
claims refers to a hydrophilic cross-linked polymer capable of
containing a large volume fraction of water. More preferably
hydrogels according to the present invention can contain greater
than about 70-90 volume % water. When a hydrophilic polymer is
formed in situ, it may inherently acquire water from its
environment or from solutions used to create the hydrogel.
[0068] The term "cross-linked" as used herein and in the appended
claims refers to a composition containing intermolecular
cross-links and optionally intramolecular cross-links arising from
the formation of covalent bonds, ionic bonds, hydrogen bonding, or
any combination thereof. "Cross-linkable" refers to a component or
compound that is capable of undergoing reaction to form a
cross-linked composition.
[0069] The polymerizable agent of the present invention may
comprise monomers, macromers, oligomers, polymers, or a mixture
thereof. The polymer compositions can consist solely of covalently
crosslinkable polymers, or ionically crosslinkable polymers, or
polymers crosslinkable by redox chemistry, or polymers crosslinked
by hydrogen bonding, or any combination thereof. The polymerizable
agent should be substantially hydrophilic and biocompatible.
[0070] The term "biocompatible" when used in relation to polymers
are art-recognized. For example, biocompatible polymers include
polymers that are neither themselves toxic to the host (e.g., an
animal or human), nor degrade (if the polymer degrades) at a rate
that produces monomeric or oligomeric subunits or other byproducts
at toxic concentrations in the host. In certain embodiments of the
present invention, biodegradation generally involves degradation of
the polymer in an organism, e.g., into its monomeric subunits,
which may be known to be effectively non-toxic. Intermediate
oligomeric products resulting from such degradation may have
different toxicological properties, however, or biodegradation may
involve oxidation or other biochemical reactions that generate
molecules other than monomeric subunits of the polymer.
Consequently, in certain embodiments, toxicology of a biodegradable
polymer intended for in vivo use, such as implantation or injection
into a patient, may be determined after one or more toxicity
analyses. It is not necessary that any subject composition have a
purity of 100% to be deemed biocompatible; indeed, it is only
necessary that the subject Compositions be biocompatible as set
forth above. Hence, a subject composition may comprise polymers
comprising 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or even less
of biocompatible polymers, e.g., including polymers and other
materials and excipients described herein, and still be
biocompatible.
[0071] To determine whether a polymer or other material is
biocompatible, it may be necessary to conduct a toxicity analysis.
Such assays are well known in the art. One example of such an assay
may be performed with live carcinoma cells, such as GT3TKB tumor
cells, in the following manner: the sample is degraded in 1M NaOH
at 37.degree. C. until complete degradation is observed. The
solution is then neutralized with 1M HCl. About 200 mL of various
concentrations of the degraded sample products are placed in
96-well tissue culture plates and seeded with human gastric
carcinoma cells (GT3TKB) at 104/well density. The degraded sample
products are incubated with the GT3TKB cells for 48 hours. The
results of the assay may be plotted as % relative growth vs.
concentration of degraded sample in the tissue-culture well. In
addition, polymers, polymer matrices, and formulations of the
present invention may also be evaluated by well-known in vivo
tests, such as subcutaneous implantations in rats to confirm that
they do not cause significant levels of irritation or inflammation
at the subcutaneous implantation sites.
[0072] Suitable hydrophilic polymers include synthetic polymers
such as poly(ethylene glycol), poly(ethylene oxide), partially or
fully hydrolyzed poly(vinyl alcohol), poly(vinylpyrrolidone),
poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide)
block copolymers (poloxamers and meroxapols), poloxamines,
carboxymethyl cellulose, and hydroxyalkylated celluloses such as
hydroxyethyl cellulose and methylhydroxypropyl cellulose, and
natural polymers such as polypeptides, polysaccharides or
carbohydrates such as Ficoll.TM., polysucrose, hyaluronic acid,
dextran, heparan sulfate, chondroitin sulfate, heparin, or
alginate, and proteins such as gelatin, collagen, albumin, or
ovalbumin or copolymers or blends thereof. As used herein,
"celluloses" includes cellulose and derivatives of the types
described above; "dextran" includes dextran and similar derivatives
thereof.
[0073] Examples of materials which can be used to form a hydrogel
include modified alginates. Alginate is a carbohydrate polymer
isolated from seaweed, which can be crosslinked to form a hydrogel
by exposure to a divalent cation such as calcium, as described, for
example in WO 94/25080, the disclosure of which is incorporated
herein by reference. Alginate is ionically crosslinked in the
presence of divalent cations, in water, at room temperature, to
form a hydrogel matrix. Modified alginate derivatives may be
synthesized which have an improved ability to form hydrogels. The
use of alginate as the starting material is advantageous because it
is available from more than one source, and is available in good
purity and characterization. As used herein, the term "modified
alginates" refers to chemically modified alginates with modified
hydrogel properties. Naturally occurring alginate may be chemically
modified to produce alginate polymer derivatives that degrade more
quickly. For example, alginate may be chemically cleaved to produce
smaller blocks of gellable oligosaccharide blocks and a linear
copolymer may be formed with another preselected moiety, e.g.
lactic acid or epsilon-caprolactone. The resulting polymer includes
alginate blocks which permit ionically catalyzed gelling, and
oligoester blocks which produce more rapid degradation depending on
the synthetic design. Alternatively, alginate polymers may be used
wherein the ratio of mannuronic acid to guluronic acid does not
produce a film gel, which are derivatized with hydrophobic,
water-labile chains, e.g., oligomers of epsilon-caprolactone. The
hydrophobic interactions induce gelation, until they degrade in the
body.
[0074] Additionally, polysaccharides which gel by exposure to
monovalent cations, including bacterial polysaccharides, such as
gellan gum, and plant polysaccharides, such as carrageenans, may be
crosslinked to form a hydrogel using methods analogous to those
available for the crosslinking of alginates described above.
Polysaccharides which gel in the presence of monovalent cations
form hydrogels upon exposure, for example, to a solution comprising
physiological levels of sodium. Hydrogel precursor solutions also
may be osmotically adjusted with a nonion, such as mannitol, and
then injected to form a gel.
