JPH09249751A - Use of hydrophobic crosslinking agent for preparing crosslinked biological material composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an injectable or transplantable crosslinked biological material composition for therapeutic use by covalently bonding a hydrophobic polymer containing succinimidyl groups to biological material.

SOLUTION: One tenth (0.1) to 2wt.%, hydrophobic polymer containing at least two succinimidyl groups and selected from among disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, dithiobis(sulfosuccinimidyl) propionate, bis(2 succinimidoxycarbonyloxy)ethyl sulfone, 3,3dithiobis(sulfosuccinimidyl propionate) and analogues and derivatives thereof is covalently bonded to at least biological material selected from among (non)fibrous collagen, gelatin and glucosanoglycans.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates generally to the use of hydrophobic crosslinkers to prepare injectable or implantable crosslinked biomaterial compositions for use in various therapeutic applications. Specifically, the present invention relates to hydrophobic cross-linking agents containing two or more succinimidyl groups (eg, disuccinimidyl suberate, bis (sulfosuccinimidyl) suberate or dithiobis (succinimidyl). Propionate)). The present invention also provides a unique crosslinked biomaterial composition prepared with a mixture of hydrophobic and hydrophilic crosslinkers. The compositions of the present invention are particularly useful in preparing shaped implants for various medical applications.

[0002]

2. Description of the Related Art U.S. Pat.No. 5,162,430 issued to Rhee et al. On November 10, 1992, owned by the same assignee as the present application, is a collagen-synthetic polymer conjugate and collagen synthesis. A method of covalently attaching to a hydrophilic polymer is disclosed. US Pat. No. 5,328,955 issued to Rhee et al. On July 12, 1994 (also owned by the same assignee) discloses various activated forms of polyethylene glycol and various linkages. However, they can be used to produce collagen-synthetic polymer conjugates with a range of physical and chemical properties. June 1994
U.S. Pat.No. 5,324,775 issued to Rhee et al. On the 28th (also owned by the same assignee) describes a biologically inert, biocompatible polymer as a synthetic hydrophilic polymer. Disclosed are biocompatible polymer conjugates prepared by covalent attachment to.

US patent application Ser. No. 08 / 146,843, filed Nov. 3, 1993, which is owned by the same assignee and pending at the time of filing this application, is a synthetic hydrophilic agent. Disclosed are conjugates containing various glycosaminoglycan species covalently linked to a polymer, which are also optionally linked to collagen. No. 08 / 147,227, filed November 3, 1993, which is owned by the same assignee and is pending at the time of filing this application, has ophthalmic or other medical uses. Disclosed is a collagen-polymer conjugate containing chemically modified collagen (e.g., succinylated collagen or methylated collagen) covalently bound to a synthetic hydrophilic polymer to produce an optically transparent material for use in are doing.

Hydrophobic cross-linking agents such as disuccinimidyl suberate, bis (sulfosuccinimidyl) suberate, and dithiobis (succinimidyl propionate) are commercially available from Pierce (Rockford, IL) in 1992. ), It has long been used to cross-link biologically active peptides.

The contents of all documents cited above and herein are incorporated herein by reference to describe and disclose the problem cited.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide injectable or implantable crosslinked biomaterial compositions for use in various therapeutic applications using hydrophobic crosslinkers. A further object of the present invention is to provide a unique crosslinked biomaterial composition using a mixture of hydrophobic and hydrophilic crosslinkers. Another object of the invention is to provide compositions that are particularly useful in the preparation of shaped implants for various medical applications.

[0007]

In the following, a crosslinked biomaterial composition prepared using various hydrophobic crosslinkers and a crosslinked biomaterial composition prepared using a mixture of a hydrophobic crosslinker and a hydrophilic crosslinker are described below. The detailed description of the preferred embodiments of the present invention, including those disclosed herein, is disclosed.

[0008] In summary the present inventors earlier patents and patent applications of the present invention, synthetic hydrophilic polymer, preferably prepared using functionally activated polyethylene glycol (PEG) as a crosslinking agent, Various crosslinked biomaterial compositions have been disclosed. In accordance with the present invention, various hydrophobic polymers having two or more succinimidyl groups (eg, disuccinimidyl suberate, bis (sulfosuccinimidyl) suberate, or dithiobis (succinimidyl prolate). We have found that pionate)) can be used to cross-link various biological materials such as collagen and glycosaminoglycans. We can also derivatize certain polymers, such as polyacids, to contain two or more succinimidyl groups, and in their derivatized form, collagen and glycosamido. It has also been found that it can be used to crosslink noglycans. In addition, the inventors have discovered that a mixture of hydrophobic and hydrophilic crosslinkers can be used to prepare unique crosslinked biomaterial composition compositions.

The present invention relates to a conjugate comprising a biomaterial covalently bound to a hydrophobic polymer, wherein the hydrophobic polymer has two or more succinimidyl groups prior to binding to the biomaterial. Have A conjugate comprising a biomaterial covalently bound to a hydrophobic polymer, wherein the hydrophobic polymer is chemically derivatized to have two or more succinimidyl groups prior to binding to the biomaterial. Such conjugates are also included in the present invention. Also disclosed are heterogeneous cross-linked biomaterial compositions containing the biomaterial (or a mixture of different species of biomaterial), a hydrophobic crosslinker, and a hydrophilic crosslinker. Furthermore, according to the present invention, shaped implants are prepared using the conjugates and compositions of the present invention.

The present invention provides a conjugate containing a biological material covalently attached to a hydrophobic polymer. Here, the hydrophobic polymer contains two or more succinimidyl groups before binding to the biological substance.

In one embodiment, the hydrophobic polymer is disuccinimidyl suberate, bis (sulfosuccinimidyl) suberate, dithiobis (succinimidyl propionate), bis (2-succinimidooxycarbonyl). (Oxy) ethyl sulfone, 3,3′-dithiobis (sulfosuccinimidyl propionate), and analogs and derivatives thereof.

In another embodiment, the hydrophobic polymer is chemically derivatized to contain two or more, preferably two, three or four succinimidyl groups. In a preferred embodiment, the hydrophobic polymer is a polyacid, which is preferably trimethylolpropane-based tricarboxylic acid, di (trimethylolpropane) -based tetracarboxylic acid, heptanedioic acid. , Octanedioic acid and hexadecanedioic acid.

In another embodiment, the biological material is
It is selected from the group consisting of collagen, gelatin, glycosaminoglycans and mixtures thereof, preferably collagen. The collagen can be fibrillar collagen or non-fibrillar collagen, preferably the non-fibrillar collagen is selected from the group consisting of succinylated collagen and methylated collagen.

In still another embodiment, the biological substance is selected from the group consisting of hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, kerato sulfate, chitin, chitosan, heparin and their derivatives. Glycosaminoglycan.

In yet another embodiment, the hydrophobic polymer is covalently bound to both collagen and one or more glycosaminoglycan species.

