Osteoimplant and method of making same

Abstract

An osteoimplant is provided which comprises a coherent aggregate of elongate bone particles, the osteoimplant possessing predetermined dimensions and shape. The osteoimplant is highly absorbent and sponge-like in nature. Also provided herein are a method of fabricating the osteoimplant and a method of repairing and/or treating bone defects utilizing the osteoimplant.

Claims

1. A method of making an osteoimplant which comprises: a. providing a quantity of elongate bone particles; b. mixing the elongate bone particles with an aqueous wetting agent to provide a fluid composition containing from about 5 to about 40 volume percent swollen, hydrated elongate bone particles; c. introducing the fluid composition into a mold; and, d. removing aqueous wetting agent thereby providing a coherent aggregate of elongate bone particles possessing dimensions and a shape of the osteoimplant.

2. The method of claim 1 wherein prior to step (d), the elongate bone particles are heated.

3. The method of claim 1 wherein at least some of the elongate bone particles are substantially fully mineralized, substantially fully demineralized, partially demineralized or superficially demineralized.

4. The method of claim 1 wherein at least some of the elongate bone particles are superficially demineralized, such elongate bone particles being bonded to each other through cross-links formed in their mutually-contacting surface-exposed collagen.

5. The method of claim 1 wherein removing the aqueous wetting agent causes mechanical entanglement of the elongate bone particles.

6. The method of claim 1 wherein removing the aqueous wetting agent comprises applying the composition to a mesh.

7. The method of claim 1 wherein removing the aqueous wetting agent comprises draining the composition through a sieve.

8. The method of claim 1 wherein removing the aqueous wetting agent comprises drying the composition at a temperature from about 30 C. to about 40 C. and the method further comprises freeze drying the composition at a temperature from about 20 C. to about 35 C. in a vacuum of from about 150 to about 100 mTorr for about 4 to 48 hours.

9. The method of claim 1 further comprising crosslinking the elongate bone particles.

10. The method of claim 1 wherein the mold is a porous tube and removing the aqueous wetting agent comprises drying and lyophilizing the composition in the porous tube.

11. The method of claim 1 wherein the mold is a cylindrical mold and the method further comprises coating walls of the mold with the composition and adding a second composition in to the mold such that the composition forms an outer surface of the osteoimplant and the second composition forms a core of the osteoimplant, the composition comprising partially and/or fully demineralized bone particles and the second composition comprising non-demineralized and/or superficially demineralized bone particles.

12. The method of claim 1 wherein the mold does not apply any pressure to the composition.

13. The method of claim 1 wherein the osteoimplant comprises an open pore, flexible, sponge-like structure and is configured to absorb fluids while generally retaining a predetermined dimension.

14. The method of claim 1 wherein the aqueous wetting agent is at least one of water and glycerol.

15. The method of claim 1 wherein the aqueous wetting agent is a liquid polyhydroxy compound.

16. A method of making an osteoimplant, the method comprising: a. mixing bone particles with an aqueous wetting agent to provide a fluid composition containing from about 5 to about 40 volume percent swollen, hydrated bone particles; b. introducing the fluid composition into a mold; and, c. removing aqueous wetting agent thereby providing a coherent aggregate of bone particles possessing a shape of the osteoimplant.

17. The method of claim 16 wherein prior to step (d), the bone particles are heated.

18. The method of claim 16 wherein at least some of the bone particles are substantially fully mineralized, substantially fully demineralized, partially demineralized or superficially demineralized.

19. The method of claim 16 wherein at least some of the bone particles are superficially demineralized, such bone particles being bonded to each other through cross-links formed in their mutually-contacting surface-exposed collagen.