[0075] Polysaccharides that are very viscous liquids or are
thixotropic, and form a gel over time by the slow evolution of
structure, are also useful. For example, hyaluronic acid, which
forms an injectable gel with a consistency like a hair gel, may be
utilized. Modified hyaluronic acid derivatives are particularly
useful. As used herein, the term "hyaluronic acids" refers to
natural and chemically modified hyaluronic acids. Modified
hyaluronic acids may be designed and synthesized with preselected
chemical modifications to adjust the rate and degree of
crosslinking and biodegradation. For example, modified hyaluronic
acids may be designed and synthesized which are esterified with a
relatively hydrophobic group such as propionic acid or benzylic
acid to render the polymer more hydrophobic and gel-forming, or
which are grafted with amines to promote electrostatic
self-assembly. Modified hyaluronic acids thus may be synthesized
which are injectable, in that they flow under stress, but maintain
a gel-like structure when not under stress. Hyaluronic acid and
hyaluronic derivatives are available from Genzyme, Cambridge, Mass.
and Fidia, Italy.
[0076] Other polymeric hydrogel precursors include polyethylene
oxide-polypropylene glycol block copolymers such as Pluronics.TM.
or Tetronics.TM., which are crosslinked by hydrogen bonding and/or
by a temperature change, as described in Steinleitner et al.,
Obstetrics & Gynecology, vol. 77, pp. 48-52 (1991); and
Steinleitner et al., Fertility and Sterility, vol. 57, pp. 305-308
(1992). Other materials which may be utilized include proteins such
as fibrin, collagen and gelatin. Polymer mixtures also may be
utilized. For example, a mixture of polyethylene oxide and
polyacrylic acid which gels by hydrogen bonding upon mixing may be
utilized. In one embodiment, a mixture of a 5% w/w solution of
polyacrylic acid with a 5% w/w polyethylene oxide (polyethylene
glycol, polyoxyethylene) 100,000 can be combined to form a gel over
the course of time, e.g., as quickly as within a few seconds.
[0077] Water soluble polymers with charged side groups may be
crosslinked by reacting the polymer with an aqueous solution
containing ions of the opposite charge, either cations if the
polymer has acidic side groups or anions if the polymer has basic
side groups. Examples of cations for cross-linking of the polymers
with acidic side groups to form a hydrogel are monovalent cations
such as sodium, divalent cations such as calcium, and multivalent
cations such as copper, calcium, aluminum, magnesium, strontium,
barium, and tin, and di-, tri- or tetra-functional organic cations
such as alkylammoniuim salts. Aqueous solutions of the salts of
these cations are added to the polymers to form soft, highly
swollen hydrogels and membranes. The higher the concentration of
cation, or the higher the valence, the greater the degree of
cross-linking of the polymer. Additionally, the polymers may be
crosslinked enzymatically, e.g., fibrin with thrombin.
[0078] Suitable ionically crosslinkable groups include phenols,
amines, imines, amides, carboxylic acids, sulfonic acids and
phosphate groups. Negatively charged groups, such as carboxylate,
sulfonate and phosphate ions, can be crosslinked with cations such
as calcium ions. The crosslinking of alginate with calcium ions is
an example of this type of ionic crosslinking. Positively charged
groups, such as ammonium ions, can be crosslinked with negatively
charged ions such as carboxylate, sulfonate and phosphate ions.
Preferably, the negatively charged ions contain more than one
carboxylate, sulfonate or phosphate group.
[0079] The preferred anions for cross-linking of the polymers to
form a hydrogel are monovalent, divalent or trivalent anions such
as low molecular weight dicarboxylic acids, for example,
terepthalic acid, sulfate ions and carbonate ions. Aqueous
solutions of the salts of these anions are added to the polymers to
form soft, highly swollen hydrogels and membranes, as described
with respect to cations.
[0080] A variety of polycations can be used to complex and thereby
stabilize the polymer hydrogel into a semi-permeable surface
membrane. Examples of materials that can be used include polymers
having basic reactive groups such as amine or imine groups, having
a preferred molecular weight between 3,000 and 100,000, such as
polyethylenimine and polylysine. These are commercially available.
One polycation is poly(L-lysine); examples of synthetic polyamines
are: polyethyleneimine, poly(vinylamine), and poly(allyl amine).
There are also natural polycations such as the polysaccharide,
chitosan.
[0081] Polyanions that can be used to form a semi-permeable
membrane by reaction with basic surface groups on the polymer
hydrogel include polymers and copolymers of acrylic acid,
methacrylic acid, and other derivatives of acrylic acid, polymers
with pendant SO.sub.3H groups such as sulfonated polystyrene, and
polystyrene with carboxylic acid groups. These polymers can be
modified to contain active species polymerizable groups and/or
ionically crosslinkable groups. Methods for modifying hydrophilic
polymers to include these groups are well known to those of skill
in the art.
[0082] The polymers may be intrinsically biodegradable, but are
preferably of low biodegradability (for predictability of
dissolution) but of sufficiently low molecular weight to allow
excretion. The maximum molecular weight to allow excretion in human
beings (or other species in which use is intended) will vary with
polymer type, but will often be about 20,000 daltons or below.
Usable, but less preferable for general use because of intrinsic
biodegradability, are water-soluble natural polymers and synthetic
equivalents or derivatives, including polypeptides,
polynucleotides, and degradable polysaccharides.
[0083] The polymers can be a single block with a molecular weight
of at least 600, preferably 2000 or more, and more preferably at
least 3000. Alternatively, the polymers can include can be two or
more water-soluble blocks which are joined by other groups. Such
joining groups can include biodegradable linkages, polymerizable
linkages, or both. For example, an unsaturated dicarboxylic acid,
such as maleic, fumaric, or aconitic acid, can be esterified with
hydrophilic polymers containing hydroxy groups, such as
polyethylene glycols, or amidated with hydrophilic polymers
containing amine groups, such as poloxamines.
