U.S. patent application number 10/997246 was filed with the patent office on 2005-07-14 for use of hydrophobic crosslinking agents to prepare crosslinked biomaterial compositions.
This patent application is currently assigned to Cohesion Technologies, Inc.. Invention is credited to Rhee, Woonza M..
Application Number | 20050154125 10/997246 |
Document ID | / |
Family ID | 32600839 |
Filed Date | 2005-07-14 |
United States Patent
Application |
20050154125 |
Kind Code |
A1 |
Rhee, Woonza M. |
July 14, 2005 |
Use of hydrophobic crosslinking agents to prepare crosslinked
biomaterial compositions
Abstract
The present invention discloses novel crosslinked biomaterial
compositions which are prepared using hydrophobic polymers as a
crosslinking agent. Preferred hydrophobic polymers are those that
contain two or more reactive succinimidyl groups, including
disuccinimidyl suberate, bix(sulfosuccinimidyl)suberate, and
dithiobis(succinimidylpropionate). Crosslinked biomaterial
compositions prepared using mixtures of hydrophobic and hydrophilic
crosslinking agents are also disclosed. The compositions of the
present invention can be used to prepare formed implants for use in
a variety of medical applications.
Inventors: |
Rhee, Woonza M.; (Palo Alto,
CA) |
Correspondence
Address: |
REED INTELLECTUAL PROPERTY LAW GROUP
1400 PAGE MILL ROAD
PALO ALTO
CA
94304-1124
US
|
Assignee: |
Cohesion Technologies, Inc.
Palo Alto
CA
|
Family ID: |
32600839 |
Appl. No.: |
10/997246 |
Filed: |
November 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10997246 |
Nov 23, 2004 |
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09344230 |
Jun 25, 1999 |
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09344230 |
Jun 25, 1999 |
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08987467 |
Dec 9, 1997 |
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08987467 |
Dec 9, 1997 |
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08403358 |
Mar 14, 1995 |
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Current U.S.
Class: |
525/54.1 |
Current CPC
Class: |
A61L 31/042 20130101;
A61L 2400/06 20130101; A61L 27/20 20130101; A61L 24/08 20130101;
A61L 31/044 20130101; A61F 2310/00365 20130101; A61L 24/08
20130101; Y10S 514/801 20130101; C08L 5/00 20130101; A61L 27/24
20130101; A61L 26/0033 20130101; A61L 31/042 20130101; A61L 27/20
20130101; C08L 5/00 20130101; C08L 5/00 20130101 |
Class at
Publication: |
525/054.1 |
International
Class: |
C08H 001/00 |
Claims
1. A system for preparing a crosslinkable composition that
crosslinks in situ following administration to a patient to form a
heterogenous, crosslinked biomaterial composition, comprising: an
aqueous suspension of a biomaterial comprised of a biocompatible
polymer containing nucleophilic groups; and an admixture of
hydrophilic crosslinking agent and a hydrophobic crosslinking agent
containing up to about 14 carbon atoms and comprised of a polyacid
esterfied with reactive moieties selected from the group consisting
of succinimidyl groups and sulfonsuccinimidyl groups, wherein the
hydrophilici crosslinking agent and the hydrophobic crosslinking
agent are each capable of covalently crosslinking the biomaterial
but are not reactive with respect to each other.
2. A system for preparing an injectable, crosslinkable collagen
composition that crosslinks in situ following administration to a
patient to form a heterogenous, crosslinked collagen composition,
comprising: an aqueous suspension of a biomaterial selected from
the group consisting of fibrillar collagen, succinylated collagen,
methylated collagen, denatured collagen, hyaluronic acid,
chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate,
keratin sulfate, keratosulfate, chitin, chitosan, heparin and
mixtures thereof; a dry admixture of a hydrophilic crosslinking
agent and a hydrophobic crosslinking agent containing up to about
14 carbon atoms and comprised of a polyacid esterified with
reactive moieties selected from the group consisting of
succinimidyl groups and sulfosuccinimidyl groups, wherein the
hydrophilic crosslinking agent and the hydrophobic crosslinking
agent each capable of covalently crosslinking the biomaterial but
are not reactive with respect to each other.
3. A method for introducing an implant of a crosslinked biomaterial
composition into the body of a mammalian patient, comprising: (a)
admixing (i) an aqueous suspension of a biomaterial comprised of a
biocompatible polymer containing nucleophilic groups, (ii) a
hydrophilic crosslinking agent, and (iii) a hydrophobic
crosslinking agent containing up to about 14 carbon atoms and
comprised of a polyacid esterified with reactive moieties selected
from the group consisting of succinimidyl groups and
sulfosuccinimidyl groups, wherein the hydrophilic crosslinking
agent and the hydrophobic crosslinking agent are each capable of
covalently crosslinking the biomaterial but are not reactive with
respect to each other; (b) placing the crosslinkable admixture
prepared in step (a) into the body of the patient; and (c) allowing
the admixture to crosslink in situ.
4. The method of claim 3, wherein step (b) is carried out by
injection.
5. The method of claim 4, wherein the injection is
subcutaneous.
6. The method of claim 5, wherein the subcutaneous injection is at
a dermal site in need of correction.
7. The method of claim 3, wherein step (b) comprises application of
the crosslinkable admixture to a soft tissue site in need of
augmentation.
8. The method of claim 3, wherein step (b) comprises application of
the crosslinkable admixture to a hard tissue site in need of
augmentation.
9. The method of claim 3, wherein the admixture prepared in step
(a) further comprises ceramic materials, and step (b) comprises
application of the crosslinkable admixture to the stie of a bone
defect.
10. The method of claim 3, wherein the admixture prepared in step
(a) further comprises ceramic materials, and step (b) comprises
application of the crosslinkable admixture to the site of a bone
defect.
11. The method of claim 3, wherein the admixture prepared in step
(a) further comprises ceramic materials, and step (b) comprises
application of the crosslinkable admixture to the stei of a
cartilage defect.
12. A crosslinked implant prepared by the process of claim 3.
13. A method for providing a crosslinked, nonimmunogenic
biomaterial coating on the surface of a preformed synthetic
implant, comprising: (a) admixing (i) an aqueous suspension of a
biomaterial comprised of a biocompatible polymer containing
nucleophilic groups, (ii) hydrophilic crosslinking agent, and (iii)
a hydrophobic crosslinking agent containing up to about 14 carbon
atoms and comprised of a polyacid esterified with reactive moieties
selected from the group consisting of succinimidyl groups and
sulfosuccinimidyl groups, wherein the hydrophilic crosslinking
agent and the hydrophobic crosslinking agent are each capable of
covalently crosslinking the biomaterial but are not reactive with
respect to each other; (b) coating a preformed synthetic implant
with the crosslinkable admixture prepared in step (a); and (c)
allowing the coating to crosslink in place.
14. The method of claim 13, wherein step (b) is carried out by
brushing, painting, extrusion, or dipping.