20. A method of making an osteoimplant, the method comprising: a. mixing bone particles with an aqueous wetting agent to provide a fluid composition containing from about 5 to about 40 volume percent swollen, hydrated bone particles; b. introducing the fluid composition into a mold; and, c. removing aqueous wetting agent to cause mechanical entanglement of the bone particles thereby providing a coherent aggregate of bone particles possessing a shape of the osteoimplant.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIGS. 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h and 1i are non-limiting representations of particular sizes and shapes of osteoimplants prepared and used as described herein;

(2) FIGS. 2 and 2a are a non-limiting representation of an intervertebral implant, specifically, a diaphyseal ring, containing a plug in accordance with the invention;

(3) FIG. 3 is a non-limiting representation of a fusion cage whose void space is filled with a plug in accordance with the invention;

(4) FIG. 4 is a photograph of an osteoimplant produced in accordance with the present invention possessing a preformed cavity or depression; and,

(5) FIG. 5 illustrates a mold which can be utilized in the fabrication of an osteoimplant such as that shown in FIG. 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(6) The composition of the osteoimplant herein can be made up of from about 5 to about 100% fully demineralized and/or demineralized elongate bone particles. At least about 50 weight percent, more preferably at least about 60 weight percent, and most preferably at least about 90 weight percent of the bone particles present in the osteoimplant herein are of the elongate variety. Any non-elongate bone particles that are optionally included in the osteoimplant can possess a wide range of dimensions, e.g., powders, chips, etc. The elongate bone particles form a coherent aggregate, or matrix, which imparts porosity and absorbency to the osteoimplant.

(7) The bone component of the osteoimplant can be obtained from cortical, cancellous, and/or corticocancellous allogenic, xenogenic or transgenic bone tissue. In general, allogenic bone tissue is preferred as the source of the bone component. The bone component can be fully mineralized or partially or fully demineralized. Porcine and bovine bones are particularly advantageous types of xenogenic bone tissue that can be used individually or in combination as sources for the bone particles although of course other xenogenic or transgenic bone tissues can also be used. Combinations of fully mineralized and demineralized bone can also be used.

(8) The bone particles employed in the fabrication of the osteoimplant of this invention are generally characterized as elongate, i.e., they possess relatively high median length to median thickness ratios. In overall appearance, the elongate bone particles can be described as filaments, fibers, threads, slender or narrow strips, etc. Thus, e.g., the elongate bone particles can possess a median length of from about 0.05 to about 200 mm, preferably from about 1 to about 100 mm, a median width of from about 0.05 to about 50 mm, preferably from about 0.1 to about 20 mm, and a ratio of median length to median width of from about 10:1 to about 2000:1, preferably of from about 20:1 to about 600:1. If desired, the elongate bone particles can be graded into different sizes to reduce or eliminate any less desirable size(s) of particles that may be present.

(9) The elongate bone particles can be readily obtained by any one of several methods, e.g., by milling or shaving the surface of an entire bone or relatively large section of bone. Employing a milling technique, one can obtain a mass of elongate bone particles containing at least about 20 weight percent of bone particles coming within the aforesaid range of dimensions.

(10) Another procedure for obtaining the elongate bone particles herein, particularly useful for pieces or sections of bone of up to about 100 mm in length, is the bone processing mill described in commonly assigned U.S. Pat. No. 5,607,269. Use of this bone mill results in the production of long, thin strips which quickly curl lengthwise to provide tubular-like elongate bone particles. The elongate bone particles are optionally subjected to demineralization in accordance with known and conventional procedures in order to reduce their inorganic mineral content. Such demineralization can occur prior to or after forming the elongate particles. Demineralization methods remove the inorganic mineral component of bone by employing acid solutions. Such methods are well known in the art, see for example, Reddi et al., Proc. Nat. Acad. Sci. 69, pp 1601-1605 (1972), incorporated herein by reference. The strength of the acid solution, the shape of the bone particles and the duration of the demineralization treatment will determine the extent of demineralization. Reference in this regard may be made to Lewandrowski et al., J. Biomed Materials Res, 31, pp. 365-372 (1996), also incorporated herein by reference.

(11) As used herein, the expression superficially demineralized refers to bone particles which have undergone surface demineralization as a result of which they possess one or more regions of surface-exposed collagen. The expression partially demineralized bone refers to bone possessing less than its original mineral content but not less than about 8 weight percent of its original mineral content. As previously stated, substantially fully demineralized bone refers to bone containing less than about 8 weight percent, and usually less than about 3 weight percent, of its original mineral content. Mixtures of one or more of the foregoing types of demineralized bone particles can be employed. Moreover, one or more of the foregoing types of demineralized bone particles can be employed in combination with nondemineralized bone particles, i.e., bone particles that have not been subjected to demineralization. It will be understood by those skilled in the art that fully demineralized bone particles yield a more porous mass compared to whole bone or superficially demineralized bone particles.

