Patent Description:
Natural tissue bioimplants are gaining acceptance as advantageous alternatives to synthetic implants in many surgical procedures. Among other advantages, bioimplants more closely resemble in size, shape and performance the biological structures that they are designed to replace than do synthetic implants. Thus bioimplants are, in many circumstances, considered the devices of choice for replacement or structural augmentation of internal tissues and organs.

The sources of bioimplants include non-human and human donors. In general, the choice of donor depends on a number of factors, including the relative sizes of the donor and recipient. For example, as an alternative to a human cadaver, a sheep, pig, cow or horse may serve as a donor. In some cases, the donor and recipient may be the same. Immunogenic limitations are overcome by crosslinking the tissues to mask antigenic molecules in the tissue. Sterilization is generally effected by contacting the tissue with a chemical sterilizing agent. In many cases, crosslinked and sterilized bioimplants provide many of the features of natural tissue, while avoiding to a great degree the problem of xeno-tissue rejection that is characteristic of live tissue implantation.

In many cases, bioimplants provide additional advantages over synthetic implants. For example, many bioimplants permit infiltration of the recipient's own cells into the bioimplant. In particular, the infiltrating cells can use the bioimplant as a template or scaffold for re-constructing organ or tissue structures comprising the recipient's own cells. In some cases, all or part of the bioimplant can be replaced by the recipient body's own cells. This process, which is referred to as remodeling, is advantageous in that it can improve the integration of the bioimplant into the implant site. Due to these advantages, it is considered advantageous to promote remodeling of bioimplant tissue.

While some bioimplants can stimulate remodeling by themselves due to their natural origin and their possession of a collagen matrix that acts as a scaffold for tissue regrowth, it is sometimes considered advantageous to stimulate remodeling by administering to a bioimplant recipient one or more agents that stimulate tissue growth. For example, bone morphogenic proteins (BMPs) have been used experimentally to promote bone regrowth in spinal fusion surgery. For example, a resorbable collagen sponge infused with recombinant bone morphogenic protein-<NUM> (rhBMP-<NUM>) has been approved for use in spinal surgery. It is believed that release of rhBMP-<NUM> from the sponge stimulates osteoblast infiltration, proliferation and organization. As the collagen sponge is resorbable, eventually regrown host tissue replaces the sponge. The use of the rhBMP-<NUM> infused collagen sponge in spinal surgery has been credited with greatly reducing the failure rate of spinal surgery.

Despite the improvements in surgical outcomes that have already been provided by growth factor infused bioimplants, many challenges remain to be overcome. For example, infusion of bioimplants is only useful where the bioimplant is absorbent, that is where soaking of the tissue in a solution containing the growth factor results in there being enough growth factor infused into the tissue to stimulate tissue growth after it has been implanted into a recipient. Thus, the infusion method is not considered effective for less porous bioimplant devices such as heart valves, skin grafts, tendon, bone and ligament repair tissues, etc. Another limitation is that release of the growth factor is by diffusion. While diffusion can in some instances be a useful method of release, in other circumstances diffusion may result in too high an initial rate of release and thus too low a later rate of release. Thus, one disadvantage of diffusive release is that the effective release period may be shorter than desired, unless excess growth factor is infused into the bioimplant at the start. However, this may not always be feasible or even possible. Moreover, even if it were possible to infuse excess growth factor into the bioimplant, a disadvantage arising out of this approach may be that the local concentration of growth factor may cause diffusion of the growth factor into surrounding tissue, including capillaries, veins and arteries, where it may bring about deleterious local or systemic effects. In some cases, such diffusion may even give rise to new tissue growth in an area distal to the area where new growth is desired.

There is thus a need for a device that overcomes the limitations of the prior art growth factor infused collagen sponge. There is a need for a bioimplant device that is capable of delivering growth factor to a desired area, wherein the growth factor is released from the bioimplant device at a rate that is less than the diffusive rate of release from the prior art growth factor-infused collagen sponge. There is likewise a need for a bioimplant device that has associated with it a growth factor that is subject to degradation of the growth factor to a lesser degree than is the growth factor-infused collagen sponge of the prior art. There is also a need for a bioimplant device that has associated with it a growth factor that is covalently bonded to the bioimplant. There is likewise a need for processes of making such bioimplant devices. These and other needs are met by embodiments of the invention.

There is also a need for a bioimplant device that carries an adjunct. There is also a need for a bioimplant device that is capable of delivering an adjunct to a desired area, wherein the adjunct is released from the bioimplant at a rate that is less than the diffusive rate of release of the adjunct from an infused collagen sponge. There is likewise a need for a bioimplant device that has associated with it an adjunct that is subject to degradation to a lesser degree than an adjunct in an adjunct-infused collagen sponge. There is also a need for a bioimplant device that has associated with it an adjunct molecule that is covalently bonded to the bioimplant. There is likewise a need for processes of making such bioimplant devices. These and other needs are met by embodiments of the invention. <CIT> relates to a porous delivery scaffold and method. <CIT> relates to a tissue repair fabric. <CIT> relates to peracetic acid crosslinked non-antigenic ICL grafts.

The foregoing and further needs are met by embodiments of the invention, which provide a chemically sterilized bioimplant according to claim <NUM>.

According to the claimed invention, the sterilization of the bioimplant is carried out in the presence of a penetration enhancer, whereby the penetration enhancer is a C<NUM> -C<NUM> alkanol, and most particularly isopropanol.

The chemically sterilized biological tissue is sterilized with a carbodiimide, such as EDC, in the presence of a C2 -C4 alkanol. especially isopropanol. Additionally, the chemically sterilized biological tissue is crosslinked with a carbodiimide, optionally in the presence of a bifunctional crosslinking agent. The biological tissue comprises processed tissue in native form, i.e. the starting tissue is not subjected to a processing step other than decellularization and/or defatting prior to conjugation of the heparin to the tissue. The biological tissue is selected from the group comprising decellularized crushed bone fragments, decellularized and/ or defatted bone, tendon, ligament, fascia, and combinations thereof, and/or bone-connective tissue combinations. In some embodiments, the adjunct retains at least some of its native activity after it has been conjugated to the biological tissue. In some embodiments, the adjunct is adapted to be released in vivo and the adjunct, once released in vivo possesses at least some of its native activity.

A better understanding of the features and advantages of embodiments of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:.

The present invention provides bioimplants. Generally speaking, the bioimplants comprise biological tissues that are chemically sterilized, and have adjunct molecules covalently attached (conjugated) thereto. According to the present invention, the adjunct molecule is heparin. Such bioimplants have notable advantages over prior art biological tissues, such as improved wound healing, tissue remodeling, tissue growth and tissue regrowth. Additionally, because the adjuncts are conjugated to the biological tissues, in some embodiments they are released at a rate that is generally less than the rate of diffusion of the adjunct from similar biological tissues wherein the adjuncts are merely infused into the biological tissues. This is especially advantageous for growth factors and other adjuncts that have activity at very low concentrations, but that are advantageously delivered over a long period of time. This is also advantageous for small molecules, which being of lower molecular weight, diffuse relatively rapidly out of tissues when they are merely infused therein. Covalent conjugation of the small molecules to the biological tissue permits a slower, more regulated release of the adjunct, thereby providing an effective local activity of the adjunct over a longer period of time. In some embodiments, the adjunct molecules retain native activity when conjugated to the biological tissue; and in some embodiments the adjunct molecules regain native activity when released from the biological tissue. Other advantages of the bioimplant of the present invention will become apparent to the person of skill in the art upon consideration of the disclosure herein.

As used herein, the term "bioimplant" refers to a device comprising a biological tissue that has been subjected to one or more process steps to render it amenable to implantation. In general, the bioimplant is a chemically sterilized bioimplant having conjugated thereto at least one adjunct. In particular embodiments, the bioimplant is chemically sterilized with a water soluble carbodiimide, such as <NUM>-ethyl-<NUM>-(<NUM>-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The biological tissue is crosslinked with a carbodiimide. Carbodiimide-mediated crosslinking is described in detail in <CIT> and U. Patent Publication No. <CIT>. In some particular embodiments, the invention specifically excludes bioimplants comprising biological tissue that has been treated with glutaraldehyde, especially glutaraldehyde solution or vapor.