In yet another embodiment, the hydrophobic polymer is covalently bound to two or more glycosaminoglycan species.

The present invention also provides a conjugate containing a biological material covalently attached to a hydrophobic polymer. Here, the hydrophobic polymer has been chemically derivatized to contain two or more, preferably two, three or four succinimidyl groups before binding to the biological material. There is.

In one embodiment, the hydrophobic polymer is a polyacid, which is preferably a trimethylolpropane-based tricarboxylic acid, a di (trimethylolpropane) -based tetracarboxylic acid, It is selected from the group consisting of heptanedioic acid, octanedioic acid and hexadecanedioic acid.

In yet another embodiment, the biological material is selected from the group consisting of collagen, gelatin, glycosaminoglycans and mixtures thereof, preferably collagen. The collagen may be fibrillar collagen or non-fibrillar collagen, preferably the non-fibrillar collagen is a chemically derivatized collagen selected from the group consisting of succinylated collagen and methylated collagen. is there.

In yet another embodiment, the biological material is selected from the group consisting of hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, kerato sulfate, chitin, chitosan, heparin and their derivatives. Glycosaminoglycan.

In yet another embodiment, the hydrophobic polymer is covalently bound to both collagen and one or more glycosaminoglycan species.

In yet another embodiment, the hydrophobic polymer is covalently attached to two or more glycosaminoglycan species.

The present invention also provides a heterogeneously crosslinked biomaterial composition containing a biomaterial, a hydrophobic crosslinker and a hydrophilic crosslinker.

In one embodiment, the hydrophobic crosslinker contains 2 or more, preferably 2, 3 or 4 succinimidyl groups, preferably the hydrophobic crosslinker is disuccine. Imidil suberate, bis
(Sulfosuccinimidyl) suberate, dithiobis (succinimidyl propionate), bis (2-succinimidooxycarbonyloxy) ethyl sulfone, 3,3′-dithiobis (sulfosuccinimidyl propionate), and Selected from the group consisting of their analogs and derivatives.

In another embodiment, the hydrophobic crosslinker contains a hydrophobic polymer chemically derivatized to contain two or more succinimidyl groups. In a preferred embodiment, the hydrophobic polymer is
Is a polyacid, which is trimethylolpropane
-Based tricarboxylic acid, di (trimethylolpropane) -based tetracarboxylic acid, heptanedioic acid, octanedioic acid and hexadecanedioic acid.

In another embodiment, the hydrophilic crosslinker is a synthetic hydrophilic polymer, preferably the synthetic hydrophilic polymer is a functionally activated polyethylene glycol. Preferably, the functionally activated polyethylene glycol is a polyfunctional, preferably bifunctionally activated polyethylene glycol.

[0027] In yet another embodiment, the biological material is selected from the group consisting of collagen, gelatin, glycosaminoglycans and mixtures thereof, preferably collagen. The collagen can be fibrillar collagen or non-fibrillar collagen, preferably the non-fibrillar collagen is a chemically derivatized collagen selected from the group consisting of succinylated collagen and methylated collagen. .

In yet another embodiment, the biological material is selected from the group consisting of hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, keratosulfate, chitin, chitosan, heparin and their derivatives. Glycosaminoglycan.

In yet another embodiment, the biological material contains a mixture of collagen and one or more glycosaminoglycan species.

In yet another embodiment, the biological material contains a mixture of two or more glycosaminoglycan species.

The present invention also provides a molded implant containing the above conjugate or the above composition.

The compositions of the present invention have many unique and unexpected characteristics compared to previously disclosed crosslinked biomaterial compositions prepared using only hydrophilic crosslinkers. An important feature of the compositions of the present invention (compared to conventional cross-linked biomaterial compositions) is their slow degradation, resulting in higher chemical stability, which is their in vivo persistence. Can lead to an increase in Further features and advantages of the invention include
It will become apparent by reading the detailed description below.

[0033]

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein and in the appended claims, the singular forms "a", "an" and "the" refer to plural unless the context clearly dictates otherwise. Note that it includes an indication of things. For example, the designation "a conjugate" includes one or more conjugate molecules and
The term "n article)" includes one or more different types of products well known to those skilled in the art, and the term "the collagen" includes a mixture of multiple collagens of different types, etc. .

Certain terminology of particular importance to the description of the present invention is defined below: The term "atelopeptide collagen" refers to the telopeptide region, which in humans
Collagen that has been chemically treated or engineered to remove collagen from other animals (eg, known to cause an immune response against collagen).

The term "biological material" as used herein refers to
Generally, collagen, gelatin and polysaccharides (e.g.,
(Including glycosaminoglycan) means a biocompatible polymer of natural origin.

As used herein, the terms "chemically attached" and "attached" mean attached via a chemical covalent bond. In practicing the present invention, the hydrophobic polymer and the biological material may be covalently bonded to each other by the reactive succinimidyl group on the hydrophobic polymer.

As used herein, "chemical crosslinker" means
It refers to any chemical reagent capable of covalently binding biological materials (eg, collagen, glycosaminoglycans and mixtures thereof) to form a network of cross-linked biological materials.

The term "collagen" as used herein refers to all types and shapes of collagen, including recombinantly produced and naturally derived raw materials.
(E.g., extracted from bovine dermis or human placenta),
Included are those that have been treated or those that have been modified.

The term "collagen suspension" refers to an aqueous carrier (eg water or phosphate buffered saline (PBS) solution).
It means a suspension of crosslinked or noncrosslinked collagen fibers therein.

The term "collagen-synthetic polymer" refers to
By collagen is meant covalently attached to a synthetic hydrophilic polymer. For example, "PEG-collagen" refers to a composition of the invention in which collagen molecules are covalently attached to polyethylene glycol (PEG) molecules.

The term "bifunctionally activated" refers to
Synthetic hydrophilicity chemically derivatized to have two functional groups that can react with primary amino groups on biocompatible polymer molecules (eg, collagen or diacetylated glycosaminoglycans) Means a polymer molecule. The two functional groups on the bifunctionally activated synthetic hydrophilic polymer are generally located at opposite ends of the polymer chain. Each functionally activated group on the bifunctionally activated synthetic hydrophilic polymer molecule can be covalently bound to the biocompatible polymer molecule, thereby allowing these biocompatible polymer molecules to Cross-linking occurs between molecules.

The term "dry" means that substantially all unbound water has been removed from the material.

The term "fibrous collagen" means collagen in which triple-helical molecules are aggregated by intermolecular charge and hydrophobic interactions to form dense fibers.

The term "functionally activated" means that it is capable of reacting with a primary amino group on a biocompatible polymer molecule.
It means a synthetic hydrophilic polymer molecule that is chemically derivatized to have one or more functional groups.

The term "in situ" as used herein means "at the site of administration".