(12) When prepared in whole or in part from bone particles that are only superficially demineralized or nondemineralized, the osteoimplant will tend to possess a fairly high compression strength, e.g., one approaching that of natural bone. Accordingly, when an osteoimplant exhibiting rigidity, e.g., a compression strength of on the order of from about 5 to about 200 MPa, preferably from about 20 to about 100 MPa and more preferably from about 25 to about 75 MPa, is desired, superficially demineralized bone particles and/or nondemineralized bone particles are advantageously employed.

(13) In a preferred demineralization procedure, relatively large mineralized bone piece(s) from which demineralized bone particles are subsequently obtained, or fully mineralized bone particles obtained from such relatively large mineralized bone piece(s), are subjected to a defatting/disinfecting step which is followed by an acid demineralization step. A preferred defatting/disinfectant solution is an aqueous solution of ethanol, the ethanol being a good solvent for lipids and the water being a good hydrophilic carrier to enable the solution to penetrate more deeply into the bone particles. The aqueous ethanol solution also disinfects the bone by killing microorganisms and viruses. Ordinarily, at least about 10 to 40 percent by weight of water (i.e., about 60 to about 90 weight percent of defatting agent such as alcohol) should be present in the defatting disinfecting solution to produce optimal lipid removal and disinfecting within the shortest period of time. The preferred concentration range of the defatting solution is from about 60 to about 85 weight percent alcohol and most preferably about 70 weight percent alcohol. Following defatting, the bone particles are immersed in acid over time to effect their demineralization. Acids which can be employed in this step include inorganic acids such as hydrochloric acid and organic acids such as peracetic acid. After acid treatment, the demineralized bone particles are rinsed with sterile water for injection to remove residual amounts of acid and thereby raise the pH. The elongate bone particles used in the manufacture of the plug naturally entangled or may be mechanically entangled employing, e.g., the wet laying procedure, akin to a paper-making process, described in aforementioned U.S. Pat. No. 5,507,813 to Dowd et al., to provide a sheet-like coherent mass of bone particles which can thereafter be shaped, e.g., by cutting, molding, etc., before or after drying and/or other processing into configurations corresponding to those desired for the bone plug of this invention.

(14) If desired, the bone particles before or after their being gathered into a coherent aggregate can be modified in one or more ways, e.g., their protein content can be augmented or modified as described in U.S. Pat. Nos. 4,743,259 and 4,902,296, the contents of which are incorporated by reference herein. The elongate bone particles can also be admixed with one or more substances such as binders/fillers, plasticizers, biostatic/biocidal agents, surface active agents, and the like, prior to, during, or after shaping the elongate bone particles into a desired configuration and size. One or more of such substances can be combined with the bone particles by soaking or immersing the elongate bone particles in a solution or dispersion of the desired substance, by physically admixing the elongate bone particles and the desired substance, co-extrusion of the substance and particles, and the like.

(15) Suitable binders/fillers include cyanoacrylates, epoxy-based compounds, dental resin sealants, dental resin cements, calcium phosphate and calcium sulfate self-setting cements, glass ionomer cements, polymethyl methacrylate, gelatin-resorcinol-formaldehyde glues, protein and collagen-based glues, acrylic resins, cellulosics, bioabsorbable polymers such as polyglycolide, polylactide, glycolide-lactide copolymers, polycaprolactone, polyanhydrides, polycarbonates, polyorthoesters, polyamino acids, polyarylates, polycyanoacrylates, polyhydroxybutyrate, polyhydroxyvalyrate, polyphosphazenes, and polyvinylpyrrolidone, carbohydrate polymers, polyiminocarbonates, polypropylene fumarates, polyanhydride esters, polytetrafluoroethylene, hexacryl, Hyaluronic acid, fibrin, fibrin-collagen, polyethylene glycol glues, mucopolysaccharides, mussel adhesive proteins, fatty acids and fatty acid derivatives, etc.