The bioimplants of the present invention comprise a biological tissue that is chemically sterilized. In particular, the present invention provides a bioimplant that comprises a biological tissue that has been sterilized with a chemical agent capable of reducing the population of bacteria and/or spores in the biological tissue by at least <NUM> logs, especially at least about <NUM> logs, more especially at least <NUM> logs, and particularly at least about <NUM> logs, whereby the sterilizing agents include a carbodiimide such as <NUM>-ethyl-<NUM>-(<NUM>-dimethyl aminopropyl)carbodiimide hydrochloride (EDC). The use of EDC as a sterilizing agent is described in detail e.g. in <CIT>. In some particular embodiments, the invention specifically excludes those bioimplants sterilized with γ-radiation. In other particular embodiments, the invention excludes tissues sterilized solely with γ-radiation.

The bioimplants of the present invention have covalently bonded to them at least one "adjunct molecule," also referred to herein simply as an "adjunct. " According to the claimed invention, this at least one adjunct molecule is heparin. The adjunct molecule is a non-endogenous molecule that promotes tissue healing, remodeling, growth or regrowth in the recipient body. The adjunct molecule exerts this tissue healing, remodeling, growth or regrowth activity in the conjugated state (that is while bound to the bioimplant) or upon release into the immediate environment of the bioimplant. The adjunct molecule is non-endogenous in the sense that it is outside the body of the bioimplant recipient when it is conjugated to, and becomes part of, the bioimplant. Thus adjunct molecules specifically exclude growth factors and other molecules that are within the body of the bioimplant recipient at the time of implantation and become covalently bonded to the bioimplant only incidentally and only during or after implantation of the bioimplant. However, adjunct molecules specifically include molecules from within the body of the intended recipient that are isolated, purified or otherwise treated to enhance their concentration, purity, activity or a combination thereof, before being conjugated to the biological tissue outside the recipient's body.

As used herein, the terms "conjugated" and "attached" and their various linguistic forms, mean that the adjunct is covalently attached to the biological tissue, either directly (according to the claimed invention) or indirectly (not forming part of the claimed invention). A direct covalent attachment between the adjunct and the biological tissue is a covalent bond formed between a side chain of the biological tissue and a side chain of the adjunct. An indirect covalent attachment formed through an intermediate "linker. " The linker is a moiety, which is the residue of a multifunctional (e.g. a bifunctional) molecule capable of forming covalent bonds with side chains on both the biological tissue and the adjunct. Thus, an indirect covalent bond between the adjunct and the biological tissue is a covalent linkage comprising a first covalent bond between a side chain of the adjunct and a reactive group on a linking moiety and a second covalent bond between the linking moiety and a side chain of the biological tissue. Suitable covalent bonds are formed as amides, esters, ethers, ureas, carbamates, carbonates, anhydrides and other covalent bonds. Especially suitable covalent bonds are amides. An amide may be formed directly between an acid group on an adjunct and an amine on a protein or glycosamine of the biological tissue or between an amine on an adjunct and an acid group on a protein in the biological tissue. An indirect covalent attachment comprising an amide bond may be formed, for example, through a diacid linker, an amino acid linker, or a diamine linker using a conjugation agent.

In the present invention, the adjunct is heparin. The source of such adjuncts can be biological tissues or synthetic sources. The adjuncts promote tissue healing, remodeling, growth or regrowth while bound to the bioimplant (conjugated state), after release from the bioimplant or both.

The present invention provides bioimplants comprising sterilized biological tissues that have conjugated to them one or more adjunct molecules. Suitable biological tissues are those tissues amenable to implantation in a host body to repair or replace injured or removed host tissue or to promote healing, remodeling, growth or regrowth of host tissue. The biological tissue comprises processed tissue in native form. The tissues may be autogenic, allogenic or xenogenic in origin. The term "native tissue" means that the tissue from which the implant is prepared ("starting tissue") is not processed prior to conjugating the adjunct to it. In particular, "native tissue" is not defatted, decellularized or crosslinked prior to conjugating the adjunct to it. Suitable native tissues include bone, tendon, ligament, fascia, and combinations thereof, such as bone-connective tissue combinations, including bone-tendon combinations and bone-ligament-bone combinations. The term "processed tissue in native form" means that the starting tissue is subjected to one or more processing steps, selected from decellularization, defatting or crosslinking prior to conjugation of the adjunct to the tissue, but otherwise remains in substantially the same form as in the native tissue. Suitable processed tissues in native form include decellularized crushed bone decellularized and/or defatted bone, tendon, ligament, fascia and combinations thereof, and/or bone-connective tissue combinations, e.g. bone-tendon or bone-ligament-bone combinations. The term "processed tissue in non-native form" means tissue that has been processed in such a way that the tissue is no longer in its native form, e.g. through solubilization, reconstitution or some other process that changes its form from its native form. Suitable processed tissues in non-native form include solubilized or purified collagen from connective tissue, gelatin from mammals or fish or demineralized bone. A "composite" is a combination of two or more members of the group of native tissues, processed tissues in native form and processed tissues in non-native form. Suitable composites include combinations of native tissues, processed tissues in native form and/or processed tissues in non-native form, such as pericardium, with gelatin, bone with gelatin, purified collagen with gelatin or demineralized bone with solubilized or purified collagen. A "complex composite" is a combination of one or more native tissues, processed tissues in native form, processed tissues in non-native form, and composites with a biocompatible material, such as a synthetic or non-mammalian (e.g. crustacean- or plant-derived) biocompatible material. Suitable complex composite comprises native tissue, processed tissue in native form, processed tissue in non-native form or a composite of native tissue, processed tissue in native form and/or processed tissue in non-native form with a biocompatible material such as a hydrogel, an alginate and/or chitosan.

Tissue may be decellularized by an art-recognized method, such as treatment with trypsin or sodium dodecylsulfate (SDS). <NPL>); <NPL>). In some embodiments, SDS treatment may leave some cells on the scaffold. Reider et al. In some embodiments, such residual cells or cell fragments may not be deleterious, as crosslinking and/or sterilization would be expected to neutralize such residual structures. In any case, should treatment with trypsin and/or SDS fail to produce a suitable starting material, decellularization may be effected with another known decellularization method, such as treatment with a combination of tert-octylphenylpolyoxyethylene and sodium deoxycholate (Rieder et al. , <NUM>) or a non-ionic detergent, such as Triton-X <NUM> (Kasimir, <NUM>). The person skilled in the art will recognize that other decellularization methods may be employed and are thus within the scope of the present invention.

Bone tissue, such as solid bone and bone fragments, may be demineralized by an art-recognized method. Such demineralization may be in conjunction with, or independent of, decellularization as described above. Such demineralization may be partial or complete. Partial demineralization is often used to modify cortical bone grafts to enhance their osteoinductive properties. Bone tissue may also be demineralized by treatment with a weak acid, such as acetic acid. Demineralization removes mineral content (such as calcium carbonate) of the bone, leaving osteoinductive agents in tact in the demineralized bone matrix. Demineralized bone matrix has been shown to induce new bone formation in vivo. <NPL>), accessed at http://www. org/<NUM>/<NUM>/bj3640465. Thus, bone tissue, such as whole bone or bone fragments may be demineralized in the presence of a weak acid solution, such as <NUM> to <NUM> HCl or other mineral acid, or in the presence of a weak acid such as acetic acid. The resulting partially demineralized bone or fully demineralized bone matrix (DBM) may then be conjugated with an adjunct molecule and sterilized and crosslinked as described in more detail below.

The invention provides a bioimplant comprising a chemically sterilized biological tissue and at least one adjunct, wherein the adjunct is covalently conjugated to the biological tissue. The bioimplant is chemically sterilized with a water soluble carbodiimide, such as EDC. The sterilization is carried out in the presence of a penetration enhancer, which is C<NUM>-C<NUM> alkanol, and most particularly isopropanol. Other alcohols that may be mentioned in this regard include ethanol, n-propanol, n-butanol, i-butanol, t-butanol, and s-butanol. The chemically sterilized biological tissue is also crosslinked with a carbodiimide, optionally in the presence of a divalent crosslinking agent and/or a coupling enhancer, such as N-hydroxysuccinimide (NHS) or N-hydroxy-<NUM>-sulfosuccinimidé (Sulfo-NHS).