As used herein, “in situ cross-linking”
The term means cross-linking a biocompatible polymer implant after implantation at a tissue site on a human or animal subject, where at least one functional group on the synthetic polymer is cross-linked. The group is covalently attached to a biocompatible polymer molecule within the implant, and at least one functional group on the synthetic polymer is free and is not compatible with other biocompatible polymer within the implant. It can be covalently bound to a coalesced molecule or a collagen molecule within the patient's own tissue.

The term "molecular weight" as used herein means the weight average molecular weight of many molecules in any given sample, as is commonly used in the art. Therefore, a sample of PEG 2000 contains, for example, a statistical mixture of polymer molecules in the weight range 1500-2500, with one molecule slightly different from its neighbors over a range. Specifying a molecular weight range means that the average molecular weight is a value within the specified range, but may include molecules outside of these ranges. Therefore, about 800 to about 20,000
The average molecular weight range of at least about 800 to about 20,000.

The term "polyfunctionally activated" refers to
To have two or more functional groups (which are located at various sites along the polymer chain) capable of reacting with a primary amino group on a biocompatible polymer molecule,
Means chemically derivatized synthetic hydrophilic polymer molecules. Each functional group on the polyfunctionally activated hydrophilic synthetic polymer can be covalently bound to a biocompatible polymer molecule, which results in cross-linking between these biocompatible polymer molecules. . Types of polyfunctionally activated synthetic hydrophilic polymers include difunctionally activated polymers, tetrafunctionally activated polymers, and star-branched polymers. Is mentioned.

The term "non-crosslinked collagen" means collagen that has not been previously crosslinked with a chemical crosslinking agent. Such non-crosslinked collagen includes both fibrillar and non-fibrillar collagen.

"Non-fibrous collagen" means, among them,
A composition containing non-fibrillar collagen is meant to be optically transparent, meaning collagen in which the triple helical molecules are not aggregated to form dense fibers.

The terms "synthetic hydrophilic polymer" or "synthetic polymer" mean a synthetically produced hydrophilic polymer, but not necessarily water soluble. Examples of synthetic hydrophilic polymers that may be used in practicing the present invention include polyethylene glycol (PEG), polyoxyethylene, polymethylene glycol, polytrimethylene glycol, polyvinylpyrrolidone, polyoxyethylene-polyoxypropylene. There are block polymers and copolymers, and their derivatives. Naturally derived polymers (e.g., protein, starch, cellulose, heparin, hyaluronic acid,
And their derivatives) are explicitly excluded from the scope of this definition.

The term "tissue augmentation" as used herein refers to
By replacing or repairing defects in soft or hard tissues in the human body.

Except as 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 methods and materials similar or equivalent to those described herein are useful in the practice or testing of the present invention, only preferred methods and materials are described below.

Detailed Description of the Preferred Embodiments of the Present Invention In accordance with the present invention, a unique crosslinked biomaterial composition is prepared using a hydrophobic crosslinker, or a mixture of hydrophobic and hydrophilic crosslinkers. . In order to prepare the crosslinked biomaterial composition of the present invention, it is first necessary to prepare one or more biomaterials and a hydrophobic crosslinking agent.

Preferred Biomaterials According to the method of the present invention, biomaterials having a primary amino group (—NH ₂ ) which can be bound with a hydrophobic or hydrophilic crosslinker, or which have such groups. Biomaterials that can be chemically derivatized to can be used to prepare the crosslinked biomaterial compositions of the present invention. Preferred biomaterials for use in practicing the present invention include all types of collagen and glycosaminoglycans, and mixtures thereof.

In practicing the present invention, collagen from any source can generally be used. For example, collagen can be extracted and purified from human or other mammalian sources, or produced recombinantly or otherwise. Any type of collagen can be used, including but not limited to types I, II, III, IV or combinations thereof, although type I is generally preferred. Either atelopeptide collagen or telopeptide-containing collagen can be used,
When using collagen from xenograft sources (eg, bovine collagen), atelopeptide collagen is generally preferred. This is because atelopeptide collagen has a lower immunogenicity than telopeptide-containing collagen. This collagen can be taken up by the human body without any significant immune response,
It should be in pharmaceutically pure form.

The collagen used in the present invention may be in a fibrous or non-fibrous form. Fibrous collagen is generally preferred for tissue augmentation applications because of its high in vivo durability. Non-fibrillar collagen, including chemically modified collagen (eg, succinylated collagen or methylated collagen) is preferred for certain situations (eg, ophthalmic applications requiring optically clear materials). Succinylated collagen and methylated collagen can be prepared according to the methods described in US Pat. No. 4,164,559, the contents of which are incorporated herein by reference. The non-crosslinked collagen used in the present invention is usually about 20 mg / ml and about 120 mg in an aqueous suspension.
Concentrations between mg / ml, preferably about 30 mg / ml and about 80 mg / m
present in a concentration between l. Fibrous collagen at various collagen concentrations in suspension was prepared using Zyderm® I collagen (35 mg / ml) and Zyderm II collagen (65 mg / ml).
ml), commercially available from Collagen Corporation.

Native collagen contains a lysine residue having a primary amino group capable of covalently bonding to the hydrophobic and hydrophilic crosslinkers of the present invention, and is therefore desired according to the method of the present invention. It need not be chemically modified in any manner prior to reaction with the crosslinker.

Although intact collagen is preferred, denatured collagen, which is commonly known as gelatin, can also be used in preparing the compositions of the present invention.

The glycosaminoglycans used in the present invention include hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, keratosulfate, chitin, chitosan, heparin and their derivatives and mixtures. , But not limited to these. Glycosaminoglycans are prepared according to the method of the present invention.
It must generally be modified to have primary amino groups that can be attached to functional groups on the hydrophobic and hydrophilic crosslinkers, for example by deacetylation or desulfation. A method for chemically modifying glycosaminoglycans by deacetylation and / or desulfation is described in Nov. 1993.
Pending US patent application Ser. No. 08 / 146,843
(This application is owned by the same assignee as this application). In general, glycosaminoglycans can be deacetylated, desulfated, or both by adding a strong base thereto, such as sodium hydroxide. Deacetylation and / or desulfation results in primary amino groups on this glycosaminoglycan that can be covalently bound to hydrophobic or hydrophilic crosslinkers according to the method of the invention.

A mixture of various glycosaminoglycan species,
Different collagens, or mixtures of different glycosaminoglycans with collagen, can be used to prepare the crosslinked biomaterial compositions of the present invention.

If the end product is intended to be incorporated into the body of a human or animal subject, the biological material used in the present invention may be in pharmaceutically pure form or pharmaceutically pure. It must be able to be refined into various shapes.