(16) Other suitable biners/fillers include bone powder, demineralized bone powder, porous calcium phosphate ceramics, hydroxyapatite, tricalcium phosphate, Bioglass and other calcium phosphate materials, calcium sulfate or calcium carbonate particles, etc.

(17) Suitable plasticizers include liquid poly hydroxy compounds such as glycerol, monoacetin, diacetin, hydrogels, etc.

(18) Suitable biostatic/biocidal agents include antibiotics, povidone, sugars, mucopolysaccharides, etc.

(19) Suitable surface-active agents include the biocompatible nonionic, cationic, anionic and amphoteric surfactants. It will be understood by those skilled in the art that the foregoing list is not intended to be exhaustive and that other materials may be admixed with bone particles within the practice of the disclosure herein such as disclosed in U.S. Pat. No. 5,073,373, the contents of which are incorporated by reference herein.

(20) Any of a variety of bioactive substances can be incorporated in, or associated with, the bone particles before, during or after fabrication of the osteoimplant. Thus, one or more of such substances can be combined with the elongate bone particles by soaking or immersing them in a solution or dispersion of the desired substance(s). Bioactive substances include physiologically or pharmacologically active substances that act locally or systemically in the host.

(21) Representative classes of bioactive factors which can be readily combined with the bone particles include, e.g., trophic factors, analgesics, anti-cancer agents, vaccines, adjuvants, antibodies, neuroleptics, genes and genetic elements for transfection including viral vectors for gene therapy, cells or cellular components, etc. A list of more specific examples would therefore include, collagen, insoluble collagen derivatives, etc., and soluble solids and/or liquids dissolved therein, e.g., antiviricides, particularly those effective against HIV and hepatitis; antimicrobials and/or antibiotics such as erythromycin, bacitracin, neomycin, penicillin, polymicin B, tetracyclines, biomycin, chloromycetin, and streptomycins, cephalosporins, ampicillin, azactam, tobramycin, clindamycin and gentamicin, etc.; biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids, peptides, vitamins, inorganic elements, co-factors for protein synthesis; hormones; endocrine tissue or tissue fragments, synthesizers; enzymes such as collagenase, peptidases, oxidases, etc., polymer cell scaffolds with parenchymal cells, angiogenic drugs and polymeric carriers containing such drugs; collagen lattices; antigenic agents; cytoskeletal agents; cartilage fragments, modified living cells such as chondrocytes, bone marrow cells, mesenchymal stem cells, natural extracts, genetically engineered living cells or otherwise modified living cells, DNA delivered by plasmid or viral vectors, genes or genetic elements, tissue transplants, demineralized bone powder, autogenous tissues such as blood, serum, soft tissue, bone marrow, etc.; bioadhesives; non-collagenous proteins such as osteopontin, osteonectin, bone sialo protein, laminin, fibrinogen, vitronectin, thrombospondin, proteoglycans, decorin, beta glycan, biglycan, aggrecan, versican, tenascin, matrix gla protein, hyaluronan, amino acids, amino acid residues, peptides, bone morphogenic proteins (BMPs); osteoinductive factor (OIF); fibronectin (FN); endothelial cell growth factor (ECGF); cementum attachment extracts (CAE); ketanserin; human growth hormone (HGH); animal growth hormones; epidermal growth factor (EGF); interleukin-1 (IL-1); human alpha thrombin; transforming growth factor (TGF-beta); insulin-like growth factor (IGF-1) (IGF-2); platelet derived growth factors (PDGF); fibroblast growth factors (FGF, aFGF, bFGF, etc.); periodontal ligament chemotactic factor (PDLGF); somatotropin; bone digestors; antitumor agents; immuno-suppressants; fatty acids (including polar and non-polar fatty acids); permeation enhancers, e.g., fatty acid esters such as laureate, myristate and stearate monoesters of polyethylene glycol, enamine derivatives, alpha-keto-aldehydes, etc.; and nucleic acids; inorganic elements, inorganic compounds, cofactors for protein synthesis, hormones, soluble and insoluble components of the immune system; soluble and insoluble receptors including truncated forms; soluble, insoluble and cell surface bound ligands including truncated forms; chemokines, bioactive compounds that are endocytosed; endocrine tissue or tissue fragments, growth factor binding proteins, e.g., insulin-like growth factor binding protein (IGFBP-2) (IGFBP-4) (IGFBP-5) (IGFBP-6); angiogenic agents, bone promoters, cytokines, interleukins, genetic material, genes encoding bone promoting actions, cells containing genes encoding bone promoting action; growth hormones such as somatotrophin; bone digestors; antitumor agents; cellular attractants and attachment agents; immuno suppressants; bone resorption inhibitors and stimulators; angiogenic and mitogenic factors; bioactive factors that inhibit and stimulate secondary messenger molecules; cell adhesion molecules, e.g., cell-matrix and cell-cell adhesion molecules; secondary messengers, monoclonal antibodies specific to cell surface determinants on mesenchymal stem cells, clotting factors; externally expanded autograft or xenograft cells, nucleic acids and any combination thereof. The amounts and types of such optionally added substances can vary widely with optimum levels and combinations being readily determined in a specific case by routine experimentation.