In preferred embodiments of the invention, the adjunct retains at least some of its native activity when conjugated to the biological tissue. In other preferred embodiments, the adjunct is released in vivo or under in vitro conditions designed to imitate in vivo conditions. In such preferred embodiments, the released adjunct has at least some native activity. As used herein, the term "at least some" means at least about <NUM>%. Thus, in embodiments of the invention the conjugated adjunct has at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, in particular about <NUM> to about <NUM>%, about <NUM> to about <NUM>%, about <NUM> to about <NUM>%, or about <NUM> to about <NUM>% of its native activity, either when bound to the biological tissue or when released from the biological tissue into the surrounding tissue in vivo or into an in vitro environment designed to simulate the in vivo environment. As used herein the term "native activity" means that activity possessed by the adjunct prior to being conjugated to the biological tissue. In general, native activity is tested under in vivo conditions or under in vitro conditions designed to simulate in vivo conditions.

A process of making the bioimplant is described, comprising: (a) contacting a biological tissue with an adjunct to form a combination; and (b) contacting the combination with a chemical sterilizing agent to form the bioimplant. The bioimplant is chemically sterilized with a water soluble carbodiimide, such as EDC, whereby the sterilization is carried out in the presence of a penetration enhancer, which is a C<NUM> -C<NUM> alkanol, and most particularly isopropanol. Other alcohols that may be mentioned in this regard include ethanol, n-propanol, n-butanol, i-butanol, t-butanol, and s-butanol. The chemically sterilized biological tissue is also crosslinked with a carbodiimide, optionally in the presence of a divalent crosslinking agent and/or a coupling enhancer, such as N-hydroxysuccinimide (NHS) or N-hydroxy-<NUM>-sulfosuccinimide (Sulfo-NHS).

In preferred embodiments of the invention, the adjunct retains at least some of its native activity when conjugated to the biological tissue. In other preferred embodiments, the adjunct is released in vivo or under in vitro conditions designed to imitate in vivo conditions. In such preferred embodiments, the released adjunct has at least some native activity. In some embodiments of this process, the conjugated adjunct has at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, in particular about <NUM> to about <NUM>%, about <NUM> to about <NUM>%, about <NUM> to about <NUM>%, or about <NUM> to about <NUM>% of its native activity, either when bound to the biological tissue or when released from the biological tissue into the surrounding tissue in vivo or into an in vitro environment designed to simulate the in vivo environment.

The adjunct molecule (heparin) promotes healing, remodeling, growth and/or regrowth of biological tissue.

Described are methods of making sterilized bioimplants having adjunct molecules linked thereto. The methods comprise conjugating an adjunct molecule to a biological tissue and sterilizing the biological tissue. In general, the conjugation and sterilization can take place in any order, or preferably simultaneously, although it is preferred that a sterilization step be the ultimate step prior to packaging the bioimplant, e.g. in a sealed polymer bag.

The sterilization is carried out in the presence of a chemical sterilizing agent, and in the presence of a penetration enhancer. The sterilizing agent is a carbodiimide, in particular a water-soluble carbodiimide, such as EDC. The sterilizing agent is preferably used in a concentration of at least about <NUM>, especially from about <NUM> to about <NUM>, more specifically from about <NUM> to about <NUM>, and most preferably from abut <NUM> to about <NUM>.

In some embodiments, the sterilizing agent is accompanied by a sterilization enhancer, such as N-hydroxysuccinimide (NHS) or <NUM>-sulfo-N-hydroxysuccinimide (Sulfo-NHS). In some embodiments, the ratio of sterilization enhancer to sterilizing agent is in the range of about <NUM>:<NUM> to about <NUM>:<NUM>, especially about <NUM>:<NUM> to <NUM>:<NUM>, preferably in the range of about <NUM>:<NUM> to <NUM>:<NUM>, and more preferably in the range of about <NUM>:<NUM> to <NUM>:<NUM>. Particularly, the sterilization enhancer is at a concentration of about <NUM> to about <NUM>, preferably about <NUM> to about <NUM>.

A penetration enhancer is used in the sterilization process in order to enhance penetration of the sterilizing agent into the microbes to be killed. Suitable penetration enhancers are C2-C4 alkanols, e.g.ethanol, isopropanol, n-propanol, isobutanol, n-butanol, s-butanol, t-butanol, or combinations of any of the foregoing. In this regard, isopropanol is especially preferred. In some embodiments, preferred concentrations of penetration enhancer are about <NUM> % (vol/vol) to about <NUM>% (vol/vol), especially about <NUM>% (vol/vol) to about <NUM> % (vol/vol) and even more preferably about <NUM> %, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>% or about <NUM>% (vol/vol). A particularly preferred penetration enhancer is about <NUM>% (vol/vol) of isopropanol.

In some preferred embodiments, the sterilization and conjugation are carried out simultaneously in the presence of a sterilizing agent, and preferably in the presence of a sterilization enhancer and/or a penetration enhancer. Although within some embodiments of the invention there may be used a bifunctional linking molecule as a conjugating agent, in some preferred embodiments the sterilization agent and optionally sterilization enhancer and/or penetration enhancer are considered sufficient to conjugate the adjunct to the biological tissue, sterilize the combined tissue and adjunct, and optionally crosslink the biological tissue. In some preferred embodiments, the biological tissue and adjunct are combined to form an intermediate, which is then frozen and lyophilized. The resulting lyophilized intermediate is then contacted with the sterilizing agent and optionally a sterilization enhancer and/or a penetration enhancer (with or, preferably without, a conjugating agent).

In some embodiments, the adjunct is conjugated to the biological tissue prior to sterilization. In such embodiments, the biological tissue and adjunct are contacted with a conjugation solution to affect conjugation. The conjugation solution comprises a conjugating agent, such as a water soluble carbodiimide or NHS. The conjugation solution also optionally comprises a conjugation enhancer, such as Sulfo-NHS. In addition, the conjugation solution also optionally comprises a bifunctional conjugating agent, such as a lower alkane diamine, diacid or amino acid.

The bioimplants of the invention are especially advantageous in that they promote postoperative tissue healing, remodeling, growth and/or regrowth. In some embodiments, the bioimplants present on their surface or release adjuncts that directly stimulate tissue healing, remodeling, growth and/or regrowth. In some embodiments, the bioimplants present on their surface or release adjuncts, which indirectly promote tissue healing, remodeling, growth and/or regrowth by inhibiting the bioactivity of one or more microbes in the vicinity of the bioimplant, thereby allowing the recipient's body to heal, remodel, grow or regrow tissue without, or with attenuated, interference from one or more microbes.

In other particular embodiments, the bioimplant releases the adjunct molecule into the vicinity of the bioimplant by cleaving, e.g. by hydrolysis (whether enzyme mediated or non-enzyme mediated), one or more covalent bonds conjugating the adjunct to the biological tissue. The thus cleaved covalent bonds may be amide, ester, anhydride or urea bonds, depending upon the conjugating agent used to conjugate the adjunct molecule to the biological tissue. In particular embodiments, in which the conjugating agent is a diamine, the covalent bonds are amide bonds, which are cleaved by hydrolysis to form a free amine chain and a carboxyl group.

The bioimplants of the invention are useful in a variety of surgical procedures. In some embodiments, the bioimplants are soft tissues that may be used to augment repair, such as suturing, closure of surgical wounds, or repair or replacement of tendons, ligaments, skin, etc. In some embodiments, the bioimplants are heart valves useful in the alleviation of vascular or arterial valve occlusion (stenosis) or other valve malfunction. In some embodiments, the bioimplants are partially or completely demineralized bone or bone fragments useful in bone repair, bioimplantation (e.g. hip, knee or other joint implantation) or spinal fusion surgery.

The sections below describe in more detail sterilization of biological tissue, conjugation of adjunct molecules to the biological tissue, and optional tissue crosslinking procedures.

Bioimplants of the invention comprise biological tissues from natural sources The types of tissue that are used include tendon, ligament, fascia and bone fragments. In some embodiments, soft tissues include multiple components, such as living cells and collagen. Collagen is fibrous proteinaceous biopolymer (scaffolding) that forms the matrix that provides structural integrity to soft tissues. Collagen is also the fibrous protein constituent of cartilage and bone. Soft tissues can be decellularized to provide a decellularized scaffolding that is made up primarily of collagen. In any case, collagen and other proteins in soft tissue have numerous functional groups that can be caused to react to form bonds with other functional groups.