15. A nonimmunogenic implant prepared by the process of claim 13.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to the use of hydrophobic
crosslinking agents to prepare injectable or implantable
crosslinked biomaterial compositions for use in a variety of
therapeutic applications. Specifically, this invention relates to
crosslinked biomaterial compositions prepared using hydrophobic
crosslinking agents containing two or more succinimidyl groups,
such as disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate,
or dithiobis(succinimidylpropi- onate). Also provided are unique
crosslinked biomaterial compositions prepared using mixtures of
hydrophobic and hydrophilic crosslinking agents. The compositions
of the invention are particularly useful in the preparation of
formed implants for a variety of medical uses.
BACKGROUND OF THE INVENTION
[0002] U.S. Pat. No. 5,162,430, issued Nov. 10, 1992, to Rhee et
al., and commonly owned by the assignee of the present application,
discloses collagen-synthetic polymer conjugates and methods of
covalently binding collagen to synthetic hydrophilic polymers.
Commonly owned U.S. Pat. No. 5,328,955, issued Jul. 12, 1994, to
Rhee et al., discloses various activated forms of polyethylene
glycol and various linkages which can be used to produce
collagen-synthetic polymer conjugates having a range of physical
and chemical properties. Commonly owned U.S. Pat. No. 5,324,775,
issued Jun. 28, 1994, to Rhee et al., discloses biocompatible
polymer conjugates prepared by covalently binding biologically
inert, biocompatible polymers to synthetic hydrophilic
polymers.
[0003] Commonly owned U.S. Pat. No. 5,510,418, issued Apr. 23,
1996, discloses conjugates comprising various species of
glycosaminoglycan covalently bound to synthetic hydrophilic
polymers, which are optionally bound to collagen as well. Commonly
owned U.S. Pat. No. 5,565,519, issued Oct. 15, 1996, discloses
collagen-polymer conjugates comprising chemically modified
collagens such as, for example, succinylated collagen or methylated
collagen, covalently bound to synthetic hydrophilic polymers to
produce optically clear materials for use in ophthalmic or other
medical applications.
[0004] Hydrophobic crosslinking agents such as disuccinimidyl
suberate, bis(sulfosuccinimidyl) suberate, and
dithiobis(succinimidylpropionate) have a long history of use for
crosslinking biologically active peptides, as described in the 1992
Pierce (Rockford, Ill.) catalog.
[0005] All publications cited above and herein are incorporated
herein by reference to describe and disclose the subject matter for
which it is cited.
[0006] We now disclose a detailed description of preferred
embodiments of the present invention, including crosslinked
biomaterial compositions prepared using various hydrophobic
crosslinking agents and crosslinked biomaterial compositions
prepared using mixtures of hydrophobic and hydrophilic crosslinking
agents.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1 shows the structural formula for disuccinimidyl
suberate (DSS), and a reaction product obtained by reacting DSS
with collagen.
[0008] FIG. 2 shows the structural formula for
dithiobis(succinimidyl propionate) (DSP), and a reaction product
obtained by reacting DSP with collagen.
[0009] FIG. 3 shows the structural formula for
bis(sulfosuccinimidyl)suber- ate (BS.sup.3), and a reaction product
obtained by reacting BS.sup.3 with collagen.
[0010] FIG. 4 shows the structural formula for
bis(2-succinimidooxycarbony- loxy)ethyl sulfone (BSOCOES), and a
reaction product obtained by reacting BSOCOES with collagen.
[0011] FIG. 5 shows the structural formulas for
3,3'-dithiobis(sulfosuccin- imidyl propionate) (DTSSP), and a
reaction product obtained by reacting DTSSP with collagen.
[0012] FIG. 6 shows a reaction scheme for derivatizing
trimethylpropane to its tricarboxylic acid form, then further
derivatizing this tricarboxylic acid from by reacting it with
N-hydroxysuccinimide (NHS) in the presence of
N,N'-dicyclohexylcarbodiimide (DCC to produce a trifunctional
crosslinking agent.
[0013] FIG. 7 shows a reaction scheme for derivatizing
di(trimethylpropane) to its tetracarboxylic acid form, then further
derivatizing this tetracarboxylic acid form by reacting it with NHS
in the presence of DCC to produce a tetrafunctional crosslinking
agent.
[0014] FIG. 8 is a bar graph illustrating the wet weight recovery
of implants of various compositions at day 7 (solid bars), day 14
(hatched bars, heavy lines), day 28 (stippled bars) and day 90
(hatched bars, light lines).
[0015] FIG. 9 depicts the method used to measure the mechanical
force required to dislodge implants of various compositions from
the surrounding tissues. The arrows indicate the direction of the
applied force.
[0016] FIG. 10 is a graph showing the anchoring force, in newtons,
of implants of various compositions removed at days 7, 14, 28 and
90.
SUMMARY OF THE INVENTION
[0017] In our earlier patents and applications, we disclosed
various crosslinked biomaterial compositions prepared using
synthetic hydrophilic polymers, preferably functionally activated
polyethylene glycols (PEGs), as the crosslinking agent. In
accordance with the present invention, we have since discovered
that various hydrophobic polymers containing two or more
succinimidyl groups, such as disuccinimidyl suberate,
bis(sulfosuccinimidyl) suberate, or
dithiobis(succinimidylpropionate), can be used to crosslink various
biomaterials such as collagen and glycosaminoglycans. We have also
discovered that certain hydrophobic polymers, such as polyacids,
can be derivatized to contain two or more succinimidyl groups and,
in the derivatized form, can be used to crosslink collagen and
glycosaminoglycans. Furthermore, we have discovered that unique
crosslinked biomaterial compositions can be prepared by using a
mixture of hydrophobic and hydrophilic crosslinking agents.
[0018] The present invention pertains to conjugates comprising
biomaterials covalently bonded to hydrophobic polymers, wherein the
hydrophobic polymer contains two or more succinimidyl groups prior
to bonding with the biomaterial. Included in the invention are
conjugates comprising biomaterials covalently bonded to hydrophobic
polymers, in which the hydrophobic polymer has been chemically
derivatized to contain two or more succinimidyl groups prior to
bonding with the biomaterial. Heterogeneous crosslinked biomaterial
compositions are also disclosed which comprise a biomaterial (or
mixtures of different species of biomaterials), a hydrophobic
crosslinking agent, and a hydrophilic crosslinking agent. Further,
in accordance with the invention, formed implants are prepared
using the conjugates and compositions of the invention.
[0019] The compositions of the present invention have many unique
and unexpected features when compared with the previously disclosed
crosslinked biomaterial compositions prepared using only
hydrophilic crosslinking agents. An important feature of the
compositions of the present invention (when compared to previous
crosslinked biomaterial compositions) is slower degradation,
resulting in greater chemical stability, which may lead to
increased in vivo persistence. Additional features and advantages
of the invention will become apparent upon reading the detailed
description of the invention which follows.
[0020] Definitions and Nomenclature
[0021] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an", and "the" include
plural referents unless the context clearly dictates otherwise. For
example, reference to "a conjugate" includes one or more conjugate
molecules, reference to "an article" includes one or more different
types of articles known to those skilled in the art and reference
to "the collagen" includes mixtures of different types of collagens
and so forth.