Covalently Crosslinkable Polymer Solutions
[0084] Covalently crosslinkable hydrogel precursors also are
useful. For example, a water soluble polyamine, such as chitosan,
can be cross-linked with a water soluble diisothiocyanate, such as
polyethylene glycol diisothiocyanate. The isothiocyanates will
react with the amines to form a chemically crosslinked gel.
Aldehyde reactions with amines, e.g., with polyethylene glycol
dialdehyde also may be utilized. A hydroxylated water soluble
polymer also may be utilized.
[0085] Alternatively, polymers may be utilized which include
substituents which are crosslinked by a radical reaction upon
contact with a radical initiator. For example, polymers including
ethylenically unsaturated groups which can be photochemically
crosslinked may be utilized, as disclosed in WO 93/17669, the
disclosure of which is incorporated herein by reference. In this
embodiment, water soluble macromers that include at least one water
soluble region, a biodegradable region, and at least two free
radical-polymerizable regions, are provided. The macromers are
polymerized by exposure of the polymerizable regions to free
radicals generated, for example, by photosensitive chemicals and or
light. Examples of these macromers are PEG-oligolactyl-acrylates,
wherein the acrylate groups are polymerized using radical
initiating systems, such as an eosin dye, or brief exposure to
ultraviolet or visible light. Additionally, water soluble polymers
which include cinnamoyl groups which may be photochemically
crosslinked may be utilized, as disclosed in Matsuda et al., ASAIO
Trans., vol. 38, pp. 154-157 (1992).
[0086] The term "active species polymerizable group" is defined as
a reactive functional group that has the capacity to form
additional covalent bonds resulting in polymer interlinking upon
exposure to active species. Active species include free radicals,
cations, and anions. Suitable free radical polymerizable groups
include ethylenically unsaturated groups (i.e., vinyl groups) such
as vinyl ethers, allyl groups, unsaturated monocarboxylic acids,
unsaturated dicarboxylic acids, and unsaturated tricarboxylic
acids. Unsaturated monocarboxylic acids include acrylic acid,
methacrylic acid and crotonic acid. Unsaturated dicarboxylic acids
include maleic, fumaric, itaconic, mesaconic or citraconic acid. In
one embodiment, the active species polymerizable groups are
preferably located at one or more ends of the hydrophilic polymer.
In another embodiment, the active species polymerizable groups are
located within a block copolymer with one or more hydrophilic
polymers forming the individual blocks. The preferred polymerizable
groups are acrylates, diacrylates, oligoacrylates, dimethacrylates,
oligomethacrylates, and other biologically acceptable
photopolymerizable groups. Acrylates are the most preferred active
species polymerizable group.
[0087] In general, the polymers are at least partially soluble in
aqueous solutions, such as water, buffered salt solutions, or
aqueous alcohol solutions. Methods for the synthesis of the other
polymers described above are known to those skilled in the art.
See, for example Concise Encyclopedia of Polymer Science and
Polymeric Amines and Ammonium Salts, E. Goethals, editor (Pergamen
Press, Elmsford, N.Y. 1980). Many polymers, such as poly(adylic
acid), are commercially available.
[0088] Naturally occurring and synthetic polymers may be modified
using chemical reactions available in the art and described, for
example, in March, "Advanced Organic Chemistry," 4th Edition, 1992,
Wiley-Interscience
[0089] Publication, New York. Such methods may be used to, for
example, introduce acrylate groups as described herein.
[0090] Preferably, the hydrophilic polymers that include active
species or crosslinkable groups include at least 1.02 polymerizable
or crosslinkable groups on average, and, more preferably, each
includes two or more polymerizable or crosslinkable groups on
average. Because each polymerizable group will polymerize into a
chain, crosslinked hydrogels can be produced using only slightly
more than one reactive group per polymer (i,e., about 1.02
polymerizable groups on average). However, higher percentages are
preferable, and excellent gels can be obtained in polymer mixtures
in which most or all of the molecules have two or more reactive
double bonds. Poloxamines, an example of a hydrophilic polymer,
have four arms and thus may readily be modified to include four
polymerizable groups.
Source of Cells
[0091] The hydrogel can be used for delivery of cells. Cells can be
obtained directed from a donor, from cell culture of cells from a
donor, or from established cell culture lines. In the preferred
embodiment, cells of the same species and preferably same
immunological profile are obtained by biopsy, either from the
patient or a close relative, which are then grown to confluence in
culture using standard conditions and used as needed. If cells that
are likely to elicit an immune reaction are used, such as human
muscle cells from immunologically distinct individual, then the
recipient can be immunosuppressed as needed, for example, using a
schedule of steroids and other immunosuppressant drugs such as
cyclosporine. However, in the most preferred embodiment, the cells
are autologaus. Cells may also be obtained from the blood of the
patient, for example by apheresis.
[0092] In the preferred embodiments, cells are obtained directly
from a donor, washed and implanted directly in combination with the
polymeric material. The cells are cultured using techniques known
to those skilled in the art of tissue culture. Cells obtained by
biopsy are harvested and cultured, passaging as necessary to remove
contaminating cells. Isolation of chondrocytes and muscle cells is
demonstrated in WO 94/25080, the disclosure of which is
incorporated herein.
[0093] Cell attachment and viability can be assessed using scanning
electron microscopy, histology, and quantitative, assessment with
radioisotopes. The function of the implanted cells can be
determined using a combination of the above-techniques and
functional assays. For example, in the case of hepatocytes, in vivo
liver function studies can be performed by placing a cannula into
the recipient's common bile duct. Bile can then be collected in
increments. Bile pigments can be analyzed by high pressure liquid
chromatography looking for underivatized tetrapyrroles or by thin
layer chromatography after being converted to azodipyrroles by
reaction with diazotized azodipyrroles ethylanthranilate either
with or without treatment with P-glucuronidase. Diconjugated and
monoconjugated bilirubin can also be determined by thin layer
chromatography after alkalinemethanolysis of conjugated bile
pigments. In general, as the number of functioning transplanted
hepatocytes increases, the levels of conjugated bilirubin will
increase. Simple liver function tests can also be done on blood
samples, such as albumin production.