Bioimplants comprising naturally occurring biological tissues, such as tissues extracted from a human, porcine, ovine, bovine, caprine, murine, canine, feline or other source, are generally unstable if left in their natural state. Having been extracted from the living environment within the body, such tissues will soon degrade unless stabilized. Microbes that naturally occur within the biological tissues, or which infest the biological tissues after they have been excised, will soon begin to break down the biological tissue as food. Also, naturally occurring antigens on the tissue, especially on the part of the tissue that is exposed to the interstitial fluid surrounding the bioimplant, attract components of the recipient body's immune system, which gradually break down the tissue and eventually give rise to tissue rejection. Also, protein on the surface of the untreated (fresh) biological tissue is easily denatured, which can lead to its gradual erosion within the recipient body. In non-decellularized soft tissue, cells within the tissue may undergo lysis (cell membrane rupture), thereby disturbing the structural integrity of the tissue and potentially exposing various antigens to the bioimplant surface. Thus, it is desirable to sterilize the biological tissue during the course of preparing the bioimplant of the present invention. Such sterilization kills microbes associated with the biological material and in some embodiments masks antigenic sites, provides structural integrity to the bioimplant, and/or retains the bioimplant in its natural shape.

In embodiments of the invention, the bioimplant is an organ or tissue derived in whole or in part from a human or an animal, or which is produced from other organic tissue, and which is to be implanted, either by itself or as part of a bioprosthesis, in a human or in an animal. In some embodiments, a bioimplant can be a xenograft, an allograft or an autograft. In some embodiments, the bioimplants also include bone tissue, especially bone fragments.

The term "crosslinking", as used herein, refers to the formation of links of various lengths within the tissue - that is within and/or between the molecules (especially the proteins) of the tissue, such links resulting from bond formation either (a) between two reactive moieties of the tissue, thus forming short covalent links within and between the molecules of the tissue, or (b) between reactive moieties on the tissue and a covalently bound bifunctional crosslinking agent. While crosslinking and "conjugating" are diverse events (the former forming bonds within the biological tissue, the latter forming bonds between the biological tissue and an adjunct molecule), in many instances crosslinking and conjugating may be carried out in the same process step, as discussed in more detail herein.

The term "crosslinking agent" is used herein to describe a bifunctional reagent capable of reacting with two or more functional groups in the biological tissue. A bifunctional reagent is a reagent having at least two functional groups capable of reacting with reactive groups in the biological tissue. Such functional groups include amines, acids hydroxyls and thiols (sulfhydryl groups). Thus, bifunctional reagents include diamines, diacids, or a β-, γ-, δ-, ε-, ζ-, or higher order amino acid, or other bifunctional agents, such as anhydrides (e.g. succinic anhydride). The bifunctional reagents are chosen to react with reactive groups in the biological tissue. Such reactive groups include amines (e.g. N-terminal amines and generally ubiquitous lysine groups), other -NH<NUM> groups (such as those on guaninyl groups of arginine residues), free carboxyl groups (e.g. those at the C-terminus and aspartic and glutamic acid groups), hydroxyl groups (e.g. those found on serine, threonine and tyrosine) and thiols (e.g. those found in cysteine residues). Thus, in some embodiments, the crosslinks comprise covalent bonds, such as amides (formed between bifunctional reagent amines and carboxyl groups in the biological tissue, or between bifunctional reagent acids or anhydrides and protein amines). In some embodiments, the crosslinks comprise esters, such as those formed between carboxyl groups on the bifunctional reagent and hydroxyl groups in the protein or those formed between hydroxyl groups on the bifunctional reagent and carboxyl groups in the protein. In still further embodiments, the crosslinks comprise disulfide bonds, e.g. between thiols on the bifunctional reagent and cysteine in the protein. Of the types of bonds that can be formed, amides are particularly advantageous as they can be easily formed using methods described herein using a coupling agent, optionally in conjunction with a coupling enhancer, as described in more detail herein.

In some embodiments, the crosslinking agent is a straight chain or a branched compound having from <NUM> to <NUM> carbon atoms. In some embodiments, the crosslinking agent is a carbocyclic compound in which the reactive functional groups are on the carbocyclic ring or are attached to the carbocyclic ring by an intervening carbon chain. In some embodiments, the reactive functional groups can include amines, hydroxyl groups, carboxylic acids, anhydrides, acid chlorides, thiols, etc. In particular embodiments, the crosslinking agent is a C<NUM>-C<NUM> alkanediamine, alkenediamine, alkynediaminealkane, aminoalkanoic acid, aminoalkenoic acid or aminoalkynoic acid. In specific embodiments, the crosslinking agent is <NUM>,<NUM>-diaminohexane, <NUM>,<NUM>-diaminoheptane, succinic acid (C<NUM>), glutaric acid (C<NUM>), adipic acid (C<NUM>) or pimelic acid (C<NUM>), or one of the anhydrides selected from: succinic, glutaric, adipic and pimelic anhydride. In other particular embodiments, the crosslinking agent is <NUM>,<NUM>,<NUM>-triaminobenzene, <NUM>,<NUM>-diaminobenzene, o-phthalic acid, p-phthalic acid, <NUM>-aminobenzoic acid and phthalic anhydride. In some embodiments, a di- or triamino crosslinking agent that has a molecular weight of about <NUM> or less, and about <NUM> or less, is employed so as to assure adequate penetration into the fresh tissue. In particular embodiments, the crosslinking agent is a straight chain from <NUM> to <NUM> carbon atoms in length with one reactive amine located at each end. Although the crosslinking agent may have optional substitutions along its length, in specific embodiments, it is a hydrocarbon that is substituted only with the reactive amines, e.g. a straight chain alkane having amines at each extremity. Exemplary crosslinking agents are <NUM>,<NUM>-hexanediamine and <NUM>,<NUM>-heptanediamine.

The terms "coupling agent" and "coupling enhancer," as used herein, refer to reagents that respectively promote and enhance the formation of bonds, especially amide bonds, between proteins within the bioimplant tissue or between functional groups (e.g. amines or carboxyls) on the proteins and the crosslinking agent. These bonds may be formed between a reactive amine and a reactive carboxyl (COOH or COO-) on the tissue (thus linking two such closely located reactive groups), or between a reactive amine or carboxyl on a crosslinking agent and a reactive carboxyl or amine on or within the tissue. Those of skill in the peptide synthesis and related art will be familiar with such reagents, e.g. carbodiimides and succinimides, especially water-soluble varieties thereof.

In some embodiments, the crosslinking reaction is facilitated by use of a coupling agent. In some more particular embodiments, the coupling agent is <NUM>-ethyl-<NUM>(<NUM>-dimethyl aminopropyl)carbodiimide hydrochloride (EDC), although other suitable coupling agents such as N-hydroxysuccinimide (NHS) can also be used. In particular embodiments, the coupling agent is used in conjunction with a coupling enhancer, such N-hydroxysulfosuccinimide (sulfo-NHS) although other suitable coupling enhancers, such as <NUM>-hydroxy- benzotriazole (HOBt) and dimethylaminopyridine (DMAP), can also be used. The concentration of the coupling agent and of the coupling enhancer can vary. However, appropriate concentrations are readily determinable by those of skill in the art. In some embodiments, the coupling agent is used in a concentration between about <NUM> and <NUM>, at a concentration of <NUM> or less, especially at a concentration of between about <NUM> and <NUM>. In some embodiments, the coupling enhancer is employed at a concentration of between <NUM> and about <NUM>, especially at a concentration of about <NUM> or less.

In some embodiments of the present invention, the crosslinking agents, the coupling agent and the coupling enhancer as well as their reaction products are water soluble. In particular embodiments, the selected crosslinking agent, coupling agent and coupling enhancer to optimize crosslinking of the tissue, while minimizing the risks of damage to the biological tissue during the crosslinking process, and of toxicity, inflammation, calcification, etc, after implantation. In specific embodiments, all solutions used for crosslinking are filtered before use, e.g. through <NUM> or less filters to remove microbial contaminants, and thereby reduce the risk of contaminating the tissue during crosslinking and/or sterilization.