(22) Various shapes of osteoimplant can be made by using extrusion or injection molding techniques, compression molds, pre-formed molds in which the material can be placed to obtain its final shape, or pre-formed shapes which can be used to cut desired shape from pre-formed material. Further to this, devices can be used that allow introduction of various agents in to the molding devices or treatments of the molds to assist in the forming of various shapes, such as, but not limited to, cross linking agents or heat or cooling of the molds and/or during the forming process. Employing such procedures, various sizes and shapes of osteoimplant can be provided such as those illustrated in FIGS. 1a-h.

(23) In one embodiment herein, an osteoimplant as previously described is made by forming demineralized elongate bone particles into a coherent aggregate and thereafter either cutting the osteoimplant from the aggregate or, preferably, to reduce waste, molding the aggregate into an osteoimplant of the desired size and configuration. To fabricate the coherent mass of elongate bone particles, a quantity of elongate bone particles with or without one or more optional materials is mixed with a suitable biocompatible fluid component, e.g., water, organic protein solvent, physiological saline, concentrated saline solution, ionic solution of any kind, aqueous sugar solution, liquid polyhydroxy compound such as glycerol or glycerol ester, hydrogel, etc., or mixtures thereof. The suitable biocompatible fluid can optionally contain one or more substances such as binder, filler, plasticizer, biostatic/biocidal agent, surface active agent, bioactive substance, etc., as previously described to form a slurry or paste. Excess fluid is then removed from the slurry or paste, e.g., by applying the slurry or paste to a mesh or screen and draining away excess fluid. Functionally, the biocompatible fluid provides a coherent aggregate of elongate bone particles whose consistency can be described as shape-sustaining but readily deformable, e.g., putty-like.

(24) If desired, the elongate bone particles can be dried, e.g., at from about 30 to about 80 C. and preferably from about 40 to about 50 C., for from about 1 to about 3 hours, and then lyophilized under conditions that are well known in the art, e.g., at a shelf temperature of from about 20 to about 35 C., a vacuum of from about 150 to about 100 mTorr and for a period of time ranging from about 4 to about 48 hours. The drying and lyophilization steps will result in the production of a coherent mass of entangled elongate bone particles that is relatively strong when dry and flexible when wetted or hydrated.

(25) In another embodiment of the general method described above, the coherent aggregate of elongate bone particles can be subjected to a compressive force, e.g., of up to about 100,000 psi, during and/or after the step of removing excess liquid and/or while the drained-but-still-wet bone particles are being dried. If desired, the compressed coherent mass can be lyophilized to provide an especially strong and rigid mass.