[0094] Analogous organ function studies can be conducted using
techniques known to those skilled in the art, as required to
determine the extent of cell function after implantation. For
example, islet cells of the pancreas may be delivered in a similar
fashion to that specifically used to implant hepatocytes, to
achieve glucose regulation by appropriate secretion of insulin to
cure diabetes. Other endocrine tissues can also be implanted.
Studies using labelled glucose as well as studies using protein
assays can be performed to quantitate cell mass on the polymer
scaffolds. These studies of cell mass can then be correlated with
cell functional studies to determine what the appropriate cell mass
is. In the case of chondrocytes, function is defined as providing
appropriate structural support for the surrounding attached
tissues.
[0095] This technique can be used to provide multiple cell types,
including genetically altered cells, within a three-dimensional
scaffolding for the efficient transfer of large number of cells and
the promotion of transplant engraftment for the purpose of creating
a new tissue or tissue equivalent. It can also be used for
immunoprotection of cell transplants while a new tissue or tissue
equivalent is growing by excluding the host immune system.
[0096] Examples of cells which can be implanted as described herein
include chondrocytes and other cells that form cartilage,
osteoblasts and other cells that form bone, muscle cells,
fibroblasts, and organ cells. As used herein, "organ cells"
includes hepatocytes, islet cells, cells of intestinal origin,
cells derived from the kidney, and other cells acting primarily to
synthesize and secret, or to metabolize materials.
Biologically Active Materials Added
[0097] The hydrogel can be used for drug delivery. Examples of
materials to be incorporated into hydrogels are proteins,
polysaccharides, nucleic acid molecules, and synthetic organic or
inorganic molecules. These may be useful for therapeutic,
prophylactic or diagnostic purposes. Drugs may include antibiotics,
antivirals, chemotherapeutic agents, anti-angiogenic agents,
hormones, drugs having an effect on vascular flow,
anti-inflammatories, and many others routinely used.
[0098] The polymeric matrix can be combined with humoral factors to
promote cell transplantation and engraftment. For example, the
polymeric matrix can be combined with angiogenic factors,
antibiotics, antiinflammatories, growth factors, compounds which
induce differentiation, and other factors which are known to those
skilled in the art of cell culture.
[0099] For example, humoral factors could be mixed in a
slow-release form with the cell-polymer suspension prior to
formation of implant or transplantation. Alternatively, the
hydrogel could be modified to bind humoral factors or signal
recognition sequences prior to combination with isolated cell
suspension.
Blends of Ionically and Covalently Crosslinkable Polymers
[0100] In a preferred embodiment, the polymer solution is formed of
two or more polymers, which crosslink to form a
semi-interpenetrating network. For example, the blend could include
PEO, which is ionically crosslinkable, and diamethacrylated PEO, in
a range of between 10 and 40% by weight covalently crosslinkable
polymer in the preferred embodiment. Alternatively, blends of two
covalently crosslinkable polymers can be used, selected on the
basis that they form a network of crosslinked homopolymers, not to
each other. Advantages of the semi-interpenetrating networks
include that the diffusion of non-crosslinked polymer can provide
advantages degradation properties, and enhance mechanical
properties, especially for use in plastic surgery.
Cell Suspensions
[0101] Preferably the polymer is dissolved in an aqueous solution,
preferably a 0.1 M potassium phosphate solution, at physiological
pH, to a concentration forming a polymeric hydrogel. The isolated
cells are suspended in the solution to a concentration of between 1
and 50 million cells/ml, most preferably between 10 and 20 million
cells/ml.
Methods of Implantation
[0102] In a preferred embodiment, the molecules or cells to be
delivered are mixed with the polymerizble agent and injected
directly into a site where it is desired to implant the molecules
or cells, prior to crosslinking of the polymer to form the
hydrogel.
[0103] The site, or sites, where molecules or cells are to be
injected is determined based on individual need, as is the
requisite amount of molecules or number of cells. For cells having
organ function, for example, hepatocytes or islet cells, the
mixture can be injected into the mesentery, subcutaneous tissue,
retroperitoneum, properitoneal space, and intramuscular space. For
formation of cartilage, the cells are injected into the site where
cartilage formation is desired. One could also apply an external
mold to shape the injected solution. Additionally, by controlling
the rate of polymerization, it is possible to mold the
cell-hydrogel injected implant like one would mold clay.
Alternatively, the mixture can be injected into a mold, the
hydrogel allowed to harden, then the material implanted.
[0104] The suspension can be injected via a syringe and needle
directly into a specific area wherever a bulking agent is desired,
i.e., a soft tissue deformity such as that seen with areas of
muscle atrophy due to congenital or acquired diseases or secondary
to trauma, burns, and the like. An example of this would be the
injection of the suspension in the upper torso of a patient with
muscular atrophy secondary to nerve damage.
[0105] The suspension can also be injected as a bulking agent for
hard tissue defects, such as bone or cartilage defects, either
congenital or acquired disease states, or secondary to trauma or
burns. An example of this would be an injection into the area
surrounding the skull where a bony deformity exists secondary to
trauma. The injunction in these instances can be made directly into
the needed area with the use of a needle and syringe under local or
general anesthesia.
[0106] The suspension could also be injected percutaneously by
direct palpation, such as by placing a needle inside the vas
deferens and occluding the same with the injected bulking
substance, thus rendering the patient infertile. The suspension
could also be injected through a catheter or needle with
fluoroscopic, sonographic, computed tomography, magnetic resonance
imaging or other type of radiologic guidance. This would allow for
placement or injection of this substance either by vascular access
or percutaneous access to specific organs or other tissue regions
in the body, wherever a bulking agent would be required.
[0107] Further, this substance could be injected through a
laparoscope or thoracoscope to any intraperitoneal or
extraperitoneal or thoracic organ. For example, the suspension
could be injected in the region of the gastroesophageal junction
for the correcting of gastroesophageal reflux. This could be
performed either with a thoracoscope injecting the substance hi the
esophageal portion of the gastroesophageal region, or via a
laparoscope by injecting the substance in the gastric portion of
the gastroesophageal region, or by a combined approach.