Reaction conditions for the crosslinking of the biological tissue may vary, depending on the crosslinking, coupling and enhancing agents employed. In general, the crosslinking process is carried out in an aqueous buffer selected from among those well known to those of ordinary skill in this art as to provide the most efficacious crosslinking reaction, while minimizing risks of calcification. Examples of suitable buffers include, but are not limited to, N-<NUM>-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES) and <NUM>-(N-morpholino)propanesulfonic acid (MOPS), and the like.

The pH and concentration of the buffered solution can vary, again depending upon the crosslinking, coupling and enhancing agents employed. The buffer concentration and pH are chosen to provide the most effective crosslinking reaction while being the least harmful to the biological tissue. For example, with EDC as the coupling agent and sulfo-NHS as the coupling enhancer, the pH of the treatment solution is maintained at between about <NUM> to about <NUM>. The reaction temperature may be between about <NUM> and <NUM>; e.g. between about <NUM> and <NUM>. Acceptable pH buffers for use in embodiments of the invention include the commonly known HEPES, TRIS and MOPS pH buffers.

Generally, a fresh or adjunct conjugated biological tissue to be crosslinked according to the present invention is kept on ice until it can be rinsed several times in ice-cold <NUM>% saline or some other suitable solution. In general, such washing or rinsing is carried out immediately after fresh biological tissue has been excised from the donor animal, or within <NUM> hours thereafter. In the case of adjunct conjugated biological tissue, the rinsing step may be skipped if the tissue is to be immediately crosslinked after the adjunct has been conjugated to the biological tissue. If additional storage time is needed, the rinsed tissue can be stored for not longer than for <NUM> hours, in an appropriate buffer at a low temperature, such as about <NUM>.

In some embodiments, the concentration of the diamine crosslinking agent is between about <NUM> and about <NUM> millimolar, between about <NUM> and <NUM> millimolar, between about <NUM> and <NUM> millimolar, or between about <NUM> and <NUM> millimolar. In particular embodiments, the diamine crosslinking agent has a carbon chain length not greater than <NUM> carbon atoms, e.g. between <NUM> and <NUM> carbon atoms. In specific embodiments, the crosslinking agent is a straight chain alkane having amine groups at its respective ends, especially <NUM>,<NUM>-hexanediamine. Treatment of the biological tissue is carried out by contacting the tissue with a solution, especially an aqueous solution, containing the coupling agent, the coupling enhancer and the crosslinking diamine. The concentrations of the coupling agent, EDC, and the coupling enhancer, Sulfo-NHS, are as previously discussed, e.g. between about <NUM> and about <NUM> of EDC and between about <NUM> and about <NUM> of sulfo-NHS.

As discussed above, bioimplants of the invention are made from naturally occurring biological tissue, such as tendon, ligament, fascia, as well as bone fragments and/or defatted bone. All tissues contemplated within the scope of the invention have therein one or more proteins, such as collagen. Non-decellularized tissues also comprise other components, such as living cells, which have various cell surface proteins, such as receptors, ion channels and other proteins that have numerous functional groups that can be caused to react with various reagents to form covalent bonds.

Various adjunct molecules also have one or more functional groups that can be caused to react with a reagent to form covalent bonds. Exemplary adjunct molecules include proteins, small peptides, ribonucleic acids, deoxyribonucleic acids, polysaccharide, glycosaminoglycan (GAG)s and antibiotics. Each of these classes of adjunct molecules possesses members having at least one reactive group capable of forming an intermolecular covalent attachment (conjugation) between the adjunct molecules and proteins in the biological tissue. Such reactive groups include carboxyl groups, sulfonates, amines, ureas, carbamates, guanidyls, thiols (sulfhydryls) and hydroxyls. Of these reactive groups, the most favorable for preparing bioimplants of the invention are considered to be carboxyl groups and amine groups, as these may be used to form in vivo labile amide groups with carboxyl on protein or bifunctional crosslinking agent.

It will be understood that a functional group of a particular type can be transformed into a different type by methods known in the art. For example, an amine group can be converted to a carboxyl group by reacting the amine with, for example, a diacid or diacid anhydride. Reaction of the amine with one carboxyl group of the diacid, or ring opening of the anhydride of the diacid anhydride, results in an amide bond being formed between the reagent molecule and the amine as well as a free acid group that may be caused to react with another reagent. As another example, a cysteine group may be transformed into a carboxylic acid reactive site by coupling a thiolalkanoic acid to the cysteine group, thereby forming a disulfide bridge between the adjunct molecule and the reagent, while at the same time providing an acid group as a functional group on the adjunct molecule. In some embodiments, an adjunct molecule may have a functional group that is an ester of an acid that can be liberated by hydrolysis of the ester bond to produce a free acid, which can then be conjugated through an amine conjugating agent. This approach is especially useful where the free acid represents the active metabolite in vivo. Thus, as used herein, the term "adjunct molecule" includes such derivatized molecules, especially where release of the adjunct molecule post implantation is effected by cleavage of the bonds formed by the reagents and the adjuncts in vivo, thereby releasing an active molecule.

In general, conjugation entails contacting an adjunct with at least one conjugation reagent. Such conjugation reagents include one or more of the following: crosslinking agents, coupling agents and/or coupling enhancers. In some embodiments, the conjugation reagent includes crosslinking agent only. In other, preferred, embodiments the conjugation reagent includes coupling agent (such as EDC). In more preferred embodiments, the conjugation reagent includes coupling agent (such as EDC) and a coupling enhancer (such as NHS or Sulfo-NHS). In some embodiments, the conjugation reagent includes coupling agent and coupling enhancer but no crosslinking agent. In other embodiments, the conjugation reagent includes coupling agent, coupling enhancer and crosslinking agent. In some embodiments, the crosslinking agent, coupling agent and coupling enhancer are the same as those described above with respect to crosslinking of biological tissue.

In some preferred embodiments, the conjugation reagent comprises a coupling agent and optionally a coupling enhancer. In some particularly preferred embodiments, the coupling agent is a carbodiimide, such as EDC. In some particularly preferred embodiments, the coupling enhancer is NHS or Sulfo-NHS. Coupling agents and coupling enhancers are described in more detail above. Use of a coupling agents alone or in combination with coupling enhancers result in direct amide bonds between the adjunct molecules and the proteins in the biological tissue.

The person skilled in the art will recognize that the coupling reagent and the coupling enhancer disclosed herein for coupling the adjunct to the biological tissue are, in some embodiments, the same as the sterilizing agent and the sterilizing enhancer, respectively, as disclosed above. One of skill in the art will recognize that a preferred embodiment entails sterilization and conjugation in the same step, using a sterilizing agent as a coupling agent and optionally a sterilizing enhancer as a coupling enhancer. In some preferred embodiments, then, coupling is also carried out in the presence of a penetration enhancer, as disclosed above. In some preferred embodiments, the tissue and the adjunct are combined and then contacted with a sterilization solution comprising sterilizing agent and optionally sterilizing enhancer and/or penetration enhancer. In particularly preferred embodiments, the tissue and adjunct are combined, frozen, lyophilized and then contacted with said sterilization solution. In other preferred embodiments, the tissue is contacted with a sterilization solution comprising adjunct molecule and sterilizing agent and optionally a sterilizing enhancer and/or penetration enhancer. In particularly preferred embodiments, the tissue is first frozen and lyophilized and then contacted with a sterilization solution comprising adjunct molecule and sterilizing agent and optionally a sterilizing enhancer and/or penetration enhancer.

In some embodiments, a crosslinking agent is used. In such cases, at least one functional group on the crosslinking agent is capable of forming a covalent bond with a functional group on the adjunct molecule. At least one other functional group is capable of forming a covalent bond with a functional group on the protein of the biological tissue. In such cases, the active group on the adjunct molecule is one that readily forms a covalent bond with a functional group on the crosslinking agent. Exemplary active groups that may be found on adjunct molecules include carboxyl groups, sulfonates, amines, guanines, ureas, carbamates, amides, imides and thiols (e.g. cysteine groups on proteins, such as growth factor proteins). Especially suitable active groups include carboxyl groups and amines.

In some embodiments, suitable crosslinking agent functional groups include functional groups that form amide or ester bonds with at least one carboxyl group on the adjunct. Such functional groups include amines and hydroxyls. In some embodiments, suitable conjugation agent functional groups include functional groups that form amide or ester bonds with at least one amine or hydroxyl on the adjunct molecule. Such functional groups include carboxyl groups and acid anhydrides.