(26) In yet a further embodiment disclosed herein, the elongate bone particles, in combination with bone particles possessing other geometries such as mineralized and demineralized bone powders and pieces, can be combined with a wetting agent as described above to produce a flowable composition containing from about 5 to about 100%, preferably from about 20 to about 60%, volume percent of bone particles of all types, the remainder of the composition comprising wetting agent. The wetting agent can optionally comprise one or more biocompatible components as previously described. The wetting agent will cause the demineralized elongate bone particles to swell and increase in flexibility. The fluid composition will possess a consistency ranging from a slurry or paste to a wet dough, depending on the amount of wetting agent used. The critical aspect is that the elongate bone particles be suspended in and evenly distributed throughout the fluid composition. This is to be contrasted with the wet laying procedure of commonly assigned U.S. Pat. No. 5,507,813 in which wetting agent is substantially removed to produce a dense mat of bone particles.

(27) In this embodiment, the fluid composition is formed by mixing the bone particles and wetting agent to form a liquid slurry, stirring the slurry for a suitable period of time sufficient to allow the wetting agent to penetrate the demineralized elongate bone particles, and removing enough wetting agent, e.g., by draining through a sieve, sufficient to provide a fluid composition containing from about 5 to about 25, preferably from about 10 to about 15, volume percent bone particles. Substantial mechanical entanglement of the elongate bone particles will occur. Suitable wetting agents include biocompatible liquids and/or hydrogels such as previously described. Optionally, the wetting agent can comprise dissolved or admixed therein one or more biocompatible substances such as previously described.

(28) Preferred wetting agents for forming the wetted mass of bone particles include water, liquid polyhydroxy compounds and their esters, and polyhydroxy compounds in combination with water and/or surface active agents. Specific polyhydroxy compounds of the foregoing type include glycerol and its monoesters and diesters derived from low molecular weight carboxylic acids, e.g., monoacetin and diacetin (respectively, glycerol monoacetate and glycerol diacetate), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol, trimethylolethane, trimethylolpropane, pentaerythritol, sorbitol, polyethylene glycol, polyoxyalkylenes, e.g., Pluronics, and the like. The preferred polyhydroxy compounds possess up to about 12 carbon atoms and, where their esters are concerned, are preferably the monoesters and diesters. Of these, glycerol is especially preferred as it improves the handling characteristics of the bone particles wetted therewith and is biocompatible and easily metabolized. Most preferred are solutions of polyhydroxy compounds in water, with glycerol/water solutions in weight ratios ranging from about 40:60 to about 5:95, respectively, being especially preferred. Mixtures of polyhydroxy compounds or esters, e.g., sorbitol dissolved in glycerol, glycerol combined with monoacetin and/or diacetin, etc., are also useful.

(29) Where the bone particles have a tendency to quickly or prematurely separate or to otherwise settle out from the fluid composition such that formation of a homogeneous suspension of bone particles in wetting agent is rendered difficult, it can be advantageous to include within the composition a suspension aid. Thus, e.g., where the wetting agent is water and/or glycerol and separation of bone particles occurs to an excessive extent where a particular application is concerned, a thickener such as a solution of polyvinyl alcohol, polyvinylpyrrolidone, cellulosic ester such as hydroxypropyl methylcellulose, carboxy methylcellulose, pectin, xanthan gum, food-grade texturizing agent, gelatin, dextran, collagen, starch, hydrolyzed polyacrylonitrile, hydrolyzed polyacrylamide, polyelectrolyte such as polyacrylic acid salt, hydrogels, chitosan, other materials that can suspend particles, etc., can be combined with the wetting agent in an amount sufficient to significantly improve the suspension-keeping characteristics of the composition. Furthermore, suspension aids that generate gas bubbles inside the fluid composition can be employed. The gas bubbles reduce the tendency of the bone particles to settle out and include peroxides and bicarbonate.

(30) As stated previously, the fluid composition is preferably placed in a mold which optionally is configured and dimensioned in the shape of the final osteoimplant. FIG. 5 depicts mold 10 and lid 20 for mold 10, lid 20 possessing protruding indentations 30. The mold can be optionally configured and dimensioned in the shape of the final osteoimplant, e.g., the osteoimplant shown in FIG. 4. Care must be taken to ensure that minimal, if any, pressure is applied to the composition in the mold which would effect compaction of the elongate bone particles. This is in contrast to the wet-lay procedure described in U.S. Pat. No. 5,507,813. The composition is then dried at a temperature of from about 30 C. to about 80 C., preferably from about 30 C. to about 40 C. to effect removal of water and provide a shaped material. Following the drying step, the shaped material is dried e.g., freeze-dried, employing a shelf temperature of from about 20 to about 35 C. and a vacuum of from about 150 to about 100 mTorr applied for from about 4 to about 48 hours. The resulting shaped material is porous and absorbent and maintains its shape and cohesiveness upon absorption of fluid. The implant can be easily cut with scissors in either the dry or rehydrated state.