[0108] The material can also be used to treat vesicoureteral
reflux. In addition to its use for the endoscopic treatment of
reflux, the system of injectable autologous muscle cell may also be
applicable for the treatment of other medical conditions, such as
urinary and rectal incontinence, dysphonia, plastic reconstruction,
and wherever an injectable permanent biocompatible material is
needed. Methods for using an injectable polymer for delivering
isolated cells via injection are described for example in WO
94/25080.
[0109] In addition to the use of the cell-polymer suspension for
the treatment of reflux and incontinence, the suspension can also
be applied to reconstructive surgery, as well as its application
anywhere in the human body where a biocompatible permanent
injectable material is necessary. The suspension can be injected
endoscopically, for example through a laryngoscope for injection
into the vocal chords for the treatment of dysphonia, or through a
hysteroscope for injection into the fallopian tubes as a method of
rendering the patient infertile, or through a proctoscope, for
injection of the substance in the perirectal sphincter area,
thereby increasing the resistance in the sphincter area and
rendering the patient continent of stool.
[0110] This technology can be used for other purposes. For example,
custom-molded cell implants can be used to reconstruct three
dimensional tissue defects, e.g., molds of human ears could be
created and a chondrocyte-hydrogel replica could be fashioned and
implanted to reconstruct a missing ear. Cells can also be
transplanted in the form of a thee-dimensional structure which
could be delivered via injection.
[0111] The present invention will now be described with reference
to certain instructive, non-limiting examples.
Examples and Testing of Tyrosyl-Mediated Integration
[0112] The present embodiment relates to tissue-initiated
photopolymerization wherein the tissue actively causes
light-induced gelation or photopolymerization and true
tissue-biomaterial integration occurs.
[0113] An exemplary method according to the present invention for
covalently bonding a polymer to an extracellular matrix is
discussed below. Specifically a method for covalently attaching a
polymer to cartilage or "tissue-initiated photopolymerization"
using acrylate-tyrosyl chemistry is depicted in
[0114] FIG. 1. The method comprises the steps of (1) removing
polysaccharides in cartilage to expose the collagen network by
enzyme treatment; (2) in situ generation of tyrosyl radicals by
photo-oxidation of tyrosine residues on collagen with
H.sub.2O.sub.2 under low intensity UV irradiation, thereby priming
the extracellular matrix; and (3) introduction of a macromer
solution of a polymerizable agent and in situ photo-gelation via
tyrosyl radical initiation and UV-excitation. Since the reaction is
initiated by components of the tissue, the macromers are grafted
(covalently react) with the tissue in addition to crosslinking
(gelation) to form a solid, crosslinked network. If enzymatic
treatment or oxidative treatment were performed alone, no hydrogel
was formed when the macromer and cartilage were exposed to light.
However, in some environments the tissue will be minimally
glycosylated, so incorporation may proceed in some instances
without necessity of enzymatic treatment.
[0115] Collagen is a ubiquitous protein in the body, serving both
structural and biological roles, and is thus an ideal target for
biomaterial integration according to the present invention.
Crosslinked Type II collagen in cartilage is responsible for the
impressive tensile and dynamic mechanical properties of the tissue,
making it an ideal target to initiate and anchor a polymer implant.
However, collagen is not the only suitable extracellular matrix
protein for the methods of the present invention: any protein with
significant numbers of exposable tyrosine residues will be
suitable.
EXAMPLE I
[0116] An exemplary in vitro protocol is discussed below. Surfaces
of fresh fetal bovine cartilage chips were treated with
chondroitinase ABC (5 unit/ml, in Tris pH 8.1) at 37.degree. C. for
1 hr. Photo-oxidation of these surfaces was performed for 5 min
with H2O2 (5%) and UV-irradiation (365 nm; 3 mW/cm.sup.2). Excess
H.sub.2O.sub.2 was removed. Pre-argon-bubbled PEODM (poly[ethylene
glycol] dimethacrylate, 15% and 20%, w/v) were added to the
cartilage surfaces without photo-initiators. The reactants
underwent the UV-irradiation (365 nm; 8 mW/cm.sup.2) for 30
min.
[0117] Chemical and morphological analysis was performed on the
cartilage-biomaterial construct to confirm tissue-initiated
polymerization and gel formation (FIG. 2). The superficial layer of
native cartilage is a mixture of proteins, proteoglycans, and
bonded water as seen chemically by Fourier transform infrared
spectroscopy (ATR-FTIR) spectrum d. After enzymatic treatment of
the cartilage surface with chondroitinase ABC, the poly(saccharide)
peaks are diminished while the typical amide protein bands are
further revealed FITR spectrum c suggesting exposure of collagen on
the cartilage surface. Hydrogels were synthesized on the cartilage
surface using both tissue-initiated photopolymerization and a
standard photoinitiating polymerization system. The cartilage
surface at the interface was then analyzed following frozen
fracture. Chemical and morphological analysis of the cartilage
surface using the standard photoinitiating system showed little
difference compared to the enzymatically-treated surface. However,
chemical analysis of the cartilage surface subjected to
tissue-initiated photopolymerization (spectrum a) demonstrates
strong signals from the ether and ester carbonyl groups of the
crosslinked network and the characteristic band of the
poly(ethylene oxide) (PEO) macromer at 945 cm.sup.-1 when compared
to the standard photoinitiating polymerization system (spectrum
b).
