Stimulating Osteoinduction 5

Technical Field

This invention relates generally to a method for stimulating osteoinduction and more specifically to a process for adapting soluble 'regenerating' factors to

10 effectively initiate new cartilage and/or bone growth at selected skeletal locations in humans and animals. Background Art

Regeneration of skeletal tissues is regulated by specific protein factors that are naturally present

15 within bone matrix. During the healing process, these components stimulate certain cell populations to form new cartilage and bone tissue which serve to replace that which was lost or damaged. Such protein substances, if extracted and purified, have potential use in clin-

20 ical situations where skeletal tissue regeneration is necessary to restore normal function, for exa.tiple, at fracture sites and at sites of periodontal defects. In addition, such a protein substance can enhance or pro¬ mote bony ingrowth into various prosthetic devices and

25 bony implants, such as allografts, processed xenogenil bone chips and the like.

Bone matrix protein is readily soluble in body fluids. Its solubility precludes direct _in_ vivo implan¬ tation at the site of a skeletal defect. For example, ^ 30 if a dissolvable capsule containing soluble bone protein is implanted at an ectopic intra-muscular site, no cart- ilage or bone induction occurs. Similarly no cartilage or bone ,induction occurs when soluble bone protein is incorporated, by lyophilization, into an inert carrier

35 (i.e., derrtineralized guanidinium chloride extracted

cancellous or cortical bone chips) and implanted at ectopic sites. Diffusion of such soluble proteins away from the implant site occurs before the 1-2 days neces¬ sary for appropriate cell populations to accumulate. Thus, special procedures are required to 'immobilize ¹ this substance in such a way that factor release coin¬ cides with the presence of sufficient numbers of respons¬ ive cell types which then will be stimulated to form cartilage and bone. Disclosure of the Invention

This application is related to copending applica¬ tion Ser. No. 591,505, under the title of Bone Purifica¬ tion Process, which discloses a process of obtaining a soluble bone matrix derived protein capable of causing undifferentiated cells to undergo chondrogenesis and to copending application Ser. No. 591,440, under the title of Process of and Material for Stimulating Growth of Cartilage and Bony Tissue at Anatomical Sites, which discloses a process in which live cells are exposed in vitro to the bone protein and then transferred _in_ vivo to cause chondro/osteogenesis. The disclosures of both copending applications are incorporated by reference.

The invention provides a method of adapting soluble bone protein for use in osteoinduction and involves combining soluble bone protein purified to a state ef¬ fective to initiate chondrogenesis with carrier means for retarding diffusion of said protein from a site of in vivo implantation.

In one embodiment of the invention, the carrier means for retarding diffusion of soluble bone protein comprises a biocompatible, biodegradable mass capable of releasing the protein in a time dependent manner. A preferred carrier of this class is a fibrin clot. A fibrin clot is useful in that it can be molded to fit the contours of small defects such as periodontal pock¬ ets.

In another embodiment the carrier means comprises bony implant means such as allografts, specially processed xenogenic bone chips and the like, treated so as to be capable of releasing the protein in a time dependent manner. The bony implant means can be treated by soaking in a surface coating solution, such as a gelatin or fibrin solution or the like, that is capable of being dried to form an adherent, biodegradable coating. Another method of immobilization involves chemical cross- linking of inert carriers into which soluble bone matrix derived protein has been incorporated. Both techniques are effective to trap the soluble bone protein and to provide for its controlled release when implanted in vivo. A preferred procedure involving demineralized, defatted, 4M guanidinium chloride extracted bone, which is typically 90% Type I collagen, comprises soaking the bone in a solution of the soluble bone protein, crying the bone, and then cross-linking it with agents such as gluteraldehyde, formaldehyde, carbodii ide or the like. The cross-linking procedure effected by these agents results in a molecular collapse of the collagenous ma¬ trix structure thereby trapping incorporated soluble bone protein. Such physical entrapment within this insolubilized matrix increases the time normally required for matrix hydration by interstitial fluid so that pro¬ tein release is sufficiently prolonged to allow for adequate host tissue ingrowth.

Another embodiment of the invention which is par¬ ticularly applicable to prosthetic devices comprises the steps of soaking the prosthesis in a surface coating solution containing soluble bone protein purified to a state effective to initiate chondrogenesis and a con¬ trolled release agent. The surface coating is dried on the prosthesis so as to form an adherent insolubilized ^•

• coating capable of releasing the protein in a time de¬ pendent manner. The controlled release agent is at least one member selected from the group consisting of gelatin and fibrin, with gelatin being preferred. The gelatin-bone protein coating, which can be applied to a variety of prosthetic devices to initiate cartilage and bone formation at the tissue-implant interface, is in¬ solubilized directly upon the implant by simple dehy¬ dration. The relatively insoluble nature of the dehy- drated complex prolongs protein release for the approp¬ riate period. Fixation of steel, ceramic and other metal alloy implant devices used for reconstruction or stabilization of damaged .bone will be .enhanced through the stimulation of a natural osseous bridge anchoring the implant within the surrounding skeletal tissue.