Preparation of Hydrophobic Crosslinking Agent To prepare the crosslinked biomaterial composition of the present invention, first, a hydrophobic polymer containing two or more succinimidyl groups, or containing them, is prepared. It is necessary to prepare a hydrophobic polymer that can be derivatized. The term "hydrophobic polymer" as used herein means a polymer containing a relatively small proportion of oxygen or nitrogen atoms. As used herein, the term "containing two or more succinimidyl groups" refers to commercially available hydrophobic polymers containing two or more succinimidyl groups as well as two or more succinimidyl groups. It is meant to include those that must be chemically derivatized to contain groups. The term "succinimidyl group" as used herein is meant to include sulfosuccinimidyl groups, or other variations of "common" succinimidyl groups. The presence of the sodium sulfite moiety on the sulfosuccinimidyl group serves to increase the solubility of the polymer.

The hydrophobic polymer used in the present invention preferably contains or contains two or more succinimidyl groups, most preferably two, three or four succinimidyl groups. Can be derivatized as follows. These succinimidyl groups are primary amino groups
(-NH ₂₎ containing biological material (e.g., collagen and various glycosaminoglycans and glycosaminoglycan derivatives) has high reactivity with.

Hydrophobic polymers already containing two or more reactive succinimidyl groups include disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS ³ ), Dithiobis (succinimidyl propionate) (DPS), bis (2-succinimidooxycarbonyloxy) ethyl sulfone (BSOCOES) and 3,3 '
-Dithiobis (sulfosuccinimidyl propionate)
(DTSSP), and analogs and derivatives thereof, but are not limited thereto. The above polymers are respectively available from Pierce (Rockford, IL) under catalog numbers 21555, 21579, 22585, 21554 and 21577. The structural formulas of the above polymers and general reaction products obtained by reacting each of these polymers with collagen are shown in the following formulas 1 to 5, respectively.

[0066]

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[0067]

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[0068]

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[0069]

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[0070]

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Certain polymers, such as polyacids, can be derivatized to contain two or more reactive succinimidyl groups. The polyacids used in the present invention include trimethylolpropane-based tricarboxylic acids, di (trimethylolpropane) -based tetracarboxylic acids, heptanedioic acid, octanedionic acid (suberic acid), and hexadecanedioic acid (sapsin). Acid (thaps
ic acid)), but is not limited thereto. Many of these polyacids are commercially available from DuPont Chemical Company.

According to the general method, the polyacid is N, N'-
In the presence of dicyclohexylcarbodiimide (DCC),
It can be chemically derivatized to contain two or more succinimidyl groups by reaction with an appropriate molar amount of N-hydroxysuccinimide (NHS).

Polyhydric alcohols (eg, trimethylolpropane and di (trimethylolpropane)) can be converted to the carboxylic acid form using various methods, as shown in Schemes 1 and 2, and then converted to the succinimidyl group. It can be further derivatized by addition.

Trimethylolpropane was derivatized to the tricarboxylic acid form, as shown in Scheme 1 below, and then further derivatized by reaction with NHS in the presence of DCC to give the trifunctional trifunctional compound. It produces a crosslinker (ie, a compound with three succinimidyl groups that is available for reaction with various biological materials).

[0075]

[Chemical 6]

Di (trimethylolpropane) is derivatized to the tetracarboxylic acid form, as shown in Scheme 2 below, and then further derivatized by reaction with NHS in the presence of DCC, Produces a tetrafunctional crosslinker.

[0077]

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Other polyacids were also prepared using methods similar to those shown in Scheme 1 or 2 for trimethylolpropane-based tricarboxylic acid and di (trimethylolpropane) -based tetracarboxylic acid, respectively. Two
It may be chemically derivatized to contain one or more succinimidyl groups. Heptanedioic acid (HOO
C- (CH ₂ ) ₅ -COOH), octanedioic acid (HOOC- (CH ₂ ) ₆ -COO
H), and hexadecanedioic acid (HOOC- (CH ₂ ) ₁₄ -COOH)
Polyacids such as are derivatized by the addition of succinimidyl groups to produce difunctional crosslinkers.

Ethylenediamine (H ₂ N-CH ₂ CH ₂ -NH ₂ ), tetramethylenediamine (H ₂ N- (CH ₂ ) _4- NH ₂ ), pentamethylenediamine (cadaverine) (H ₂ N- (CH ₂ ) _5- NH ₂ ), hexamethylenediamine (H ₂ N- (CH ₂ ) _6- NH ₂ ), bis (2-hydroxyethyl) amine (HN- (CH ₂ CH ₂ OH) ₂ ), bis (2- Polyamines such as aminoethyl) amine (HN- (CH ₂ CH ₂ NH ₂ ) ₂ ), and tris (2-aminoethyl) amine (N- (CH ₂ CH ₂ NH ₂ ) ₃ ) chemically react with polyacids. Containing 2 or more succinimidyl groups by reacting with a suitable molar amount of N-hydroxysuccinimide in the presence of DCC according to the general method described above for polyacids. Can be derivatized as follows. Many of these polyamines are commercially available from DuPont Chemical Company.

The preferred hydrophobic polymers for use in the present invention, whether commercially available containing two or more succinimidyl groups, are chemically modified to contain two or more succinimidyl groups. If derivatized, it will generally have carbon chains of about 14 carbon lengths or less. Polymers with carbon chains substantially longer than 14 carbons have very low solubilities in aqueous solutions, which alone result in very long reaction times when mixed with aqueous solutions of biological substances (e.g. collagen). .

Crosslinked biomaterial composition using hydrophobic crosslinking agent
In the general method for preparing the preparation crosslinked biological material compositions of the present invention,
A biological material containing a primary amino group, or a biological material that has been chemically derivatized to contain such a group, has two or more succinimidyl groups (which are (Which can cross-link this biological material by reacting it with a nucleophilic primary amino group) of, or a hydrophobic polymer derivatized to contain such groups. Mixed with coalesced. The hydrophobic cross-linking agent can be stored and used in either dry or solution form, but is preferably used in dry form. The cross-linking agent can be mixed with either an aqueous solvent or a hydrophobic solvent prior to mixing with the biological material. If an aqueous solvent is used, the succinimidyl group is reactive with neutrofil (eg, oxygen and water) so the crosslinker should be mixed with the solvent immediately before use. Upon prolonged exposure to an aqueous solvent, hydrolysis of this crosslinker causes it to lose its crosslinkability.

These biomaterials and the hydrophobic crosslinker (in dry form) can be stored in separate syringes and then mixed using the syringe-syringe mixing method as follows. The syringe containing the biological material is connected to the syringe containing the crosslinker using a syringe connector (eg, a three-way stopcock) and the materials are allowed to mix until they are well mixed. By moving back and forth between individual syringes (generally a minimum of about 20 passes is required, where the total amount of these substances is The biomaterial and crosslinker are mixed. During this mixing step, cross-linking starts between the molecules of the biomaterial and the cross-linking agent.