[0118] Directed covalent integration potentially provides a more
stable biomaterial-tissue interface, therefore the mechanical
functionality of the attachment was examined. After equilibration
in phosphate-buffered saline for 24 hrs, the tissue-initiated
cartilage-hydrogel complexes created using the tyrosyl-("CartGel"s)
were cut into two pieces, as shown in FIG. 3. The upper half of the
gel-layer was split off and named "Gel-1"; the same sized lower
half of the gel together with its bond cartilage matrix (the
complex) was named "Cart-Gel-2". PEODM gels (15% and 20%, w/v) were
also made on the cartilage surfaces only treated with
chondroitinase ABC (but without H.sub.2O.sub.2-treatment) by the
initiation of 0.05% photoinitiator Irgacure.RTM. D-2959
(Ciba-Giegy) and named "Cart-Gel-3." The percentage of
(radius/thickness).times.radian.times.100% was recorded as the
rotating torsion-strain. The rotation rate was programmed as
3.75.times.10.sup.4 rad/sec (strain: 0.02%/sec) for 500 sec, then
reversing -3.75.times.10.sup.-4 rad/sec for 500 sec, and finally
maintaining 0-rate relaxation for 500 sec. The stress-strain-time
profiles were automatically recorded by computer.
[0119] The results of the mechanical functionality testing are
shown in FIG. 3. FIG. 3A is a schematic of the testing procedure.
FIGS. 3B-D represent the stress-strain-time behavior of 15% gels
and 20% gels. FIGS. 3 E-H represent the rheological behavior of
"Gel-1" (squares), "Cart-Gel-2" (circles), and "Cart-Gel-3"
(triangles). FIG. 3E and 3G illustrates the stress over time of the
samples. FIGS. 3F and 3H plot intersections of various stress and
strain points of the diagram. FIG. 3I indicates the applied
strain-time conditions.
[0120] Gel-1 exhibited standard viscoelastic behavior with high
elasticity and eventual plastic distortion. The hydrogel-cartilage
constructs, Cart-Gel-2, created by tissue-initiated polymerization
exhibited similar viscoelastic behavior, with no evidence of
slipping or failure. However, the hydrogel synthesized on the
cartilage surface using a standard photoinitiating system,
Cart-Gel-3, required little torsional stress to cause slipping on
the cartilage surface. In contrast, the integrated gel is
significantly attached to the tissue surface such that the gel will
reach the limit of plastic deformation before slipping from the
cartilage surface.
EXAMPLE II
[0121] Analysis of the mechanism of the tissue-initiated
polymerization and direct covalent integration according to the
present invention was carried out in a simplified system using pure
collagen protein and hydroxyethyl methacrylate (HEMA). Collagen was
chosen as the target for reaction due to its universal presence and
mechanical stability. Collagen contains tyrosine residues which may
be oxidized to produce the initiating radical for polymerization.
While tyrosine makes up only 0.6% of human Type II collagen, 90% of
these residues are located at the C-terminus, providing a
concentrated source of radicals for polymerization.
[0122] Type II collagen was reacted with H.sub.2O.sub.2 (1%) for 15
min. After the un-reacted H.sub.2O.sub.2 was removed, HEMA (250 mM)
was added and the UV-irradiation (365 nm; 3 mW/cm.sup.2) was
performed for 5 min. The product solution was loaded into the
Sephadex (Amersham) G-25 size exclusion column to remove the excess
HEMA monomer and the potential byproduct of oligo-HEMA. As a
control, the reaction without H.sub.2O.sub.2-oxidation was also
carried out. The purified products were lyophilized for ATR-FTIR
analysis. All the solutions used in this experiment were
pre-argon-bubbled.
[0123] Conjugation of Type II collagen and HEMA was characterized
by ATR-FTIR (FIG. 4). On the left the model reaction is shown, and
on the right the ATR-FTIR spectra are shown. Type II collagen
(Curve "1"), product of Type II collagen reacting with HEMA under
UV-irradiation with (Curve "3") or without (Curve "2")
pre-oxidation. The results indicate that with H.sub.2O.sub.2
oxidation and UV-irradiation, conjugation of collagen-HEMA is
achieved with peaks from HEMA (ester carbonyl at 1718 cm.sup.-1 and
--C--O-- peaks at 1150 cm.sup.-1) and protein (amide peaks I, II,
and III present at 1650 cm.sup.-1, 1550 cm.sup.-1, and 1245
cm.sup.-1) present (Curve 3). Without oxidative treatment,
photo-conjugation did not occur (Curve 2).
[0124] To confirm the mechanism of tissue-initiated polymerization
an electron spin-trapping strategy was developed to isolate and
confirm the protein-centered tyrosyl radical source for
polymerization. Qian et al. demonstrated the ability of tyrosine
residues on protein sequence to form tyrosyl radicals after mild
oxidation (Biochemical Journal, vol. 363; pp. 281-288, (2002)).
[0125] MNP (2-methyl-2-nitrosopropane) was utilized as a spin
trapping agent and probe for the tyrosyl radical using electron
spin resonance (ESR). ESR profiles were recorded using a
spectrometer operating 9.47 GHz with modulation of 100 KHz. All the
solutions used in this experiment were pre-argon-bubbled. Bovine
collagen (Type H, 2 mg/ml), MNP (100 mM) and H2O2 (0.5%) was mixed
and allowed to react for 10 min. Similar mixtures without
H.sub.2O.sub.2 or MNP were simultaneously prepared. The products
were loaded into a Sephadex.RTM. (Amersham) G-25 size-exclusion
column (SEC) to remove the excess H.sub.2O.sub.2, un-reacted MNP
and non-protein spin adducts. A portion of the samples were
directly characterized by ESR; the others were first
non-specifically digested by pronase and then subjected to ESR in
order to determine the site of radical adduct. Cartilage powders
from the superficial layer were treated with chondroitinase ABC.
Photo-oxidation was carried out by immersing the powders into 5%
H.sub.2O.sub.2 in presence of MNP and exposing to the
UV-irradiation (365 nm; 8 mW/cm.sup.2) for 5 min. A portion of
purified products was subjected to ESR; the others were digested by
collagenase II/trypsin, and then subjected by ESR.
[0126] The results of the ESR spin-trapping are depicted
schematically in FIG. 5. Schematic illustration of the MNP/tyrosyl
ESR spin-trapping are on the left and ESR spectra are on the right.