Some crosslinking agents that may be used to conjugate an adjunct to a protein in a biological tissue include the homobifunctional, water soluble reagents: bis-(sulfosuccinimidyl)suberate, disulfosuccinimidyl tartrate, and ethylene glycol -bis-(sulfosuccinimidyl succinate). Other conjugating agents include the heterobifunctional, water soluble reagents: N-sulfosuccinimidyl(<NUM>-iodoacetyl)aminobenzoate, sulfosuccinimidyl-<NUM>-(N-maleimidomethyl)cyclohexane-<NUM>-carboxylate, sulfosuccinimidyl-<NUM>-(p-maleimidophenyl)butyrate, <NUM>-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester. While water soluble reagents are considered to be superior to non-water soluble reagents, it is also possible to use non-water soluble bifunctional crosslinking agents, such as bis-(succinimidyl)suberate, disuccinimidyl tartrate, and ethylene glycol bis-(succinimidyl succinate). Other crosslinking agents that may be mentioned include: dimethyl-<NUM>,<NUM>'-dithiobispropionimidate, dimethyl-<NUM>,<NUM>'- dithiobisbutyrimidate, and dimethyl-<NUM>-<NUM>'-dithiobiscaproimidate.

Some especially suitable bifunctional crosslinking agents include diamines, diacids and amino acids, and in particular diamines, diacids and amino acids having from <NUM> to <NUM> carbons between the functional groups. Where diamines, diacids or amino acids are used as conjugating agents, it is advantageous to use a coupling agent (such as a carbodiimide or a cyclic imide) and/or a coupling enhancer (such as a cyclic imide, HOBt or DMAP), as described in more detail herein.

Before the bioimplant can be implanted in a mammal; especially a human, sterilization must be effected, and such is normally done prior to packaging. The conditions for sterilization are discussed in detail above. In some preferred embodiments, sterilization and conjugation take place in the same process step, and in particular using the same reagent solution comprising sterilizing agent and optionally a sterilizing enhancer and/or a penetration enhancer. In some particularly preferred embodiments, the sterilization solution comprises sterilizing agent and penetration enhancer and optionally a sterilizing enhancer. In some other preferred embodiments, the sterilization solution comprises sterilizing agent, sterilizing enhacer and penetration enhancer. In other embodiments, the sterilization solution comprises sterilizing agent, a crosslinking agent and optionally sterilizing enhancer and/or penetration enhancer.

Following are descriptions of particular schemes for preparing an adjunct conjugated crosslinked and sterilized bioimplant of the invention. The person skilled in the art will recognize that other embodiments may be developed within the scope of the present invention and no disclaimer of such broader invention is intended by presentation of these illustrative examples.

As discussed above, crosslinking of biological tissue and conjugation of adjunct molecules to biological tissue may be carried out in the presence of a variety of crosslinking agents. In some embodiments, the present invention comprises crosslinking of biological tissue with a diamine, such as a C<NUM>-C<NUM> linear diamine having amine groups with at least about four carbons between them. In particular embodiments, the diamine is water soluble, although non-water soluble or slightly water soluble diamines may be used in some embodiments if a detergent, especially a non-ionic detergent, is used to aid in solubilizing the diamine. Exemplary water soluble diamines contemplated within the scope of the present invention include <NUM>,<NUM>-pentane diamine, <NUM>,<NUM>-hexane diamine and <NUM>,<NUM>-heptane diamine.

In some embodiments, the present invention comprises crosslinking of biological tissue with a diacid, such as a C<NUM>-C<NUM> linear diacid having carboxyl groups with at least about four carbons between them. In particular embodiments, the diacid is water soluble, although non-water soluble or slightly water soluble diacids may be used in some embodiments if a detergent, especially a non-ionic detergent, is used to aid in solubilizing the diacid. Exemplary water soluble diacids contemplated within the scope of the present invention include <NUM>,<NUM>-pentane dicarboxylic acid, <NUM>,<NUM>-hexane dicarboxylic acid and <NUM>,<NUM>-heptane dicarboxylic acid.

In place of a diacid, there may be substituted a diacid anhydride, such as a C<NUM>-C<NUM> diacid anhydride having carboxyl groups with at least about four carbons between them. In particular embodiments, the diacid anhydride is water soluble, although non-water soluble or slightly water soluble diacid anhydrides may be used in some embodiments if a detergent, especially a non-ionic detergent, is used to aid in solubilizing the diacid anhydride. Exemplary water soluble diacid anhydrides contemplated within the scope of the present invention include <NUM>,<NUM>-pentane dicarboxylic acid anhydride, <NUM>,<NUM>-hexane dicarboxylic acid anhydride and <NUM>,<NUM>-heptane dicarboxylic acid anhydride.

In place of a diacid or diamine, there may be substituted an amino acid, such as a C<NUM>-C<NUM> amino acid having a carboxyl group and an amine with at least about four carbons between them. In particular embodiments, the amino acid is water soluble, although non-water soluble or slightly water soluble amino acids may be used in some embodiments if a detergent, especially a non-ionic detergent, is used to aid in solubilizing the amino acid. Exemplary water soluble amino acids contemplated within the scope of the present invention include <NUM>-aminopentan-<NUM>-oic acid, <NUM>-aminohexan-<NUM>-oic acid and <NUM>-amino-heptan-<NUM>-oic acid. Other amino acids may be used as described above.

The diamines form amide bonds between carboxyl groups within the biological tissue, thus linking the carboxyl groups together through an alkylene linker. The diacids and diacid anhydrides form amide bonds with amine (including guanine) groups in proteins within the biological tissue. Likewise, the amine functions of the amino acids form amide bonds with proteinaceous carboxyl groups, while the carboxyl groups of the amino acids form amide bonds with amine groups in the biological tissue.

It is considered advantageous to use a coupling agent to aid in amide bond formation within the biological tissue and between the biological tissue and the. Suitable coupling agents include EDC or N-hydroxysuccinimide (NHS), which enhance amide bond formation when used together with diamine, diacid or amino acid. It is believed that coupling enhancer EDC increases the reaction rate between an amine and a carboxyl group by forming an active intermediate, which lowers the reaction energy barrier of amide bond formation. The formation of an amide bond using EDC is shown in Scheme <NUM>, below.

As can be seen in Scheme <NUM>, amide bond formation proceeds through an active intermediate. In particular, a carboxylic acid moiety in a molecule represented by Ra-COOH attacks the diimide carbon, forming a relatively unstable o-acylisourea intermediate. Attack of the carbonyl carbon by a free set of electrons in the amine functional group of Rb-NH<NUM> results in formation of the amide bond between Ra and Rb. In addition, there is formed as a byproduct, a water soluble isourea, which is rinsed away from the biological tissue after conjugation and/or crosslinking.

In some embodiments, Ra represents a protein in a biological tissue and Rb represents an amine-bearing adjunct. In some specific embodiments, Ra represents a protein in the biological tissue having one or more free carboxyls (COOH, or COO-), while Rb represents a protein, peptide, an antibiotic, RNA or DNA having a free amine or a polysaccharide, glycosaminoglycan (GAG) or derivatized to have a free amine. In some embodiments, Ra represents a carboxyl or other acid group on an adjunct and Rb represents a free amine (such as a lysine side chain) on a protein in the biological tissue. In some particular embodiments, Ra is a free carboxyl on a protein, a peptide, an antibiotic or a DNA, RNA or polysaccharide, glycosaminoglycan (GAG) derivatized to have a free carboxyl and Rb represents a free amine on a lysine side chain in a protein in the biological tissue.

In some embodiments, the biological tissue is coupled to the adjunct via a crosslinking agent. In some such embodiments, the acid Ra is a protein, especially a protein in the biological tissue having one or more carboxylic acid moieties (e.g. C-terminal COOH, or carboxyl groups of aspartic and/or glutamic acid residues) available for reaction. In some such embodiments, Ra is be the residue of an acid-bearing adjunct, such as a protein, a peptide or an antibiotic; or it can be a derivatized polysaccharide, glycosaminoglycan (GAG), DNA or RNA. Also, in some such embodiments, Ra can also be an acidic crosslinking agent, such as a diacid, a diacid anhydride or an amino acid. Where Ra is a protein, Rb is, in some embodiments, an amino crosslinking reagent, such as a diamine crosslinking agent. Where Ra is a crosslinking agent, Rb is, in some embodiments, a protein having exposed amine groups (e.g. N-terminal amine or side chain amines of lysine residues). Where Ra represents the residue of an acid-bearing adjunct, Rb is, in some embodiments, an amine-bearing crosslinking or coupling reagent, or Rb can be an amine-bearing protein.