[0022] Specific terminology of particular importance to the
description of the present invention is defined below:
[0023] The term "atelopeptide collagen" refers to collagens which
have been chemically treated or otherwise processed to remove the
telopeptide regions, which are known to be responsible for causing
an immune response in humans to collagens from other animal, such
as bovine, sources.
[0024] The term "biomaterial" as used herein refers in general to
biocompatible, naturally occurring polymers, including collagen,
gelatin, and polysaccharides such as glycosaminoglycans
[0025] The terms "chemically conjugated" and "conjugated" as used
herein mean attached through a covalent chemical bond. In the
practice of the invention, a hydrophobic polymer and a biomaterial
may be covalently conjugated to each other by means of a reactive
succinimidyl group on the hydrophobic polymer.
[0026] The term "chemical crosslinking agent" as used herein refers
to any chemical agent capable of covalently binding biomaterials,
such as collagen, glycosaminoglycans, and mixtures thereof, to form
a crosslinked biomaterial network.
[0027] The term "collagen" as used herein refers to all types and
forms of collagen, including those which have been recombinantly
produced, extracted from naturally occurring sources (such as
bovine corium or human placenta), processed, or otherwise
modified.
[0028] The term "collagen suspension" refers to a suspension of
noncrosslinked or crosslinked collagen fibers in an aqueous
carrier, such as water or phosphate-buffered saline (PBS)
solution.
[0029] The term "collagen-synthetic polymer" refers to collagen
covalently bonded to a synthetic hydrophilic polymer. For example,
"PEG-collagen" denotes a composition of the invention wherein
molecules of collagen are covalently bonded to molecules of
polyethylene glycol (PEG).
[0030] The term "difunctionally activated refers to synthetic
hydrophilic polymer molecules which have been, chemically
derivatized so as to have two functional groups capable of reacting
with primary amino groups on biocompatible polymer molecules, such
as collagen or deacetylated glycosaminoglycans. The two functional
groups on a difunctionally activated synthetic hydrophilic polymer
are generally located at opposite ends of the polymer chain. Each
functionally activated group on a d difunctionally activated
synthetic hydrophilic polymer molecule is capable of covalently
binding with a biocompatible polymer molecule, thereby effecting
crosslinking between the biocompatible polymer molecules.
[0031] The term "dry" means that substantially all unbound water
has been removed from a material.
[0032] The term "fibrillar collagen" refers to collagens in which
the triple helical molecules aggregate to form thick fibers due to
intermolecular charge and hydrophobic interactions.
[0033] The term "functionally activated" refers to synthetic
hydrophilic polymers which have been chemically derivatized so as
to have one or more functional group capable of reacting with
primary amino groups on biocompatible polymer molecules.
[0034] The term "in situ" as used herein means at the site of
administration.
[0035] The term "in situ crosslinking" as used herein refers to
crosslinking of a biocompatible polymer implant following
implantation to a tissue site on a human or animal subject, wherein
at least one functional group on the synthetic polymer is
covalently conjugated to a biocompatible polymer molecule in the
implant, and at least one functional group on the synthetic polymer
is free to covalently bind with other biocompatible polymer
molecules within the implant, or with collagen molecules within the
patient's own tissue.
[0036] The term "molecular weight" as used herein refers to the
weight average molecular weight of a number of molecules in any
given sample, as commonly used in the art. Thus, a sample of PEG
2000 might contain a statistical mixture of polymer molecules
ranging in weight from for example, 1500 to 2500, with one molecule
differing slightly from the next over a range. Specification of a
range of molecular weight indicates that the average molecular
weight may be any value between the limits specified, and may
include molecules outside those limits. Tlius, a molecular weight
range of about 800 to about 20,000 indicates an average molecular
weight of at least about 800, ranging up to about 20,000.
[0037] The term "multifunctionally activated" refers to synthetic
hydrophilic polymers which have been chemically derivatized so as
to have two or more functional groups which are located at various
sites along the polymer chain and are capable of reacting with
primary amio groups on biocompatible polymer molecules. Each
functional group on a multifunctionally activated synthetic
hydrophilic polymer molecule is capable of covalently binding with
a biocompatible polymer molecule, thereby effecting crosslinking
between the biocompatible polymer molecules. Types of
multifunctionally activated hydrophilic synthetic polymers include
difunctionally activated, tetrafunctionally activated, and
star-branched polymers.
[0038] The term "noncrosslinked collagen" refers to collagens that
have not been previously crosslinked using chemical crosslinking
agents. Such noncrosslinked collagens may include both fibrillar
and nonfibrillar collagens.
[0039] The term "nonfibrillar collagen" refers to collagens in
which the triple helical molecules do not aggregate to form thick
fibers, such that a composition containing nonfibrillar collagen
will be optically clear.
[0040] The terms "synthetic hydrophilic polymer" or "synthetic
polymee" refer to polymers which have been synthetically produced
and which are hydrophilic, but not necessarily water-soluble.
Examples of synthetic hydrophilic polymers which can be used in the
practice of the present invention are polyethylene glycol (PEG),
polyoxyethylene, polymethylene glycol, poly-trimethylene glycols,
polyvinylpyrrolidones, polyoxyethylene-polyoxypropylene block
polymers and copolymers, and derivatives thereof. Naturally
occurring polymers such as proteins, starch, cellulose, heparin,
hyaluronic acid, and derivatives thereof are expressly excluded
from the scope of this definition.
[0041] The term "tissue augmentation" as used herein refers to the
replacement or repair of defects in the soft or hard tissues of a
human body.
[0042] Except as otherwise defined above, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar or
equivalent to those described herein may be useful in the practice
or testing of the present invention, only the preferred methods and
materials are described below.
[0043] In accordance with the present invention, unique crosslinked
biomaterial compositions are prepared using hydrophobic
crosslinking agents, or mixtures of, hydrophilic and hydrophobic
crosslinking agents. In order to prepare the crosslinked
biomaterial compositions of the present invention, it is first
necessary to provide one or more biomaterials and a hydrophobic
crosslinking agent.
[0044] Biomaterials
[0045] Any biomaterial that has, or can be chemically derivatized
to have, primary amino (--NH.sub.2) groups capable of binding with
hydrophobic or hydrophilic crosslinking agents according to the
methods of the invention may be used to prepare the crosslinked
biomaterial compositions of the invention. Preferred biomaterials
for use in the practice of the present invention include all types
of collagen and glycosaminoglycans, and mixtures thereof.
[0046] In general, collagen from any source may be used in the
practice of the present invention; for example, collagen may be
extracted and purified from human or other mammalian source, or may
be recombinantly or otherwise produced. Collagen of any type,
including, but not limited to, types I, II, I, IV, or any
combination thereof, may be used, although type I is generally
preferred. Either atelopeptide or telopeptide-containing collagen
may be used; however, when collagen from a xenogeneic source, such
as bovine collagen, is used, atelopeptide collagen is generally
preferred, because of its reduced immunogenicity compared to
telopeptide-containing collagen. The collagen should be in a
pharmaceutically pure form such that it can be incorporated into a
human body without generating any significant immune response.