The concentration of the hydrophobic crosslinker used in practicing the present invention will depend on many factors, including the type and molecular weight of the crosslinker used, the type and concentration of the biological material used. , And the desired degree of crosslinking. In general, the inventor has discovered that a concentration of hydrophobic crosslinker in the range of about 0.1 to about 2% by weight of the final composition is preferred for preparing the crosslinked biomaterial composition of the present invention.

Mixtures of hydrophobic and hydrophilic crosslinkers
Preparation of Heterogeneous Cross-Linked Biomaterial Compositions Using a General Method of Preparing Heterogeneous Cross-Linked Biomaterial Compositions of the Invention comprises a biological material containing primary amino groups or containing such groups. The biological material, which has been chemically derivatized as described above, is combined with a mixture of hydrophobic and hydrophilic cross-linking agents and covalently bonded. Preferably, the mixture of hydrophobic and hydrophilic crosslinkers is stored and used in dry form in order to prevent loss of crosslinkability by hydrolysis. The hydrophobic cross-linking agent and the hydrophilic cross-linking agent are
Generally, they do not react with each other. Both crosslinkers contain the same reactive groups (i.e., succinimidyl groups) that preferentially bind to primary amino groups on various biological materials (e.g., collagen and derivatized glycosaminoglycans). .

In the alternative, the biomaterial is first mixed with either the hydrophobic or hydrophilic crosslinker and then (preferably rapidly and continuously before gelation occurs). , Mixed with other types of crosslinkers.

The term "hydrophobic polymer" as used herein means a polymer containing a relatively small proportion of oxygen or nitrogen atoms. Hydrophobic polymers containing two or more reactive succinimidyl groups, or chemically derivatized to contain such groups, are heterogeneous crosslinks of the present invention. It is the preferred hydrophobic cross-linking agent used to prepare the biomaterial composition.

The term "hydrophilic polymer" as used herein means a polymer containing a relatively large proportion of oxygen and / or nitrogen atoms, these atoms being linked by hydrogen bonds. Helps to attract water molecules. Synthetic hydrophilic polymers (eg, functionally activated polyethylene glycols) are the preferred hydrophilic crosslinkers used to prepare the heterogeneous crosslinked biomaterial compositions of the present invention. Various activated forms of polyethylene glycol are disclosed in U.S. Pat.No. 5,328,955, which is owned by the same assignee as the present application, the disclosure of which is incorporated herein by reference, and No. 08 / 344,040, filed November 3, 1994, which is under examination at the time of filing this application.

The synthetic hydrophilic polymer used in the present invention is
It is preferably polyfunctionally activated, and more preferably bifunctionally activated. Preferred synthetic hydrophilic polymers are PEG succinimidyl glutarate (SG-PEG), PEG succinimidyl (SE-PEG; in the bifunctionally activated form, which in U.S. Pat. Called "S-PEG"), PEG succinimidyl succinamide (SSA-PEG), and PEG succinimidyl carbonate (SC-PEG). By reacting SG-PEG with a biological substance (eg, collagen), a covalently bound conjugate containing an ester bond is obtained, and SE-PEG (n = 1 to 3)
Alternatively, the reaction of SC-PEG (n = 0) with a biological substance gives a conjugate containing an ether bond, and SSA-PEG (n
= 1 to 10) with a biological substance, a conjugate containing an amide bond is obtained. The amide and ether linkages are generally less susceptible to hydrolysis than the ester linkages, thus depending on the environment in which the implant material is placed, a crosslinked biomaterial composition with high stability and in vivo durability is obtained. Can be done. Ether bonds are susceptible to oxidation and may be susceptible to deterioration due to free radicals.

Many of the above activated forms of polyethylene glycol are now commercially available from Shearwater Polymers (Huntsville, Alabama), and Union Carbide (South Charleston, West Virginia).

Use and Administration The crosslinked biomaterial compositions of the present invention include shaped implants for use in various medical applications, including various artificial organs, and tubular implants for use as vascular grafts and / or stents. Is particularly useful for the preparation of In the general method of preparing shaped implants, the biomaterial / crosslinker reaction product prepared as described above is preferably applied before significant crosslinking between the biomaterial and the crosslinker (or mixture of crosslinkers) occurs. Then, it is extruded into molds of various sizes and shapes. This time will vary depending on both the type and concentration of the biomaterial and crosslinker used, but is generally in the range of about 5 minutes to about 60 minutes. The material should be removed from the mold only after a period of time sufficient for equilibrium crosslinking between the biomaterial and the cross-linking agent to occur. If necessary, remove any unbound residual crosslinker before placing this implant in the patient.
It may be removed from this implant.

The biomaterial / crosslinker mixture may also be used (eg by extrusion, dipping, brushing or painting), in one of the shaped synthetic implants (eg artificial bone prostheses or synthetic vascular grafts or stents). It can be applied to one or more surfaces and crosslinked in situ, thereby providing a crosslinked non-immunogenic biomaterial coating on the surface of the implant. Alternatively, all or a portion of the molded synthetic implant may be dipped into a container containing the biomaterial / crosslinking agent reaction solution.

The biomaterial / crosslinking agent mixture can be extruded in the form of threads and crosslinked in that form. Once the yarn is fully crosslinked, it can be dried to remove substantially all unbound water. This dried thread is passed through a needle to effect soft tissue augmentation, and the skin site requiring correction (e.g.,
Can be inserted into crushed scars, or wrinkles). The dried thread can also be cut into fine pieces, suspended in a non-aqueous carrier, and injected into the tissue site in need of augmentation.
This tissue site may be a skin site or other soft tissue site such as a poorly functioning sphincter (e.g., urethral sphincter,
Anal sphincter or esophageal sphincter). When exposed to body fluids, the crosslinked threads rehydrate in situ and swell to approximately 5 times their dry diameter. The dried thread may also be used as a suture material, or as a knit, knit or woven material to provide a biomaterial for tendon or ligament repair or replacement.

In order to provide a suitable material for hard tissue augmentation (eg, repair or replacement of bone or cartilage), a suitable particulate material may be added prior to mixing the biomaterial with the crosslinker.
(Eg, ceramic particles) may be mixed with the biological material. These substances may be applied in liquid form to the defect site of bone or cartilage prior to cross-linking and are similar to the methods described above for the preparation of implants that are cross-linked in situ or formed for repair of soft tissue. Molded bone or cartilage implants can be prepared using the method of 1. and then molded or cut to the desired size and shape.

The cross-linked biomaterial composition of the present invention may also be used as an injectable formulation in the augmentation of soft or hard tissues of the body. Following the mixing of the biomaterial and the cross-linking agent, the reaction product should be injected at the tissue site before significant cross-linking occurs to prevent occlusion of the needle with the cross-linking composition. If the substance is injected into the tissue site before equilibrium cross-linking occurs, the functional groups on the cross-linking agent can bind to collagen molecules within the recipient tissue, thereby allowing the bio-substance to bind to the recipient tissue. Biological mooring occurs. Implants that are "biologically anchored" in the recipient tissue can be difficult to move and therefore more durable in vivo than currently available injectable biomaterial compositions.