(31) Alternatively, a slurry of demineralized elongate bone particles can be injected into a porous tube. The bone particles can be dried and lyophilized in the tube, then removed and cut to length. Examples of suitable porous tubes are dialysis tubing, sausage casings, and rigid metal or plastic tubing perforated with a series of small holes (the holes generally being small enough that a few fibers escape, preferably 0.2 mm or less). It is also possible to use rigid tubing with large holes, and line with another tube that will contain the bone particle slurry such as a flexible dialysis tube. The slurry can be injected into the tube by any suitable means, for example a disposable plastic syringe or a slurry pump. If a thin, flexible tube is used to form the osteoimplant, the osteoimplant can be cut to length (after drying) while inside the tube by cutting through both the tube and the material inside. Alternatively, the dried aggregate elongate bone particles can be removed from the flexible tube (preferably by cutting the tube away) or from a rigid tube (preferably by pushing the material out of the tube) and then cut. Cutting is facilitated by using a cutting jig or guide similar to a cigar cutter or a small double bladed guillotine type of device where the blade spacing equals the desired plug length. Osteoimplants that are dried in a porous tube can have a tougher skin on their outer circumferential surface due to more rapid water loss from the surface than the interior. This can be advantageous in that the osteoimplant will resist insertion and handling forces better. The thickness and toughness of the skin can be influenced by a combination of drying conditions and tubing porosity.

(32) Optionally, the bone particles in the osteoimplant can be crosslinked in accordance employing well-known techniques, e.g., those disclosed in U.S. Pat. No. 6,294,187 the contents which are incorporated by reference herein. These crosslinking procedures result in the formation of chemical bonds between the surface-exposed collagen of mutually contacting surface-demineralized and/or substantially completely demineralized elongate bone particles making up, or contained in, the aggregate of naturally or mechanically entangled elongate bone particles.

(33) Where a mold, e.g., a cylindrical mold, is employed to shape the coherent mass of bone particles into the osteoimplant of this invention, the walls of the mold can be coated with a slurry or paste containing partially and/or fully demineralized bone particles followed by addition of a slurry or paste containing non-demineralized and/or superficially demineralized bone particles (or vice versa). The resulting molded osteoimplant contains at least one region, e.g., an outer surface, composed of partially and/or fully demineralized bone particles and at least one region, e.g., a core, composed of non-demineralized and/or superficially demineralized bone particles. In this manner, the differential in compressive strength, porosity, osteogenicity and other properties between partially and/or fully demineralized bone particles on the one hand and non-demineralized and/or superficially demineralized bone particles on the other hand can be exploited. For example, where the osteoimplant is employed in a load-bearing situation, non-demineralized and/or superficially demineralized bone particles can be concentrated in that region of the osteoimplant which will be subjected to an applied load at the implant site.

(34) When the osteoimplant of this invention is fashioned as a plug, the plug can assume a determined or regular form or configuration such as may be desirable for any specific cavity of a commercially available osteoimplant, e.g., spinal cages, or any other relatively well-defined cavity at a surgical site, e.g., extraction sockets, bore holes, etc. Of course, the coherent mass can be machined or shaped by any suitable mechanical shaping means. Computerized modeling can, for example, be employed to provide an intricately-shaped plug which is custom-fitted to a particular cavity of an osteoimplant with great precision. In a preferred embodiment, the plug possesses the configuration of a central cavity of a commercially available spinal cage.