[0047] Collagens for use in the present invention may be in the
fibrillar or nonfibrillar form. Fibrillar collagens are generally
preferred for tissue augmentation applications due to their
increased persistence in vivo. Nonfibrillar collagens, including
chemically modified collagens such as succinylated or methylated
collagen, may be preferable in certain situations, such as
ophthalmic applications where an optically transparent material is
required. Succinylated and methylated collagens can be prepared
according to the methods described in U.S. Pat. No. 4,164,559
(which is hereby incorporated by reference in its entirety).
Noncrosslinked collagens for use in the present invention are
normally in aqueous suspension at a concentration between about 20
mg/ml to about 120 mg/ml, preferably, between ibout 30 mg/ml to
about 80 mg/ml. Fibrillar collagen in suspension at various
collagen concentrations is commercially available from Collagen
Corporation under the trademark Zyderm.RTM. I Collagen (35 mg/ml)
and Zyderm II Collagen (65 mg/ml).
[0048] Collagen in its native state contains lysine residues having
primary amino groups capable of covalently binding with the
hydrophobicand hydrophilic crosslinking agents of the invention and
therefore need not be chemically modified in any way prior to
reaction with the desired crosslinking agent according to the
methods of the invention.
[0049] Although intact collagen is preferred, denatured collagen,
commonly known as gelatin, can also be used in the preparation of
the compositions of the invention.
[0050] Glycosaminoglycans for use in the present inventioninclude,
without limitation, hyaluronic: acid, chondroitin sulfate A,
chondroitin sulfate C, dermatan sulfate, keratan sulfate,
keratosulfate, chitin, chitosan, heparin, and derivatives or
mixtures thereof. Glycosaminoglycans must generally be modified,
such as by deacetylation or desulfation, in order to provide
primary amino groups capable of binding-with functional groups on
hydrophobic and hydrophilic crosslinking agent according to the
methods of the present invention. Methods for chemically modifying
glycosaminoglycans by deacetylation and/or desulfation are
described in commonly owned U.S. Pat. No. 5,510,418, issued Apr.
23, 1996. In general, glycosaminoglycans can be deacetylated,
desulfated, or both, as applicable, by the addition of a strong
base, such as sodium hydroxide, to the glycosaminoglycan.
Deacetylation and/or desulfation provides primary amino groups on
the glycosaminoglycan which are capable of covalently binding with
hydrophobic or hydrophilic crosslinking agents according to the
methods of the present invention.
[0051] Mixtures of various species of glycosaminoglycan, various
types of collagen, or mixtures of various glycosaminoglycans with
collagen may be used to prepare the crosslinked biomaterial
compositions of the present invention.
[0052] If the final composition is intended for incorporation into
the body of a human or animal subject, biomaterials for use in the
present invention must be in pharmaceutically pure form, or capable
of being purified to be in pharmaceutically pure form.
[0053] Preparation of Hydrophobic Crosslinking Agents
[0054] In order to prepare the crosslinked biomaterial compositions
of the present invention, it is first necessary to provide a
hydrophobic polymer which contains, or can be derivatized to
contain, two or more succinimidyl groups. As used herein, the term
"hydrophobic polymer" refers to polymers which contain a relatively
small proportion of oxygen or nitrogen atoms. As used herein, the
term "containing two or more succinimidyl groups" is meant to
encompass hydrophobic polymers which are commercially available
containing two or more succinimidyl groups, as well as those that
must be chemically derivatized to contain two or more succinimidyl
groups. As used herein, the tenn "succmmudyl group" is intended to
encompass sulfosuccinimidyl groups and other such variations on the
"generic" succinimidyl group. The presence of the sodium sulfate
moiety on the sulfosuccinimidyl group serves to increase the
solubility of the polymer.
[0055] Hydrophobic polymers for use in the present invention
preferably contain, or can be derivatized to contain, two or more
succinimidyl groups, most preferably, two, three, or four
succinimidyl groups. These succinimidyl groups are highly reactive
with biomaterials containing primary amino (--NH.sub.2) groups,
such as collagen and various glycosaminoglycans and
glycosaminoglycan derivatives.
[0056] Hydrophobic polymers which already contain two or more
reactive succinimidyl groups include, without limitation,
disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate
(BS.sup.3) dithiobis(succinimidylpr- opionate) (DSP),
bis(2-succinimidooxycarbonyloxy)ethyl sulfone (BSOCOES), and
3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSPP), and their
analogs and derivatives. The above-referenced polymers are
commercially available from Pierce (Rockford, Ill.), under catalog
Nos. 21555, 21579, 22585, 21554, and 21577, respectively.
Structural formulas for the above-referenced polymers, as well as
generalized reaction products obtained by reacting each of these
polymers with collagen, are shown in FIGS. 1 to 5,
respectively.
[0057] Certain polymers, such as polyacids, can by derivatized to
contain two or more reactive succinimidyl groups. Polyacids for use
in the present invention include, without limitation,
trimethylolpropane-based tricarboxylic acid, di(trimethylol
propane)-based tetracarboxylic acid, heptanedioic acid, octanedioic
acid (suberic acid), and hexadecanedioic acid (thapsic acid). Many
of these polyacids are commercially available from DuPont Chemical
Company.
[0058] According to a general method, polyacids can be chemically
derivatized to contain two or more succinimidyl groups by reaction
with an appropriate molar amount of N-hydroxysuccinimide (NHS) in
the presence of N,N'-dicyclohexylcarbodiimide (DCC).
[0059] Polyalcohols such as trimethylolpropane and di(trimethylol
propane) can be converted to carboxylic acid form using various
methods, then further derivatized by the addition of succinimidyl
groups, as shown in FIGS. 6 and 7.
[0060] Trimethylolpropane can be derivatized to tricarboxylic acid
form, then further derivatized by reaction with NHS in the presence
of DCC to produce a trifunctional crosslinking agent (i.e., a
compound having three succinimidyl groups available for reaction
with various biomaterials), as shown in FIG. 6.
[0061] Di(trimethylol propane) can be derivatized to
tetracarboxylic acid form, then further derivatized by reaction
with NHS in the presence of DCC to produce a tetrafunctional
crosslinking agent, as shown in FIG. 7.
[0062] Other polyacids can be chemically derivatized to contain two
or more reactive succinimidyl groups using methods similar to those
shown in FIGS. 6 and 7 for trimethylolpropane-based tricarboxylic
acid and di(trimethylolpropane)-based tetracarboxylic acid,
respectively. Polyacids; such as heptanedioic acid
(HOOC(CH.sub.2).sub.5--COOH), octanedioic acid
(HOOC--(CH.sub.2)6--COOH), and hexadecanedioic acid
(HOOC--(CH.sub.2).sub.14--COOH) are derivatized by the addition of
succinimidyl groups to produce difunctional crosslinking
agents.