The combination of tyrosine, MNP, horseradish peroxidase (HRP), and
H.sub.2O.sub.2 produced an ESR spectrum with a characteristic
hyperfine coupling (.alpha..sup.N) of the tyrosyl radical, 15.5 G
(spectrum g). A broad ESR line (>10 G) with a large molecular
rotational correlation time, .tau..sub.c, is detected when collagen
is treated with H.sub.2O.sub.2 and MNP (spectrum c), indicating
that the MNP trapping is located on a macromolecule. After
digestion with pronase (spectrum d), the ESR lines are clarified
and demonstrate an .alpha..sup.N of 15.5 G as in spectrum a with
the pure tyrosyl radical. A similar phenomenon is observed when
cartilage tissue is treated with MNP and an oxidative agent
(spectrum e), and subsequent enzymatic digestion (with Type II
Collagenase and trypsin, spectrum f). The resulting ESR curve is
equivalent to the characteristic tyrosyl radical presented in
spectrum a (Type II collagen and H.sub.2O.sub.2, without MNP). The
conditions of Type II collagen and MNP, without
H.sub.2O.sub.2-oxidation are shown in spectrum b. Radicals were not
detected in control curves from collagen treated without
H.sub.2O.sub.2 or without the MNP radical trapper. The ESR data in
this simplified model system confirmed the hypothesized production
of a tyrosyl radical on collagen in cartilage after a mild
oxidative reaction.
[0127] Compatibility of directed covalent attachment is critical
for preventing host tissue damage and for applications where
delivery and encapsulation of cells in the biomaterial is desired.
The biocompatibility of the treatment procedure and directed
biomaterial tissue attachment was investigated by determining
viability of cells in situ in the tissue and of cells encapsulated
in the attached biomaterial. The compatibility of the tissue enzyme
and oxidative treatment was investigated first using a fluorescent
live-dead cell assay. We have been aware that enzymatic treatment
of cartilage does not affect cell viability while merely cutting
the tissue causes adjacent cell death.
[0128] Following the chondroitinase ABC enzymolysis on the defect
surface of a fresh fetal bovine cartilage for 1 hr (37.degree. C.),
the photo-oxidation was performed respectively with 2%, 5% and 10%
H.sub.2O.sub.2 under UV-irradiation (365 nm; 3 mW/cm.sup.2; 5 min).
After clearance of excess H.sub.2O.sub.2, pre-argon-bubbled PEODM
solutions (15%, w/v) were added into the cartilage defect without
photo-initiators. The photo-grafting reaction was allowed to
proceed for 10 min (365 nm UV, 8 mW/cm.sup.2). Then the sticky
liquid-like premature gel was sucked off and replaced by the
mixture of macromer (15% PEODM) together with 2 million/ml Passage
4 bovine chondrocytes and 0.05% (w/w) photo-initiator D-2959 in PBS
(pH 7.4). The photopolymerization/cell-encapsulation was carried
out under UV-irradiation (365 nm, 3 mW/cm.sup.2) for 5 min. The
method is illustrated schematically. in FIG. 6. The cartilage or
cartilage-hydrogel slices from each step described above were
prepared for the qualitative cytotoxicity evaluation by Live/Dead
assay (Molecular Probes, Inc., Eugene, Oreg.). The biocompatibility
of the enzymatic treatment for covalent integration confirmed that
no additional cell death is observed. A dose-response effect of
cell death was observed with oxidative treatment, such that
increasing cell death was observed with increasing H.sub.2O.sub.2
concentration. The H.sub.2O.sub.2 concentration of 2% allows
tissue-initiated photopolymerization and integration to proceed in
a biocompatible manner, without excessive cell death.
Examples and Testing of AldehydeMmediated Integration
[0129] The below examples illustrate the method according to the
present invention.
EXAMPLE III
[0130] FIG. 7 illustrates the aldehyde priming embodiment of the
invention. The method described below may be found in more detail
in Li et al., Macromolecules vol. 36, pp. 2556-2562 (2003), the
entire disclosure of which is hereby incorporated by reference.
[0131] Chondroitin sulfate A sodium salt (CS, 10 g, .about.20 mmol
disaccharide repeating unit) was dissolved in 100 ml phosphate
buffer saline (PBS, pH 7.4), followed by addition of glycidyl
methacrylate (GMA, 3 g, .about.20 mmol) while vigorously stirring
at room temperature for 1 to 15 days. The purification was
performed by anhydrous acetone extraction twice to remove all the
compounds that failed to covalently graft onto CS chains. The
purified products were lyophilized for 48 hrs. The final yield of
chondroitin sulfate methacrylate (CS-MA) product was all 7 to 8
g.
[0132] Six hundred mg of CS-MA (0.8.about.4.2 mmol of adjacent
diol, 70% CS-A, Sigma) and 616 mg of sodium periodate (.about.2.88
mmol, NaIO.sub.4, Sigma) were dissolved together in 10 ml of
de-ionized water and protected from light. The reaction was allowed
to continue for .about.14 hr in dark with vigorous stirring. The
insoluble byproducts were removed with a 0.22 .mu.m filter and the
product was loaded into a Sephadex.RTM. G-25 size exclusion
chromatography (SEC) column, by which the product was purified from
the water-soluble byproducts and un-reacted small molecules. The
product, chondroitin sulfate-aldehyde-methacrylate (CS-ald-MA), was
obtained by lyophilization, with a yield of .about.90%. The
determination of aldehyde substitution degree was performed via a
hydroxylamine hydrochloride titration. The result was 60 to 70%.
The tissue adhesive was formulated by mixing equal volumes (20 pi)
of 25% CS-ald and 40% bovine serum albumin (BSA, Sigma). The
adhesive was used immediately after the formulation and the
reaction was completed in 2 to 5 min with the Schiff-base
mechanism.
[0133] The resulting compound provides a chondroitin with both
aldehyde and methacrylate groups to integrate a surface of an
extracellular matrix with a photopolymerizing polymer, thereby
serving as a priming agent, as illustrated in FIG. 7.