The bioactive agent (eg, a cytokine or growth factor) may be added to the present invention by simple mixing or by covalently linking the active agent to the crosslinker prior to incorporating the crosslinker into the biological material. Can be mixed with the composition. The active agent recruits cells to the implant area and also helps anchor the implant to the recipient tissue and, when applied to the wound site, may promote wound healing.

[0096]

The following examples are presented in order 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 of the invention. It is not intended to limit the scope of what the inventors regard as the 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 deviations should be accounted for.
is there. Unless otherwise indicated, parts are parts by weight and molecular weights are parts by weight.
Average molecular weight, temperature is in degrees Celsius, and pressure is at atmospheric pressure.
Or close to it.

Example 1 Preparation and Preparation of Crosslinked Collagen Composition Using Hydrophobic Crosslinking Agent
And characterization fibrous collagen (Zyderm® I collagen; Col
obtained from lagen Corporation (Palo Alto, CA) and methylated (non-fibrillar) collagen (approximately 1-3 at 21 ° C).
(Prepared by reacting fibrillar collagen with methanol for a day), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS
³ ), bifunctionally activated SE-PEG (n = 2, Mw 3800),
And cross-linked with bifunctionally activated SG-PEG (Mw 3800).

Contents of a 1 cc syringe containing 1.0 cc of Zyderm collagen (collagen concentration of 35 mg / ml) and contents of a 1 cc syringe containing one of the following cross-linking agents in the following amounts: A fibrillar collagen preparation was prepared by mixing with: 3 mg DSS; 3 mg BS ³ ; 5 mg SE-.
PEG; or 5 mg SG-PEG.

Contents of a 1 cc syringe containing 1.0 cc of methylated collagen (21 mg / ml collagen concentration) and 1 cc syringe containing one of the following cross-linking agents in the following amounts: A methylated collagen formulation was prepared by mixing with: 3 mg DSS; 3 mg BS ³ ; 10 mg
SE-PEG; or 10 mg SG-PEG.

All crosslinkers were used in dry form. The crosslinker and collagen were mixed by passing the material between the syringes using a three-way stopcock, using about 40-50 passes between the two syringes. Once this collagen and the cross-linking agent are thoroughly mixed,
Transfer this material to one syringe and at 37 ° C for approximately
Incubated for 16 hours.

Each of the crosslinked collagen materials prepared above was extruded from the plunger end of its syringe. The resulting cross-linked cylindrical gel was then divided into 5 mm thick discs. Each formulation was then evaluated according to some or all of the following test methods: differential scanning calorimetry (DSC), soluble in 1 mg / ml trypsin solution, and 3% aqueous hydrogen peroxide (H ₂ Oxidative deterioration in O ₂ ). The results of these evaluations are presented in Table 1 below.

[0102]

[Table 1]

Differential Scanning Calorimetry (DSC) is used to measure the heat denaturing transition of collagen, which can be used to assess the relative strength of the resulting crosslinks. As indicated by the above DSC results, crosslinking of fibrous collagen by hydrophobic crosslinking agent DSS and BS ³ includes a cross-linking obtained with a hydrophilic crosslinking agent SG-PEG, it is robust at least the same extent. For methylated (non-fibrillar) collagen formulations, slightly lower numbers were obtained.

Solubility in trypsin solution was carried out by incubating a 5 mm thick disc of each cross-linked material at 37 ° C. in a solution containing 1 mg trypsin in 1 ml of water to disperse the cross-linked collagen gel. It was determined by measuring the period required for As shown above, dissolving the DSS-ZI gel requires approximately twice the time (7 days each) required to dissolve the SEPEG-ZI and SGPEG-ZI gels (3 days each). Required, which indicates that DSS was more strongly cross-linked (ie, higher cross-link density) to fibrillar collagen than either SEPEG or SGPEG. Methylated collagen formulations have generally been shown to be less stable in trypsin solution, while methylated collagen formulations crosslinked with DSS are those crosslinked with either SEPEG or SGPEG. , A significant improvement in stability.

Oxidative degradation was measured by incubating a 5 mm thick disc of each crosslinked material at 37 ° C. in a 3% aqueous hydrogen peroxide solution and measuring the time required to disperse the crosslinked collagen gel. It was decided. Similar to the trypsin solubility results above, to dissolve the DSS-ZI gel, SEPEG-ZI gel (10 days) and SGPEG-ZI gel (7 days)
Approximately twice as much time (14 days) as it takes to dissolve
, Which indicated that DSS was more strongly cross-linked to fibrillar collagen than either SEPEG or SGPEG. Therefore, collagen materials cross-linked with hydrophobic cross-linkers are significantly more unexpectedly improved in terms of trypsin sensitivity and susceptibility to oxidative degradation than those cross-linked with hydrophilic cross-linkers previously described in the art. It was observed.

Example 2 In Vivo Durability of Crosslinked Collagen Composition Zyplast® Collagen (Glutaraldehyde crosslinked collagen having a collagen concentration of 35 mg / ml,
Collagen Corporation (obtained from Palo Alto, CA) and Zyderm (R) collagen mixture (weight ratio 70:30) 1.0 gram containing 1.0 gram of syringe contents.
A cross-linked collagen formulation was freshly prepared by mixing with the contents of a 1 cc syringe containing either mg DSS, 3 mg SE-PEG or 3 mg SG-PEG. Zyplast
A 70:30 weight ratio of non-crosslinked mixture of Zyderm collagen was used as a control. 24 Male Sprague-Dawley Each
Two groups of rats, according to the following schedule,
Two of the four formulations were injected with implants of 0.5 ml each.

Animal Group A: Site 1 Zyplast / Zyderm Collagen Mixture (Control) Site 2 Zyplast / Zyderm Collagen Mixture Crosslinked with DSS Animal Group B: Site 1 Zyplast / Zyderm Collagen Mixture Site Crosslinked with SG-PEG 2 Zyplast / Zyderm collagen mixture crosslinked with SE-PEG.

The materials were injected subcutaneously with a 27 gauge needle within approximately 5 minutes after mixing the collagen and crosslinker.

Six animals from each of groups A and B were selected and sacrificed at 7, 14, 28 and 90 days after transplantation. Implants were excised with surrounding tissue and examined histologically. The injected crosslinked material had a discontinuous elliptical and bolus-like morphology, while the non-crosslinked formulation was present as a more diffuse mass. Implants from 4 animals in each group,
Used for histological studies and wet weight experiments. 2 for each group
An implant from a single animal was used to measure the mechanical force required to remove the implant from the receiving tissue. The results of this histological study and wet weight experiments are described below.