[0063] Polyamines such as ethylenediamine (H.sub.2N--CH.sub.2
CH.sub.2--NH.sub.2), tetramethylenediamine
(H.sub.2N--(CH.sub.2).sub.4--N- H.sub.2), pentamethylenediamine
(cadaverine) (H.sub.2N--(CH.sub.2).sub.5--- NH.sub.2),
hexamethylenediarnine (H.sub.2N--(CH.sub.2).sub.6--NH.sub.2),
bis(2-hydroxyethyl)amine (HN--(CH.sub.2CH.sub.2OH).sub.2),
bis(2)aminoethyl)amine (HN--(CH.sub.2CH.sub.2NH.sub.2).sub.2), and
tris(2-aminoethyl)amine (N--(CH.sub.2CH.sub.2NH.sub.2).sub.3) can
be chemically derivatized to polyacids, which can then be
derivatized to contain two or more succinimidyl groups by reacting
with the appropriate molar amounts of N-hydroxysuccinimide in the
presence of DCC according to the general method described above for
polyacids. Many of these polyamines are commercially available from
DuPont Chemical Company.
[0064] Preferred hydrophobic polymers for use in the invention,
whether they are commercially available containing two or more
succinimidyl groups or must be chemically derivatized to contain
two or more succinimidyl groups, generally have a carbon chain that
is no longer than about 14 carbons. Polymers having carbon chains
substantially longer than 14 carbons generally have very poor
solubility in aqueous solutions and, as such, have very long
reaction times when mixed with an aqueous solution of a biomaterial
such as collagen.
Preparation of Crosslinked Biomaterial Compositions Using
Hydrophobic Crosslinking Agents
[0065] In a general method for preparing the crosslinked
biomaterial compositions of the invention, a biomaterial which
contains, or has been chemically derivatized to contain, primary
amino groups is mixed with a hydrophobic polymer which contains, or
has been derivatized to contain, two or more succinimidyl groups
capable of crosslinking the biomaterial by reacting with
nucleophilic primary amino groups on the biomaterial. The
hydrophobic crosslinking agent can be stored and used in either dry
form or in solution, but is preferably used in dry form. The
crosslinking agent may be mixed with either an aqueous solvent or a
hydrophobic solvent prior to mixing with the biomaterial. If an
aqueous solvent is used, the crosslinking agent should be mixed
with the solvent just prior to use, as the succinimidyl groups are
reactive with neutrophiles such as oxygen and water. Exposure to
aqueous solvents for extended periods of time will result in loss
of crosslinking ability due to hydrolysis of the crosslinking
agent.
[0066] The biomaterial and hydrophobic crosslinking agent (in dry
form) may be stored in separate syringes and then mixed using
syringe-to-syringe mixing techniques, as follows: the biomaterial
and crosslinking agent are mixed by connecting the syringe
containing the biomaterial with the syringe containing the
crosslinking agent using a syringe connector (such as a three-way
stopcock) and passing the material back and forth between the two
syringes until the material is adequately mixed (usually requiring
a minimum of about 20 passes, with one pass being counted each time
the volume of material passes through the syringe connector).
During the mixing process, crosslinking is initiated between
molecules of the biomaterial and the crosslinking agent.
[0067] The concentration of the hydrophobic crosslinking agent used
in the practice of the invention will vary depending upon a number
of factors, including the type and molecular weight of the
crosslinking agent used, the type and concentration of biomaterial
used, and the degree of crosslinking desired. In general, we have
found that hydrophobic crosslinking agent concentrations in the
range of about 0.1 to about 2 percent by weight of the final
composition are preferred to prepare the crosslinked biomaterial
compositions of the present invention.
Preparation of Heterogeneous Crosslinked Biomaterial Compositions
Using Mixtures of Hydrophobic and Hydropholic Crosslinking
Agents
[0068] In a general method for preparing the heterogeneous
crosslinked biomaterial compositions of the inventions, a
biomaterial which contains, or has been chemically derivatized to
contain, primary amino groups is combined and allowed to covalently
bond with a mixture of hydrophobic and hydrophilic crosslinking
agents. Preferably, the mixture of hydrophobic and hydrophilic
crosslinking agents is stored and used in dry form, to prevent loss
of crosslinking activity due to hydrolysis. The hydrophobic and
hydrophilic crosslinking agents will generally not react with one
other because both crosslinking agents contain the same reactive
groups (i.e., succinimidyl groups) which preferentially bind to
primary amino groups on various biomaterials such as collagen and
derivatized glycosaminoglycans.
[0069] In an alternative method, the biomaterial is mixed first
with either the hydrophobic or hydrophilic crosslinking agent, then
(preferably in rapid succession, before gellation occurs), with the
other type of crosslinking agent.
[0070] As used herein, the term "hydrophobic polymer" refers to
polymers which contain a relatively small proportion of oxygen or
nitrogen atoms. Hydrophobic polymers which contain, or have been
derivatized to contain, two or more reactive succinimidyl groups
are the preferred hydrophobic crosslinking agents for use in the
preparation of the heterogeneous crosslinked biomaterial
compositions of the invention.
[0071] As used herein, the term "hydrophilic polymer" refers to
polymers which contain a relatively large proportion of oxygen
and/or nitrogen atoms, which serve to attract water molecules for
hydrogen bonding. Synthetic hydrophilic polymers, such as
functionally activated polyethylene glycols, are the preferred
hydrophilic crosslinking agents for use in the preparation of the
heterogeneous crosslinked biomaterial compositions of the present
invention. Various activated forms of polyethylene glycol are
described in detail in commonly owned U.S. Pat. No. 5,328,955,
issued Jul. 12, 1994, and commonly owned U.S. Pat. No. 6,165,489,
issued Dec. 26, 2000.
[0072] Synthetic hydrophilic polymers for use in the present
invention are preferably multifunctionally activated and, more
preferably, difunctionally activated. Preferred synthetic
hydrophilic polymers are difunctionally activated forms of PEG
succinimidyl glutarate (SG-PEG), PEG succinimidyl (SE-PEG; referred
to only as "S-PEG" in the '955 patent), PEG succinimidyl
succinamide (SSA-PEG), and PEG succinimidyl carbonate (SC-PEG).
Reaction of SG-PEG with a biomaterial such as collagen results in
covalently bound conjugates containing an ester linkage; reaction
of SE-PEG (n=1-3) or SC-PEG (n=0) with a biomaterial results in
conjugates containing an ether linkage; and reaction of SSA-PEG
(n=1-10) with a biomaterial results in conjugates containing an
amide linkage. The amide and ether linkages are generally less
susceptible to hydrolysis than the ester linkage, and therefore may
result in crosslinked biomaterial compositions having greater
stability and persistence in vivo, depending on the environment
into which the implant material is placed. Ether linkages are
susceptible to oxidation, and may be sensitive to degradation by
free radicals.
[0073] Many of the activated forms of polyethylene glycol described
above are now available commercially from Shearwater Polymers,
Huntsville, Ala., and from Union Carbide, South Charleston, W.
Va.