Examples and Testing of Animal Models
EXAMPLE IV
[0134] In a further in vitro illustrative of the method according
to the present invention, chondral (n=4) and osteochondral (n=4)
defects were created on the medial and lateral tibial surface of
bovine knees obtained from an abattoir. Chondral and osteochondral
defects (d=1 cm) were created using a curette and drill at a depth
of approximately 2 and 5 mm, respectively. Saline was injected into
the joint and surrounding tissue to maintain hydration in the
cadaveric leg during simulation. A 1 lb. weight was placed on the
leg to further simulate natural forces and movement.
[0135] A hydrogel implant was applied. The hydrogel solution was
prepared by thoroughly mixing 10% w/v of poly(ethylene) oxide
diacrylate (Shearwater, PEODA) and the photoinitiator,
Irgacure.RTM. 2959 (Ciba, 0.05% w/v final concentration), in
sterile PBS with 100 U/ml of penicillin and 100 ug/m1 streptomycin
(Gibco). TGF-.beta.3 (RDI, 150 ng/ml) was added to the hydrogel
solutions.
[0136] All hydrogel implants remained in the chondral defects after
exposure to overnight mechanical stress (n=4). No hydrogel debris
was observed in the joint space. Fifty percent of the hydrogels in
the osteochondral defects remained in place after simulation. The
hydrogels in the larger osteochondral defects appeared to swell,
causing them to protrude from the defects. Thus, chondral defects
were used as a model to examine cartilage regeneration in the
goat.
EXAMPLE V
[0137] Mesenchymal stem cells (MSCs) were isolated from femoral
aspirates of 3 to 31/2 year old goats and expanded in Mesenchymal
Stem Cell Medium (Clonetics, MSCGM). Passage 3 cells were
trypsinized, washed in serum free medium, and then resuspended in
the hydrogel solution immediately prior to implantation.
[0138] The hydrogel solution was prepared by thoroughly mixing 10%
w/v of poly(ethylene) oxide diacrylate (Shearwater, PEODA) and the
photoinitiator, Irgacure 2959 (Ciba, 0.05% w/v final
concentration), in sterile PBS with 100 U/ml of penicillin and 100
ug/ml streptomycin (Gibco). TGF-.beta.3 (RDI, 150 ng/ml) was added
to the hydrogel solutions and MSCs were resuspended in the polymer
solution at a concentration of 20 million cells/ml and gently mixed
to make a homogeneous suspension.
[0139] Two defects were created on the medial tibial plateau and
one on the lateral femoral condyle on hind limb of two goats (2-3
years old) using a curette. Defects were critical-size (5 mm
diameter) such that natural repair would not occur. Experimental
defects (n=4) were filled with polymer, MSCs (allogeneic),
hyaluronic acid (to enhance viscosity) and TGF-.beta.3. The
cell-polymer suspension was added and polymerized until the defect
was completely filled. The samples were photo-polymerized with a 5
minute exposure to long-wave, 365 nm UV light at 4 mW/cm2
(Acticure). Control defects included an .empty defect and a defect
containing only polymer (n=2). After wound closure, the legs were
cast for two weeks after which the animals were allowed to move ad
lib. Samples were harvested for gross and histological observation
after 2 and 4 weeks of implantation.
[0140] After casts were removed at two weeks post implantation, no
swelling or infection was observed. Before harvesting at one month,
the goat demonstrated active movement without any limb favoring or
lameness. Samples harvested after two weeks grossly showed minimal
evidence of regeneration. However, samples harvested one month
after implantation demonstrated significant evidence of
regeneration. The experimental defect where the photopolymerizing
MSC-hydrogel construct was placed showed significantly more
cartilage tissue in the defect space compared to the control defect
which did not contain a gel. The femoral defects also contained
repair tissue after one month.
[0141] This study demonstrates the application of a
photopolymerizing hydrogel system for cartilage repair according to
the present invention in a large animal model. Cartilage has been
generated from goat MSCs photoencapsulated in a hydrogel and
incubated in vitro. The photopolymerizing hydrogel system has been
applied in vivo in mine and rate but has not been introduced into a
joint environment. Recently allogenic MSCs have been successfully
applied in cardiac repair systems suggesting their possible use for
cartilage repair. Allogeneic MSCs are an ideal cell source since no
additional surgical procedure is required to obtain cells before
hydrogel treatment. This in vivo work did not specifically use any
integration method but is an example of when we would use it.
[0142] While the present invention has been described with
reference to certain preferred embodiments, one of ordinary skill
in the art will recognize that additions, deletions, substitutions,
modifications and improvements can be made while remaining within
the spirit and scope of the present invention as defined by the
appended claims.
[0143] Although cartilage was used as a model tissue in examples I
and II, the embodiments pertaining to those examples will function
in other tissues containing tyrosine residues, the tyrosine
residues being disposed in either collagen or another protein of
the extracellular matrix. Likewise, while H.sub.2O.sub.2 was used
as an oxidizer in examples I and II, other oxidizing agents may be
used, for example vitamin C. Although Example I used poly(ethylene
glycol) dimethacrylate (PEODM) and Example II used hydroxyethyl
methacrylate (HEMA), any polymerizable agent with an acrylate may
be used in those embodiments, as herein disclosed.
[0144] Although Example III used albumin as a carrier, other agents
can be used besides albumin. For example, polyethlyimine (PEI) or
another agent containing amino groups may be used. Further
regarding Example III, although chondroitin sulfate was modified to
produce an agent with aldehyde and methacrylate groups, other
saccharides may also be used. Still further regarding example III,
although the priming agent contained a methacrylate group for
binding to the polymerizable agent, other chemical groups may be
used instead.
[0145] Although Examples IV and V used photoinitiators, the
tyrosyl-acrylate or aldehyde embodiments described herein may be
used. Moreover, use of the invention in living tissue is not
limited to the exact compositions described in the examples.
[0146] Although ultraviolet (UV) light is used in various instances
throughout the examples, visible light or other forms of
electromagnetic radiation may also be used to achieve the same
purposes depending on the chemistry involved.
* * * * *