Excised implants were examined histologically and 0-4 for each of the three parameters of inflammatory infiltration, fibroblast in-growth and fibroplasia.
It was scored by the grades up to. A score of 4 indicates that the maximum amount of parameters is present, and a score of 0 indicates that no specific parameter associated with the examined implant was observed (ie 0 for inflammatory infiltrates means that implant). No inflammatory infiltration was observed at the site). The results of the histological examination are presented in Tables 2, 3 and 4 and described below. Average points are shown in parentheses.

[0111]

[Table 2]

Collagen implants crosslinked with DSS showed a mild inflammatory response at days 7 and 14, which was slightly higher than other (crosslinked and non-crosslinked) collagen compositions. . By the 28th day, this DSS
Inflammatory infiltration into the cross-linked implants was minimal and was reduced to non-existence by day 90.

[0113]

[Table 3]

Unlike other cross-linked and non-cross-linked collagen formulations, the DSS cross-linked implants showed no signs of fibroblast ingrowth throughout the duration of this study. The cause is most likely due to the very rigid crosslinked collagen network obtained using DSS as the crosslinker.

[0115]

[Table 4]

Fiber growth was observed to be similar for all three cross-linked collagen compositions examined.

The weight of each implant was measured following explantation. The wet weight of the implant is shown in Figure 1 as a percentage of the initial weight of the implant for each of the four formulations at each time point. There was no significant difference between any of the formulations on days 7, 14 and 28. However, on day 90, the collagen formulations crosslinked with DSS had significantly better wet weight retention (close to 100%) than the other formulations. Histological examination showed no in-growth of fibroblasts, suggesting that the wet weight of the DSS cross-linked implant consisted essentially of the implant material itself, rather than the invading cells. To be This view was that this DSS cross-linked collagen implant was not resorbed into its target tissue as fast as other collagen implant materials, probably due to the solid cross-linking network obtained using DSS as the cross-linking agent. Means

At each of the 7th, 28th and 90th day of this study, a portion of the skin containing the implant was excised from two animals in each of the groups A and B. The skin surrounding this implant was shaped into the same rectangular shape measuring 2 cm x 4 cm. The encapsulated tissue grown on the surface of the implant was removed so that the implant appeared to simply rest on the surface of the skin.

A piece of skin containing the implant was cut into pieces using one thumbtack at each of the four corners of the skin.
Pinned to a cm x 5 cm wooden board. As shown in Fig. 2, slings (s
ling). Instron type universal testing machine
Using the Model 4202, secure the wooden board (which has the skin fragments attached) to one clamp of the Instron tester, and the other end of the sling to the other clamp. The mechanical force required to remove the implant from this tissue was measured by fixing the. The clamp holding the sling was pulled on the Instron tester until the implant broke apart from the tissue. For each of the four formulations on days 7, 28 and 90,
It is shown as a graph in FIG. There was no significant difference between the formulations crosslinked with the hydrophobic crosslinker (DSS) and either the hydrophilic crosslinker (SE-PEG; SG-PEG) .

Example 3 A rack containing a mixture of a hydrophobic crosslinker and a hydrophilic crosslinker.
Preparation and Characterization of Hashi Biomaterial Composition Fibrous Collagen (Zyderm® I Collagen, 35
Collagen concentration of mg / ml; Collagen Corporation (Palo A
(obtained from lto, CA)), disuccinimidyl suberate
(DSS), SE-PEG bifunctionally activated (n = 2, Mn 380
0), or 50:50 of SE-PEG bifunctionally activated with DSS
The mixture (by weight) was used to crosslink. The contents of a 5 cc syringe containing 5.0 grams of Zyderm collagen were
A crosslinked collagen formulation was prepared by mixing with the contents of a 5 cc syringe containing either g DSS, 15 mg SE-PEG or 15 mg DSS / SE-PEG mixture.

All crosslinkers were used in dry form. this
The DSS / SE-PEG mixture contains 5 mg each of DSS and SE-PEG.
Just before cross-linking, by placing in a cc syringe and then shaking the syringe by hand to mix the two cross-linking agents,
Prepared.

The crosslinker and collagen were mixed by passing the material between the two syringes using a three-way stopcock, using about 40-50 passes between the two syringes. Once the collagen and crosslinker were mixed well, the material was transferred to one syringe and incubated at 37 ° C for approximately 16 hours.

Each of the crosslinked collagen materials prepared above was extruded from the plunger end of its syringe. The resulting cross-linked cylindrical gel was then divided into 5 mm thick discs. The three formulations were evaluated using differential scanning calorimetry (DSC). The gel strength of each formulation is
The measurement was performed using an Instron type universal testing machine Model 4202. Each of these three crosslinked collagen formulations
The DSC and gel strength results are presented in Table 5 below.

[0124]

[Table 5]

The fact that the DSC and gel strength results are inconsistent for collagen compositions prepared with a mixture of hydrophobic and hydrophilic crosslinkers is due to some of the following factors: Possible: The two crosslinkers were not thoroughly mixed prior to mixing with collagen; the heterogeneity of the composition itself; and, perhaps, the proportion of crosslinkers was not optimal. Other factors may include that SE-PEG can cross-link collagen more quickly than DSS due to the poor solubility of DSS in aqueous solutions of collagen fibers.

Collagen compositions prepared with a mixture of hydrophobic and hydrophilic crosslinkers have a relative contribution of two different types of crosslinkers to the properties of the final composition (hydrophobic crosslinker). Agents may contribute to improved stability and hydrophilic crosslinkers may contribute to improved elasticity and overall good handling) and may be useful in certain therapeutic applications.

The present invention discloses a novel biomaterial composition prepared by using a hydrophobic polymer as a crosslinking agent. Preferred hydrophobic polymers are those having two or more reactive succinimidyl groups, such as disuccinimidyl suberate, bis (sulfosuccinimidyl).
Suberates, and dithiobis (succinimidyl propionate). Also disclosed are crosslinked biomaterial compositions prepared with a mixture of hydrophobic and hydrophilic crosslinkers. The compositions of the present invention can be used to prepare shaped implants for use in various medical applications.

The present invention is not intended to be limited by the above embodiments used for purposes of illustration. The invention is intended to have the scope defined in the appended claims.

[0129]

INDUSTRIAL APPLICABILITY According to the present invention, there are provided injectable or implantable crosslinked biomaterial compositions prepared by using a hydrophobic crosslinking agent for various therapeutic applications. Also,
According to the present invention, a unique crosslinked biomaterial composition is provided using a mixture of hydrophobic and hydrophilic crosslinkers. Additionally, the present invention provides compositions that are particularly useful in preparing shaped implants for various medical applications.

The compositions of the present invention have many unique and unexpected characteristics compared to previously disclosed crosslinked biomaterial compositions prepared using only hydrophilic crosslinkers. An important feature of the compositions of the present invention (compared to conventional cross-linked biomaterial compositions) is their slow degradation, resulting in higher chemical stability, which is their in vivo persistence. Can lead to an increase in

[Brief description of drawings]

FIG. 1 is a graph showing the wet weight of various implants recovered from rats as a percentage of their initial weight.