[0074] Utility
[0075] The crosslinked biomaterial compositions of the present
invention are particularly useful in the preparation of formed
implants for use in a variety of medical applications, including
various artificial organs and tubular implants for use as vascular
grafts and/or stents. In a general method for preparing a formed
implant, a biomaterial/crosslinking agent reaction mixture,
prepared as described above, is extruded into molds of various
sizes and shapes, preferably before significant crosslinking has
occurred between the biomaterial and the crosslinking agent (or
mixture of crosslinking agents). This period of time will vary
depending upon the type and concentration of both the biomaterial
and the crosslinking agent(s) used, but is generally within the
range of about 5 to about 60 minutes. The material should be
removed from the mold only after adequate time has elapsed to allow
for equilibrium crosslinking to occur between the biomaterial and
crosslinking agent(s). If necessary, residual unbound crosslinking
agent can be removed from the implant prior to its incorporation
into the body of a patient.
[0076] The biomaterial/crosslinking agent mixture can also be
applied to (for example, by extrusion, dipping, brushing, or
painting) onto one or more surface of a preformed synthetic
implant, such as a bone prosthesis or synthetic vascular graft or
stent, and allowed to crosslink in place, thereby providing a
crosslinked, nonimmunogenic biomaterial coating on the surface of
the implant. Alternatively, all or part of a preformed synthetic
implant can be dipped into a container holding the
biomaterial/crosslinking agent reaction solution.
[0077] The biomaterial/crosslinking agent mixture can be extruded
in the shape of a string and allowed to crosslink in that
configuration. When the strings are fully crosslinked, they can be
dried to remove substantially all unbound water. The dried strings
can be inserted through a needle to a dermal site in need of
correction (such as a depressed scar or wrinkle) in order to
provide soft tissue augmentation. The dried strings can also be
chopped into fine pieces, suspended in a nonaqueous carrier, and
injected to a tissue site in need of augmentation, which may be a
dermal site or other soft tissue site such as an inadequately
functioning sphincter (e.g., urinary, anal, or esophageal
sphincter). When exposed to biological fluids, the crosslinked
strings will rehydrate in situ and swell to approximately five
times their dried diameter. The dried strings can also be used as
suture materials, or braided, knit, or woven to provide
biomaterials for tendon or ligament repair or replacement.
[0078] A suitable particulate material, such as ceramic particles,
can be mixed with the biomaterial prior to mixing with the
crosslinking agent to provide a material suitable for hard tissue
augmentation, such as the repair or replacement of bone or
cartilage. These materials can be administered in fluid form (prior
to crosslinking) to the site of a bone or cartilage defect and
allowed to crosslink in place, or can be used to prepare formed
bone or cartilage implants (using techniques similar to those
described above for the preparation of formed implants for soft
tissue repair) which can then be molded or cut to the desired size
and shape.
[0079] The crosslinked biomaterial compositions of the invention
can also be used as injectable formulations in the augmentation of
soft or hard tissues of the body. Following mixing of the
biomaterial and the crosslinking agent(s), the reaction mixture
should be injected to a tissue site before significant crosslinking
has occurred, to prevent blockage of the syringe needle with the
crosslinked composition. If the material is injected to a tissue
site before equilibrium crosslinking has occurred, functional
groups on the crosslinking agent(s) may bind to collagen molecules
in the host tissue, thereby providing biological anchoring of the
biomaterial to the host tissue. Implants which have been
"biologically anchored" to host tissue are more difficult to
displace and therefore may show greater persistence in vivo than
currently available injectable biomaterial compositions.
[0080] Biologically active agents, such as cytokines or growth
factors, can be incorporated into the compositions of the
invention, either by simple admixture, or by covalently binding the
active agent to the crosslinking agent prior to combining the
crosslinking agent with the biomaterial. The active agents may
serve to recruit cells to the area of the implant further anchoring
the implant to host tissue, and may accelerate wound healing when
administered to a wound site.
EXAMPLES
[0081] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make the preferred embodiments of the
conjugates, compositions, and devices and are not intended to limit
the scope of what the inventors regard as their invention. Efforts
have been made to ensure accuracy with respect to numbers used
(e.g., amounts, temperature, molecular weight, etc.) but some
experimental errors and deviation should be accounted for. Unless
indicated otherwise, parts are parts by weight, molecular weight is
weight average molecular weight, temperature is in degrees
Centigrade, and pressure is at or near atmospheric.
Example 1
Preparation and Characterization of Crosslinked Collagen
Compositions Using Hydrophobic Crosslinking Agents
[0082] Fibrillar collagen (Zyderm.RTM. I Collagen, available from
Collagen Corporation, Palo Alto, Calif.) and methylated
(nonfibrillar) collagen (prepared by reacting fibrillar collagen
with methanol for approximately 1-3 days at 21.degree. C.) were
crosslinked using disucciaimidyl suberate (DSS),
bis(sulfosuccinimidyl) suberate (BS.sup.3), difunctionally
activated SE-PEG (n=2, 3800 MW), and difunctionally activated
-SG-PEG (3800 MW).
[0083] The fibrillar collagen formulations were prepared by mixing
the contents of a 1-cc syringe containing 1.0 cc of Zyderm Collagen
(35 mg/ml collagen concentration) with the contents of a 1-cc
syringe containing one of the following crosslinking agents in the
quantity specified:
[0084] 3 mg of DSS;
[0085] 3 mg of BS.sup.3;
[0086] 5 mg of SE-PEG; or
[0087] 5 mg of SG-PEG.
[0088] The methylated collagen formulations were prepared by mixing
the contents of a 1-cc syringe containing 1.0 cc of methylated
collagen (21 mg/ml collagen concentration) with the contents of a
1-cc syringe containing one of the following crosslinking agents in
the quantity specified:
[0089] 3 mg of DSS;
[0090] 3 mg of BS3;
[0091] 10 mg of SE-PEG; or
[0092] 10 mg of SG-PEG.
[0093] All of the crosslinking agents were used in dry form. The
collagen and crosslinking agent were mixed by passing the material
between the two syringes using a 3-way stopcock, employing about 40
to 50 passes of material between the syringes. Once adequate mixing
of the collagen and crosslinking agent had been achieved, the
material was transferred into one syringe and incubated at
37.degree. C. for approximately 16 hours.
[0094] Each of the crosslinked collagen materials prepared as
described above was extruded out of the plunger end of its syringe.
The resulting crosslinked cylindrical gels were then sectioned into
5-mm thick disks. Each of the formulations was then evaluated
according to some or all of the following test methods:
differential scanning calorimetry (DSC), solubilization in 1 mg/ml
trypsin solution, and oxidative degradation in 3% hydrogen peroxide
(H.sub.20.sub.2). The results of these evaluations are presented in
Table 1, below.
1TABLE 1 CHARACTERIZATION OF VARIOUS CROSSLINKED COLLAGEN
COMPOSITIONS DSC Solubilization Oxidative Degradation Material In
Tm .degree. C. in Trypsin Solution in 3% H.sub.2O.sub.2 DSS-ZI 74.3
7 days 14 days DSS-MC 57.7 2 days N/A BS.sup.3-ZI 67.6 N/A N/A
BS.sup.3-MC 58.6/64 N/A N/A SEPEG-ZI N/A 3 days 10 days SEPEG-MC
N/A 16 hours N/A SGPEG-ZI 60.8 3 days 7 days SGPEG-MC N/A 16 hours
N/A ZI = Zyderm .RTM. I Collagen (35 mg/ml collagen concentration)
MC = methylated collagen (21 mg/ml collagen concentration) DSS =
disuccinimidyl suberate BS.sup.3 = bis(sulfosuccinimidyl) suberate
SEPEG = difunctionally activated SE-PEG (n = 2, 3800 MW) SGPEG =
difunctionally activated SG-PEG (3800 MW) N/A = Data not
available.