Other features and a fuller understanding of the invention will be had from the following detailed des¬ cription of the best modes. Best Modes for Carrying Out the Invention The following examples more fully describe the invention useful for regenerating skeletal tissue by enhancing bone ingrowth into bone defects, bony implants and prosthetic devices.

Example I

An intended use for the method of the present inven¬ tion involves the direct implantation into fracture sites or periodontal defects of treated bone into which soluble bone protein has been incorporated. Bovine cancellous bone chips were demineralized in 0.6M HC1 for 4 days at 4°C. Following a cold (4°C) water wash, the bone pieces were defatted in chloroform- methanol (1:1) at room temperature and allowed to air dry overnight. A 4M guanidinium chloride extraction (3 days at 4°C) removed soluble components possessing inter-

fering biological or immunological properties. The bone pieces were then lyophilized.

Small square shaped pieces (4-5 mm Long x 1-2 mm wide) of the demineralized, defatted bone were incubated under vacuum in an aqueous solution containing 800-1000 ugs Lowry protein/ml of soluble bone protein identified as Protein γι. prepared in accordance with the process disclosed in Ser. No. 591,505. One such treated bone piece absorbed approximately 200ul of solution, thereby incorporating about 200ug of soluble bone protein. Be¬ cause the treated bone piece had a highly porous struc¬ ture, the surface area could not be accurately determined, but was estimated to be 2-3cm2, which gave a coating concentration of roughly The bone pieces were lyophilized. Some of them were further treated by cross-linking with gluteraldehyde (2.5% in water) at 4°C overnight. Excess gluteraldehyde was removed with a cold water rinse.

Other small pieces of the demineralized defatted bone were soaked in a 10% gelatin solution containing

800ug/ml of Protein PΛ J . These were air dried overnight so as to coat the external surfaces of the carrier with a thin layer of semi-solid gelatin-bone protein mixture. A 10% gelatin solution solidifies at 25°C and progres- sively dehydrates with time forming first a viscous- glue and finally a dry adherent paste.

Example 2 Soluble bone protein identified as Protein Ayi was prepared in accordance with the procedure disclosed in Ser. No. 591,505. 800 to lOOug Protein _V ι was added to 1 ml of 0.1M phosphate buffer (ph 7.4) containing 50 mg fibrinogen. To this mixture was added 20 units of _; - thrombin (20 ul of a 1000 units/ml stock solution) . Within several minutes a clot formed, trapping Protein y _j inside the clot.

The clot was implanted into a defect in the illiac crest of adult Fischer rats. This defect was created by removing a standarized section (0.5 cm) of crest bone form the illium using a pair of roungers. Three to four weeks after implantation, the site of the implant was assessed histologicall . Defect sites implanted with fibrin clots containing soluble bone protein showed increased amounts of- cartilage or bone formation compared to the amounts of cartilage or bone formed at defect- sites implanted with fibrin clots containing albumen or with fibrin clots alone.

Example 3 , Demineralized bone cnips prepared as described in Example 1, (4 to 5mm long; 1 to 2mm wide) were incubated in a 10% gelatin solution containing 800ug/ml of the Protein Ay-j. The treated bone chips were air dried and implanted at an ectopic intra muscular site in five to eight week old CBA male mice. Fourteen days after im- plantation, sites were examined by X-ray and histology. Sites implanted with gelatin-soluble bone protein coated chips showed induction of cartilage and bone. Sites implanted with gelatin-albumen coated chips showed no induction.

Example 4 Demineralized bone chips prepared as described in Example 1 were incubated under vacuum in an aqueous solution containing 800-lOOOug/ml of Protein ι« The chips were cross-linked by soaking overnight in a 2.5% solution of gluteraldehyde at 4°C. The chips were im¬ planted into ectopic intra-muscular sites as in Example 3. Fourteen days after implantation, the sites were examined by X-ray and histology. Sites implanted with cross-linked bone protein chips showed induction of

cartilage and bone formation. Sites implanted with cross-linked albumen chips showed no induction.

Example 5

Human bone chips demineralized and defatted as described in Example 1 were incubated under vacuum in an aqueous solution containing 800-1000ug/ml of Protein VJ. The chips were cross-linked with gluteraldehyde as in Example 4 were implanted in an ectopic sub-cutan¬ eous site in white leghorn chick hatchlings. Fifteen days after implantation the sites were examined histolog- ically. Sites implanted with cross-linked bone protein chips showed cartilage and bone deposits. Sites implan¬ ted with cross-linked albumen chips showed fibrous encapsu¬ lation only.

Modifications of the above invention and materials- and procedures employed therein which are obvious to persons of skill in the art are intended to be within the scope of the following claims..