In general, EDC-mediated crosslinking and conjugation are conceptually very similar, as crosslinking, conjugation or both may be carried out using a bifunctional agent (crosslinking/conjugating) capable of forming amide bonds with the desired materials.

In some embodiments, it is considered advantageous to crosslink a biological tissue in one step and conjugate the adjunct molecule to the biological tissue in another step. In some such embodiments, it is considered advantageous to use a homobifunctional linking agent, such as a diamine, a diacid or a diacid anhydride as the crosslinking agent. In some particular embodiments, the homobifunctional linking agent is a diamine, which is used in conjunction with EDC and optionally a coupling enhancer, such as sulfo-NHS or NHS. In such cases, the crosslinking of the biological tissue with a diamine blocks carboxylic acid groups in the tissue proteins, leaving amine groups on the tissue proteins available for conjugation to an adjunct molecule through a suitable conjugation linker. Such conjugation is carried out with a conjugating agent having at least two functional groups. The first functional group should be a functional group capable of reacting with the tissue protein amines to form amide bonds. Suitable conjugation agents include diacids, diacid anhydrides and amino acids, especially such conjugation agents having <NUM>-<NUM> carbons in which the two functional groups are separated by at least four carbon atoms. Where the conjugation agent is a diacid or a diacid anhydride, the adjunct should be one that has, or has been modified to have, at least one free amine for formation of an amide bond with the conjugation agent. Where the conjugation is an amino acid, the adjunct should be one that has, or has been modified to have, at least one carboxylic acid moiety available for formation of an amide bond with the conjugation agent. In any case, conjugation can favorably take place by contacting the adjunct, the crosslinked biological material and the conjugation agent, optionally together with a coupling agent (e.g. EDC or NHS) and/or a coupling enhancer (e.g. NHS or sulfo-NHS).

Schemes <NUM> and 2A, depicted in <FIG> and <FIG>, show how crosslinking followed by adjunct conjugation, is effected in a biological tissue. Referring to Scheme <NUM> in <FIG>; in the first step, the protein Pr is shown, having multiple carboxyl moieties and at least one amine moiety. The protein is crosslinked by contacting it with the crosslinking agent diam, which is a diamine, in the presence of a coupling agent (EDC) and a coupling enhancer (sulfo-NHS), which produces the crosslinked protein XLPr.

In a first alternative reaction (A) shown in Scheme <NUM>, the crosslinked protein XLPr is reacted with an adjunct molecule a-adj (R<NUM>CO<NUM>-), having a free carboxyl group, in the presence of coupling agent (EDC) and a coupling enhancer (sulfo-NHS), which produces the adjunct conjugated crosslinked protein <NUM>. The person skilled in the art will recognize that, while R<NUM>CO<NUM>- may be an adjunct molecule having a free carboxyl in its native state (such as a protein, a peptide or an antibiotic such as one of the penicillins or cephalosporins), it can also represent an adjunct molecule that has been modified to have a carboxyl group, e.g. by reacting an amine-bearing adjunct with a diacid, diacid anhydride, etc., as described above. In some embodiments, the adjunct may have a sulfonate (SO<NUM>-) group instead of a carboxyl group, in which case the expected result will be a sulfonamide bond between the protein and adjunct.

In another alternative reaction (B1) shown in Scheme <NUM>, the crosslinked protein XLPr is reacted with an amino acid conjugating agent aa (H<NUM>N-R<NUM>-CO2-) in the presence of coupling agent (EDC) and a coupling enhancer (sulfo-NHS), which produces the intermediate crosslinked protein <NUM>. This intermediate <NUM> is then reacted (B2) with a carboxyl group-bearing adjunct molecule a-adj (R<NUM>-CO<NUM>-), to form the adjunct conjugated crosslinked protein <NUM>. The person of skill in the art will recognize that the two reaction steps can be collapsed into a single step, as illustrated in the first reaction sequence (B) illustrated in Scheme 2A of <FIG>, wherein the crosslinked protein XLPr is reacted with the amino acid aa and the carboxyl bearing adjunct a-adj in the presence of EDC and sulfo-NHS to form the adjunct conjugated crosslinked protein <NUM>.

In yet another alternative reaction shown in Scheme <NUM>, the crosslinked protein XLPr is reacted (C1) with a diacid dia or diacid anhydride diaan in the presence of EDC and sulfo-NHS to form the intermediate <NUM>, which has a free carboxyl group available for further reaction. This free carboxyl group can form an amide bond (C2) with the amine in an adjunct molecule b-adj (R<NUM>-NH<NUM>), e.g. in the presence of EDC and sulfo-NHS to form the adjunct conjugated crosslinked protein <NUM>. The person skilled in the art will recognize that these two steps can be carried out in the same reaction mixture, as depicted in the second reaction sequence (C) shown in Scheme 2A in <FIG>.

Schemes <NUM> and 3A, depicted in <FIG> and <FIG>, respectively, show how crosslinking followed by adjunct conjugation, is effected in a biological tissue. Referring first to Scheme <NUM> in <FIG>, in the first step, the protein Pr is shown, having multiple amine moieties and at least one carboxyl moiety. The protein is then crosslinked by contacting it with the crosslinking agent diacid, which is a diacid (or, as may be appreciated by the person skilled in the art, a dianhydride), in the presence of a coupling agent (EDC) and a coupling enhancer (sulfo-NHS), which produces the crosslinked protein XLPr.

In a first alternative reaction (A) shown in Scheme <NUM>, the crosslinked protein XLPr is reacted with an adjunct molecule b-adj (R<NUM>-NH2), in the presence of coupling agent (EDC) and a coupling enhancer (sulfo-NHS), which produces the adjunct conjugated crosslinked protein <NUM>. The person skilled in the art will recognize that, while R<NUM>-NH<NUM> may be an adjunct molecule having a free amine in its native state (such as a protein or a peptide), it can also represent an adjunct molecule that has been modified to have an amine group, e.g. by reacting an carboxyl-bearing adjunct (such as a penicillin or cephalosporin) with a diamine as described above.

In another alternative reaction depicted in Scheme <NUM>, the crosslinked protein XLPr is reacted (B1) with an amino acid conjugating agent aa (H<NUM>N-R<NUM>-CO2-), in the presence of coupling agent (EDC) and a coupling enhancer (sulfo-NHS), which produces the intermediate crosslinked protein <NUM>. This intermediate <NUM> is then reacted (B2) with an adjunct molecule b-adj (R<NUM>-CO<NUM>-), to form the adjunct conjugated crosslinked protein <NUM>. The person of skill in the art will recognize that the two reaction steps can be collapsed into a single step, as illustrated in the first reaction sequence (B) illustrated in Scheme 3A of <FIG>.

In yet another alternative reaction depicted in Scheme <NUM>, the crosslinked protein XLPr is reacted (C1) with a diamine diam in the presence of EDC and sulfo-NHS to form the intermediate <NUM>, which has a free amine group available for further reaction. This free amine group can form an amide bond (C2) with the carboxyl group in an adjunct molecule a-adj (R<NUM>-NH<NUM>), e.g. in the presence of EDC and sulfo-NHS to form the adjunct conjugated crosslinked protein <NUM>. The person skilled in the art will recognize that these two steps can be carried out in the same reaction mixture, as depicted in the second reaction sequence (C) in Scheme 3A in <FIG>.

In preferred embodiments, crosslinking of the tissue and conjugation of the adjunct to the tissue take place in the same step. While a crosslinker may be used in such processes, in some embodiments it is considered preferably to directly conjugate the adjunct to the protein. As proteins in tissues tend to have both free carboxyl groups and free amines, it is considered possible to conjugate adjuncts having at least one carboxyl, at least one amine, or both at least one carboxyl and at least one amine as side chains.