[0095] Differential scanning calorimetry (DSC) is used to measure
denaturational transitions in collagen, which can be used to assess
the relative strength of crosslinking achieved. As indicated by the
DSC results above, crosslinking of fibrillar collagen by the
hydrophobic crosslinking agents DSS and BS.sup.3 is at least as
strong as that achieved using the hydrophilic crosslinking agent
SG-PEG. Slightly lower numbers were obtained for the methylated
(nonfibrillar) collagen formulations.
[0096] Solubilization in trypsin solution was determined by
incubating a 5-mm thick disk of each crosslinked material at
37.degree. C. in a solution comprising 1 mg trypsin in 1 ml water
and measuring how much time was required to disperse the
crosslinked collagen gel. As shown above, approximately twice as
much time (7 days) was required to solubilize the DSS-ZI gel as was
required to solubilize the SEPEG-ZI and SGPEG-ZI gels (3 days
each), indicating that DSS achieves stronger crosslinking (i.e.,
increased crosslinking density) to fibrillar collagen than do
either SE-PEG or SG-PEG. The methylated collagen formulations
demonstrated less stability in trypsin solution in general, out the
methylated collagen formulations crosslinked using DSS showed
considerable improvement in stability over those crosslinked using
either SE-P-EG or SG-PEG.
[0097] Oxidative degradation was determined by incubating a 5-mm
thick disk of each crosslinked material at 37.degree. C. in a 3%
solution of hydrogen peroxide in water and measuring how much time
was required to disperse the crosslinked collagen gel. As with the
results of the trypsin solubilization described above, nearly twice
as much time (14 days) was required to solubilize the DSS-ZI gel as
was required to solubilize the SEPEG-ZI (10 days) and SGPEG-ZI gels
(7 days), indicating that DSS achieves stronger crosslinking to
fibrillar collagen than do either SE-PEG or SG-PEG. Thus, with
regard to trypsin sensitivity and susceptibility to oxidative
degradation, the collagen materials crosslinked using hydrophobic
crosslinking agents showed considerable and unexpected improvement
over those crosslinked with the hydrophilic crosslinking agents
previously described in the art.
Example 2
In Vivo Persistence of Crosslinked Collagen Compositions
[0098] Crosslinked collagen formulations were prepared fresh by
mixing the contents of a 1-cc syringe containing 1.0 grain of a
mixture of Zyplast.RTM. (glutaraldehydecrosslinked collagen having
a collagen concentration of 35 mg/ml, available from Cohesion
Technologies, Palo Alto, Calif.) and Zyderm.RTM. Collagens (in a
70:30 weight/weight ratio) with the contents of a 1 cc syringe
containing either 3 mg of DSS, 3 mg of SE-PEG, or 3 mg of SG-PEG. A
noncrosslinked mixture of Zyplast and Zyderm Collagens in a 70:30
weight ratio was used as the control. Two groups consisting of 24
male Sprague-Dawley rats each were injected with implants
consisting of 0.5 milliliters each of two of the four formulations,
according to the schedule below.
[0099] Animal Group A:
[0100] Site 1 Zyplast/Zyderm Collagen mixture (control)
[0101] Site 2 Zyplast/Zyderm Collagen mixture crosslinked using
DSS
[0102] Animal Group B:
[0103] Site 1 Zyplast/Zyderm Collagen mixture crosslinked using
SG-PEG
[0104] Site 2 Zyplast/Zyderm Collagen mixture crosslinked using
SE-PEG
[0105] The materials were injected subcutancously through a
27-gauge needle within approximately 5 minutes of mixing the
collagen and crosslinking agent.
[0106] Six animals from each of Groups A and B were sacrificed at
each of the 7, 14, 28, and 90 day post-implantation time points.
The implants with surrounding tissue were excised and examined
histologically. The injected crosslinked materials had assumed a
discrete, football-shaped, bolus-like configuration, whereas the
noncrosslinked formulation was present as a more diffuse mass. The
implants from four animals out of each group were used for
histology studies and wet weight experiments. The implants from two
animals out of each group were used to measure the mechanical force
required to dislodge the implant from the host tissue. The results
of the histology studies and wet weight experiments are discussed
below.
[0107] The excised implants were examined histologically and scored
on a scale of 0 through 4 on each of three parameters: inflammatory
infiltrate, fibroblast ingrowth, and fibrosis. A score of 4
indicated the presence of a maximum amount of a parameter; a score
of 0 indicated that the particular parameter was not observed in
connection with the implant being examined (i.e., a score of 0 on
inflammatory infiltrate indicates that no inflammatory infiltrate
was observed in the implant site). Results of the histological
examinations are presented in Tables 2, 3, and 4, and discussed
below. Average scores are listed in parentheses.
2TABLE 2 INFLAMMATORY INFILTRATE Implant Material Day 7 Day 14 Day
28 Day 90 Z/Z 0, 2, 2, 1 2, 0, 0 0, 0, 0, 1 0, 0, 0, 0 (1.25)
(0.67) (0.25) (0) Z/Z + DSS 1, 2, 2, 3, 2 3, 3, 1 1, 1, 1, 1 0, 0,
0, 0 (2.0) (2.3) (1.0) (0) Z/Z + SG-PEG 1, 1, 1 0, 1, 3, 1 1, 0, 2,
1 0, 0, 0, 0 (1.0) (1.25) (1.0) (0) Z/Z + SE-PEG 1, 1, 1, 2 0, 1,
1, 1 1, 0, 2, 2 0, 0, 0, 0 (1.25) (0.75) (1.25) (0) Z/Z = mixture
of Zyplast .RTM. and Zyderm .RTM. I Collagens in a 70:30
weight/weight ratio DSS = disuccinimidyl suberate BS.sup.3 =
bis(sulfosuccinimidyl) suberate SEPEG = difunctionally activated
SE-PEG (n = 2, 3800 MW) SGPEG = difunctionally activated SG-PEG
(3800 MW)
[0108] At days 7 and 14, the collagen implants crosslinked using
DSS showed a moderate inflammatory response, slightly greater than
the responses observed for the other (crosslinked and
noncrosslinked) collagen compositions. By day 28, inflammatory
infiltrate into the DSS-crosslinked implant was minimal,
diminishing to nonexistent by day 90.