FIG. 2 is a schematic diagram showing a method of testing to measure the mechanical force required to remove an implant from receiving tissue.

FIG. 3 is a graph showing the force with which an implant is anchored to a recipient tissue.

Claims (46)

[Claims] 1. A conjugate containing a biological substance covalently bound to a hydrophobic polymer, wherein the hydrophobic polymer is:
A conjugate containing two or more succinimidyl groups prior to binding to the biological material. 2. The hydrophobic polymer is disuccinimidyl suberate, bis (sulfosuccinimidyl) suberate, dithiobis (succinimidyl propionate), bis (2-succinimidooxycarbonyloxy) ethyl. The conjugate of claim 1 selected from the group consisting of sulfone, 3,3'-dithiobis (sulfosuccinimidyl propionate), and analogs and derivatives thereof. 3. The conjugate of claim 1, wherein the hydrophobic polymer is chemically derivatized to contain two or more succinimidyl groups. 4. The conjugate according to claim 3, wherein the hydrophobic polymer is a polyacid. 5. The polyacid is trimethylolpropane
A conjugate according to claim 4, which is selected from the group consisting of: -based tricarboxylic acids, di (trimethylolpropane) -based tetracarboxylic acids, heptanedioic acid, octanedioic acid and hexadecanedioic acid. 6. The conjugate according to claim 1, wherein the hydrophobic polymer contains 2, 3 or 4 succinimidyl groups before binding to the biological substance. 7. The conjugate according to claim 1, wherein the biological material is selected from the group consisting of collagen, gelatin, glycosaminoglycans and mixtures thereof. 8. The conjugate according to claim 7, wherein the biological substance is collagen. 9. The conjugate according to claim 8, wherein the collagen is fibrous collagen. 10. The conjugate according to claim 8, wherein the collagen is non-fibrillar collagen. 11. The conjugate according to claim 10, wherein the non-fibrillar collagen is a chemically derivatized collagen selected from the group consisting of succinylated collagen and methylated collagen. 12. The biological material is hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, keratosulfate, chitin, chitosan,
The conjugate according to claim 7, which is a glycosaminoglycan selected from the group consisting of heparin and derivatives thereof. 13. The conjugate of claim 7, wherein the hydrophobic polymer is covalently bound to both collagen and one or more glycosaminoglycan species. 14. The conjugate of claim 7, wherein the hydrophobic polymer is covalently bound to two or more glycosaminoglycan species. 15. A conjugate containing a biomaterial covalently bound to a hydrophobic polymer, wherein the hydrophobic polymer is two or more succinimidyls prior to binding with the biomaterial. A conjugate that has been chemically derivatized to contain a group. 16. The hydrophobic polymer is a polyacid.
The conjugate according to claim 15. 17. The polyacid is selected from the group consisting of trimethylolpropane-based tricarboxylic acid, di (trimethylolpropane) -based tetracarboxylic acid, heptanedioic acid, octanedioic acid and hexadecanedioic acid. The conjugate according to claim 16. 18. The method of claim 15, wherein the hydrophobic polymer is chemically derivatized to contain 2, 3 or 4 succinimidyl groups prior to binding with the biological material. The conjugate as described. 19. The conjugate according to claim 15, wherein the biological material is selected from the group consisting of collagen, gelatin, glycosaminoglycans and mixtures thereof. 20. The biological material is collagen.
The conjugate according to claim 19. 21. The conjugate according to claim 20, wherein the collagen is fibrous collagen. 22. The conjugate according to claim 20, wherein the collagen is non-fibrillar collagen. 23. The conjugate according to claim 22, wherein the non-fibrillar collagen is a chemically derivatized collagen selected from the group consisting of succinylated collagen and methylated collagen. 24. The biological substance is hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, keratosulfate, chitin, chitosan,
20. The conjugate of claim 19, which is a glycosaminoglycan selected from the group consisting of heparin and their derivatives. 25. The conjugate of claim 19, wherein the hydrophobic polymer is covalently bound to both collagen and one or more glycosaminoglycan species. 26. The conjugate of claim 19, wherein the hydrophobic polymer is covalently bound to two or more glycosaminoglycan species. 27. A heterogeneously crosslinked biomaterial composition comprising a biomaterial, a hydrophobic crosslinker and a hydrophilic crosslinker. 28. The composition of claim 27, wherein the hydrophobic crosslinker contains two or more succinimidyl groups. 29. The hydrophobic crosslinking agent is disuccinimidyl suberate, bis (sulfosuccinimidyl) suberate, dithiobis (succinimidyl propionate),
29. The composition of claim 28, selected from the group consisting of bis (2-succinimidooxycarbonyloxy) ethyl sulfone, 3,3'-dithiobis (sulfosuccinimidyl propionate), and analogs and derivatives thereof. Stuff. 30. The composition of claim 27, wherein the hydrophobic crosslinker comprises a hydrophobic polymer chemically derivatized to contain two or more succinimidyl groups. 31. The hydrophobic polymer is a polyacid.
The composition of claim 30. 32. The polyacid is selected from the group consisting of trimethylolpropane-based tricarboxylic acid, di (trimethylolpropane) -based tetracarboxylic acid, heptanedioic acid, octanedioic acid and hexadecanedioic acid. 32. The composition according to claim 31. 33. The composition of claim 28 or 30, wherein the hydrophobic crosslinker contains 2, 3 or 4 succinimidyl groups. 34. The composition of claim 27, wherein the hydrophilic crosslinker is a synthetic hydrophilic polymer. 35. The synthetic hydrophilic polymer is a functionally activated polyethylene glycol.
The composition according to 4. 36. The composition of claim 35, wherein the functionally activated polyethylene glycol is a polyfunctionally activated polyethylene glycol. 37. The composition of claim 36, wherein the polyfunctionally activated polyethylene glycol is a bifunctionally activated polyethylene glycol. 38. The composition of claim 27, wherein the biological material is selected from the group consisting of collagen, gelatin, glycosaminoglycans and mixtures thereof. 39. The biological material is collagen.
39. The composition of claim 38. 40. The composition of claim 39, wherein the collagen is fibrillar collagen. 41. The composition of claim 39, wherein the collagen is non-fibrillar collagen. 42. The composition of claim 41, wherein the non-fibrillar collagen is a chemically derivatized collagen selected from the group consisting of succinylated collagen and methylated collagen. 43. The biological material is hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, keratosulfate, chitin, chitosan,
39. The composition of claim 38, which is a glycosaminoglycan selected from the group consisting of heparin and their derivatives. 44. The biological material is collagen or 1
39. The composition of claim 38, which comprises a mixture of one or more glycosaminoglycan species. 45. The composition of claim 38, wherein the biological material contains a mixture of two or more glycosaminoglycan species. 46. A molded implant containing the conjugate of claim 1 or 15 or the composition of claim 27.