The following examples are presented as illustrative, non-limiting embodiments of the present invention. Although these examples are directed toward specific adjuncts, tissue types, and methods of attaching the adjuncts to tissues, the person skilled in the art will recognize that the present invention is not limited to these illustrative examples, and may be practiced with additional adjuncts, tissue types and attachment methods as described herein.

Pericardial tissue was stabilized (cross-linked) using the techniques defined in <CIT> , and <CIT>. Following the cross-linking step, pericardial tissues were exposed to a solution of the glycosaminoglycan (GAG) chondroitin sulfate (<NUM>%-<NUM>%) in the presence of EDC for defined periods of time (<NUM>-overnight). Tissues were subsequently rinsed, and sterilized using the methods described in <CIT> ; <CIT> ; and <CIT>. The levels of GAG (glycosaminoglycan) attachment in the tissues were assessed by histological means (differential staining of sections with PAS-Alcian Blue, where GAGs stain blue, while collagen stains pink). The pictures in <FIG> , indicate attachment of GAGs across the pericardial membrane. <FIG> shows essentially no blue staining, which is consistent with no GAG being attached to the pericardium. <FIG> shows mixed blue and pink staining, which is consistent with partial GAG attachment to the pericardium. <FIG> shows nearly complete blue staining, which is indicative of complete GAG attachment to the pericardium.

The various processing steps (cross-linking, sterilization, etc.,) were then evaluated to determine the tissue processing steps during which the adjuncts could be attached to the tissues. Specifically, this example indicates the various methods of generating a sterile tissue with stably attached adjuncts. A Type <NUM> collagen sponge that had been subjected to various intermediary processing steps was contacted with a solution of chondroitin sulfate (as an adjunct example) and EDC. <FIG> show low- and high-magnification histological sections of GAG-attached Type <NUM> collagen sponge, wherein GAG-attachment was carried out during tissue sterilization. <FIG> shows GAG-attached Type <NUM> collagen sponge, wherein GAG was attached to the tissue during cross-linking. As is apparent from <FIG> , attachment of GAG to collagen sponge during sterilization resulted in a diffused attachment, whereas attachment of GAG to tissue during cross-linking produced a close-fitting attachment.

In order to demonstrate the applicability of the methods of the present invention to various collagen-based tissue types, three different tissue types were treated in accordance with the present invention. Three different tissue types (pericardium, demineralized cancellous bone, and a collagen sponge made from solubilized Type I collagen) were first cross-linked and subsequently exposed to chondroitin sulfate in the presence of EDC. Tissues were subsequently rinsed, and sterilized using the methods described in <CIT> ; <CIT> ; and <CIT>. The levels of GAG attachment in the tissues were assessed by histological means (differential staining of sections with PAS-Alcian Blue). The pictures in <FIG> indicate uniform attachment of GAGs in cancellous bone and collagen sponge tissues.

In order to demonstrate that the methods according to the invention may be used to attach various adjuncts to tissues, chondroitin sulfate and hyaluronic acid (HA) were attached to stabilized collagen matrix. The results of attachment of chondroitin sulfate to collagen matrix are discussed above in reference to <FIG>. Hyaluronic acid was attached to collagen matrix essentially as described above in Examples <NUM>-<NUM> for chondroitin sulfate, with hyaluronic acid being substituted for chondroitin sulfate in the attachment step. Histological sections showing hyaluronic acid attached to collagen sponge are shown in <FIG> , with <FIG> representing a high-magnification image of the result shown in <FIG>. A histological section of hyaluronic acid attached to pericardium is shown in <FIG>.

As a further example of attaching an adjunct to a collagen matrix, IGF-<NUM>, which is considered representative of growth factors, was attached to a collagen sponge, essentially by the methods described above with reference to chondroitin sulfate in Examples <NUM>-<NUM>. Successful attachment of IGF to collagen sponge was measured using a reverse ELISA assay. In this model, a anti-IGF-<NUM> antibody solution was incubated in contact with a non-IGF-<NUM> attached (control) collagen sponge and an IGF-<NUM> attached collagen sponge. Successful attachment of IGF-<NUM> to the collagen sponge was indicated by a reduction in ELISA signal for the anti-IGF-<NUM> antibody solution after the incubation period. As can be seen in <FIG> , the reverse ELISA assay demonstrates a depressed IGF-<NUM> signal for the IGF-<NUM> attached sponge as compared to the control sponge. Thus, the results shown in <FIG> demonstrate that IGF-<NUM> was successfully attached to collagen sponge in the presence of EDC.

In order to demonstrate the stability of collagenous tissues modified with adjuncts according to the present invention, the stability of chondroitin sulfate attached to crosslinked and sterilized pericardium (prepared essentially as in Example <NUM>, above) was evaluated after <NUM> months of storage at room temperature. Histological stains specific for GAGs (i.e. PAS-Alcian Blue) indicate the successful retention of the attached GAGs to the crosslinked and sterilized membrane. This demonstrates that the method methods according to a reference example allow one to prepare, sterilize and store collagenous tissues with attached adjuncts that are stable in a hydrated form at room temperature. These results are shown in <FIG>.

The following experiment was conducted to demonstrate the biocompatibility of a tissue prepared by methods according to a reference example. In particular, the biocompatibility was evaluated for sterilized collagen sponge having adjunct attached thereto by methods according to a reference example. Crosslinked and sterilized collagen sponge having chondroitin sulfate attached thereto was produced by methods of a reference example and was subjected to cell culture-based assessments. Primary chondrocytes were seeded onto sterilized collagen sponges with or without an adjunct (chondroitin sulfate) attached. The seeded collagen sponges were then incubated in a humidified incubator (<NUM>, <NUM>% CO<NUM>, basal MEM media with <NUM>% fetal calf serum). Viability and metabolic activity of the seeded cells were assessed by the MTT assay at defined time points thereafter. <FIG> shows results from a MTT assay, which measures the mitotic activity (and thus the viability) of the attached cells. The results in <FIG> demonstrate a cognizable advantage in cell growth, which is conferred by the presence of chondroitin sulfate (with both human and bovine chondrocytes, at <NUM> days and <NUM> days of culture, respectively).

<FIG> show low and high magnification microscopic images, respectively, of the cultures after addition of MTT. Viable cells are stained purple, and the presence of newly synthesized matrix is seen around the cells as a thin fibrinous layer. <FIG> is a high magnification image of the sponge showing the appearance of newly synthesized matrix, which has been produced by the seeded chondrocytes.

Stability and compatibility of the tissues with added adjuncts were also tested following implantation in small animals. Briefly, selected samples from the foregoing examples were implanted subcutaneously into rats. Explants were retrieved <NUM> weeks later, and sections prepared from the explants. These sections were histologically processed and stained to assess the presence of the adjuncts, as well as for the nature of cellular infiltrates. <FIG> demonstrates the response from a chondroitin sulfate-modified collagen sponge. Differential staining by PAS-Alcian Blue indicates the continued presence of the GAG even at <NUM> weeks post implantation. Slow release of the GAG is evident by diffuse appearance of the blue stain in select areas ( <FIG> ). The nature of cellular infiltrates into the samples indicates a biocompatible response from the host (i.e. absence of an overt and active inflammation, appearance of new matrix, and blood vessels between the collagen stands of the tissues ( <FIG> ).

The present invention permits the covalent bonding (attachment) of various adjuncts to various types of biological tissues. The biological tissues may be fully-, partially- or un-crosslinked. The adjunct-attached biological tissues are stable over time and are biocompatible. The adjunct-attached biological tissues encourage tissue remodeling, regeneration and healing. Thus, the adjunct-attached biological tissues according to the present invention are useful as implants for a variety of therapeutic uses. The methods of attaching adjuncts to biological tissues according to the invention thus find use in a variety of therapeutic settings.

Claim 1:
A chemically sterilized bioimplant comprising a biological tissue and heparin, the biological tissue being selected from the group comprising decellularized crushed bone fragments, decellularized and/or defatted bone, tendon, ligament, fascia and combinations thereof, and/or bone-connective tissue combinations, wherein the starting tissue is not subjected to a processing step other than decellularization and/or defatting prior to conjugation of the heparin to the tissue;
wherein the heparin is covalently conjugated directly to the biological tissue, and
the bioimplant is sterilized with a sterilizing agent, the sterilizing agent being a carbodiimide in the presence of a C<NUM>-C<NUM> alkanol, and crosslinked with a carbodiimide.