3TABLE 3 FIBROBLAST INGROWTH Implant Material Day 7 Day 14 Day 28
Day 90 Z/Z 0, 0, 0, 0 1, 1, 1 1, 0, 0, 1 0, 0, 1, 1 (0) (1.0) (0.5)
(0.5) Z/Z + DSS 0, 0, 0, 0, 0 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 (0) (0)
(0) (0) Z/Z + SG-PEG 0, 0, 0 1, 1, 1, 1 1, 0, 2, 1 0, 1, 1, 1 (0)
(1) (1.0) (0.75) Z/Z + SE-PEG 0, 0, 0, 0 1, 1, 1, 0 1, 0, 2, 2 0,
0, 0, 1 (0) (0.75) (1.25) (0.25)
[0109] Unlike the other crosslinked and noncrosslinked collagen
formulations, the DSS-crosslinked implants showed no evidence of
fibroblast ingrowth throughout the entire duration of the study.
This is most likely due to the very tight crosslinked collagen
network achieved using DSS as a crosslinking agent.
4TABLE 4 FIBROSIS Implant Material Day 7 Day 14 Day 28 Day 90 Z/Z
0, 1, 1, 2 1, 0, 0 0, 0, 1, 0 0, 0, 0, 0 (1.0) (0.33) (0.25) (0)
Z/Z + DSS 1, 0, 2, 1, 2 1, 0, 1 0, 0, 1, 1 0, 0, 0, 0 (1.2) (0.67)
(0.5) (0) Z/Z + SG-PEG 0, 1, 1 1, 1, 1, 1 1, 1, 1, 1 0, 0, 0, 0
(0.67) (1.0) (1.0) (0) Z/Z + SE-PEG 1, 0, 2, 1 0, 1, 1, 1 0, 1, 1,
1 0, 0, 0, 0 (1.25) (0.75) (0.75) (0)
[0110] Fibrosis was observed to be similar in all three of the
crosslinked collagen compositions examined.
[0111] Each of the implants was weighed following explantation. Wet
weight of the implant as a percentage of the original weight of the
implant is shown in FIG. 8 for each of the four formulations at
each time point. There were no significant differences between any
of the formulations at the 7, 14, and 28 day time points; however,
at the 90-day time point, the collagen formulation crosslinked
using DDS showed significantly better retention of wet weight
(close to 100 percent) than the other formulations. Due to the lack
of fibroblast ingrowth seen during histological examination, the
wet weight of the DSS-crosslinked implant is believed to consist
substantially of the implant material itself rather than invading
cells. This observation indicates that the DSS-crosslinked collagen
implants were not resorbed into the host tissue as quickly as the
other collagen implant materials, possibly due to the tightly
crosslinked network achieved using DSS as a crosslinking agent.
[0112] At each of the 7, 28, and 90-day time points of the study,
the portion of the skin containing the implant was excised from two
animals from each of Groups A and B. The skin surrounding the
implant was trimmed into a uniform rectangular shape having
dimensions of 2 cm.times.4 cm. The encapsulated tissue that had
grown over the surface of the implant was removed so that the
implant now appeared to be merely resting on the surface of the
dermis.
[0113] The piece of skin containing the implant was pinned to a 3
cm.times.5 cm wooden board using one thumbtack at each of the four
corners of the skin. As illustrated in FIG. 9, a sling was placed
externally around the perimeter of the implant. The mechanical
force required to dislodge the implant from the tissue was measured
using the Instron Universal Tester, Model 4202, by holding the
wooden board (to which the piece of skin was attached) in one of
the Instron's clamps and holding the end of the sling in the other
clamp. The Instron pulled on the clamp holding the sling until the
implant broke free from the tissue. Force anchoring to tissue is
depicted graphically in FIG. 10 for each of the four formulations
at the 7, 28, and 90-day time points. There were no significant
differences between the formulations crosslinked using the
hydrophobic crosslinking agent (DSS) and the formulations
crosslinking using either of the hydrophilic crosslinking agents
(SE-PEG; SG-PEG).
Example 3
Preparation and Characterization of Crosslinked Biomaterial
Compositions Containing Mixtures of Hydrophobic and Hydrophilic
Crosslinking Agents
[0114] Fibrillar collagen (Zyderm.RTM. I Collagen, 35 mg/ml
collagen concentration available from Collagen Corporation, Palo
Alto, Calif.) was crosslinked using disuccinimidyl suberate (DSS),
difunctionally activated SE-PEG (n=2, 3800 MW), and a 50:50
(weight/weight) mixture of DSS and difunctionally activated SE-PEG.
The crosslinked collagen formulations were prepared by mixing the
contents of a 5-cc syringe containing 5.0 grams of Zyderm Collagen
with the contents of a 5-cc syringe containing either 15 mg of DSS,
15 mg of SE-PEG, or 15 mg of the DSS/SE-PEG mixture.
[0115] All of the crosslinking agents were used in dry form. The
DSS/SE-PEG mixture was prepared immediately prior to crosslinking
by placing 7.5 mg each of DSS and SE-PEG into a 5-cc syringe, then
shaking the syringe by hand to mix the two crosslinking agents.
[0116] The collagen and crosslinking agent were mixed by passing
the material between the two syringes using a 3-way stopcock,
employing about 40 to 50 passes of material between the syringes.
Once adequate mixing of the collagen and crosslinking agent had
been achieved, the material was transferred into one syringe and
incubated at 37.degree. C. for approximately 16 hours.
[0117] Each of the three crosslinked collagen materials prepared as
described above was extruded out of the plunger end of its syringe.
The resulting crosslinked cylindrical gels were then sectioned into
5-mm thick disks. The three formulations were evaluated using
differential scanning calorimetry (DSC). The gel strength of each
formulation was measured using the Instron Universal Tester, Model
4202. DSC and gel strength results for each of the three
crosslinked collagen formulations are presented in Table 5,
below.
5TABLE 5 DSC AND GEL STRENGTH RESULTS FOR VARIOUS CROSSLINKED
COLLAGEN COMPOSITIONS Average Gel Crosslinking DSC Gel Strength
Strength Agent in .degree. C. in Newtons in Newtons S.D. DSS 74.3
54.1 46.5 6.0 40.5 45.0 41.5 51.1 SE-PEG 59.5 59.4 58.6 2.5 56.4
56.2 58.7 62.4 DSS/SE-PEG 53-65* 28.1 41.7 8.2 65-80* 43.9 44.2
50.3 42.3 *Broad main peak. **Broad shoulder peak.
[0118] The inconsistency in the DSC and gel strength results for
the collagen composition prepared using a mixture of hydrophobic
and hydrophilic crosslinking agents may be due to several factors,
among them: insufficient mixing of the two crosslinking agents
prior to mixing with collagen, the heterogeneous nature of the
composition itself, and, possibly, a ratio of crosslinking agents
that had not been optimized. Another factor may be that the SE-PEG
is able to crosslink collagen more quickly than DSS due to the
lower solubility of the DSS in the aqueous solution in which the
collagen fibers are suspended.
[0119] Collagen compositions prepared using mixtures of hydrophobic
and hydrophilic crosslinking agents may be useful in certain
therapeutic applications due to the relative contributions of the
two different types of crosslinking agent to the properties of the
final composition: the hydrophobic crosslinking agent, increased
stability; the hydrophilic crosslinking agent, increased elasticity
and better overall handling properties.
[0120] It is not intended that the invention be limited by the
preferred embodiments described above, which are used for purposes
of illustration. The invention is intended to have the scope
defined by the attached claims.
* * * * *