(35) A plug fabricated in accordance with this invention preferably possesses a bone particle content of at least about 5 to about 100 weight percent, preferably at least about 20 weight percent and more preferably at least about 60 weight percent, based on the weight of the entire mass. It will be understood by those skilled in the art that plugs possessing a putty-like consistency will possess lower amounts of bone particles, on a weight-by-weight basis, compared to plugs which are subjected to the drying and lyophilizing and/or compression steps described above.

(36) The foregoing plug can be easily inserted into a cavity of an implant or bone defect site. The plug will often tend to swell somewhat upon contact with irrigation fluids such as those that are commonly used during surgical procedures or upon contact with bodily fluids normally present at the implant site. This swelling has the advantage of providing a tighter fit with the cavity of the osteoimplant or bone defect thus assuring that the plug will remain in place.

(37) The plug can be used for implantation at a surgical site in a variety of ways and for treatment of a variety of bone defects. In certain embodiments, the osteoimplant can be manufactured to contain the plug prior to its packaging and storage. Alternatively, the plug can be provided in a form suitable for placement in an osteoimplant at the time of the implantation of the latter. In yet a further embodiment, the plug described herein is provided in a form suitable for association with one or more osteoimplants or one or more cavities of an osteoimplant after the osteoimplant has been placed at an implant site. In yet a further embodiment, the preformed plug described herein is provided in a form suitable for placement in a relatively well-defined defect site such as, e.g., an extraction socket, bore hole, etc. Of course, any combination of the above-described embodiments may be used depending upon the intended use and/or specific defect site.

(38) In any of the above-described plug embodiments, the plug can be used alone, associated with standard rehydrating solutions such as previously described, associated with autograft tissue such as iliac crest, local bone and/or marrow, blood, plasma or serum obtained at the time of implantation or sometime prior to implantation as well as combinations thereof. In certain embodiments, the plug will be placed at an implant site in the as-packaged condition. In alternative embodiments, the plug can be rehydrated just prior to implantation.

(39) As previously stated, the plug form of the osteoimplant herein can be inserted into the cavity of the implant as is or, if desired, it can be suitably compressed prior to insertion into the implant or defect site. When the plug is compressed just prior to its insertion, any excess liquid or moisture will be displaced from the material as the volume of the plug is temporarily reduced. In this embodiment, the compressed plug, being smaller than its receiving cavity, is easily inserted into such cavity. After relaxation of the applied compressive force, the plug will substantially return to its original volume thus assuring-its retention in the cavity.

(40) The foregoing description and embodiments were chosen and described to best explain the principles of the invention and its practical applications, thereby enabling others skilled in the act to best utilize the invention in its various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, the foregoing descriptions of the preferred embodiments of the disclosure herein have been presented for purposes of illustration and description and are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many other modifications and variations are possible in light of the above teachings. For example, the plug can be configured to fit a predetermined defect caused at an implant site, e.g., a bore hole of defined diameter and depth. Optionally, the plug can be contacted with fluids such as blood, plasma, serum, bone marrow, etc. obtained from the recipient immediately prior to implantation. Such modifications are also envisioned as being within the scope of the invention herein.

(41) The following examples illustrate the practice of the present invention and in no way limit the scope of the claims appended hereto.

Example 1

(42) Process of Making a Species-Specific Osteoimplant with Defined Dimensions

(43) Species-specific (Rhesus Monkey) long bones were aseptically cleaned. The cortical bone was processed in the bone milling apparatus described in U.S. Pat. No. 5,607,269 to yield 65 grams of elongate bone particles. The elongate bone particles were placed in a reactor and allowed to soak for 5-10 minutes in 0.6N HCl plus 20-2000 ppm nonionic surfactant solution. Following drainage of the HCl/surfactant, 0.6N HCl at 15 ml per gram of total bone was introduced into the reactor along with the elongate bone particles. The reaction proceeded for 40-50 minutes. Following drainage through a sieve, the resulting demineralized elongate bone particles were rinsed three times with sterile, deionized water at 15 ml per gram of total bone, being replaced at 15 minute intervals. Following drainage of the water, the bone particles were covered in alcohol and allowed to soak for at least 30 minutes. The alcohol was then drained and the bone particles were rinsed with sterile deionized water. The bone particles were then contacted with a mixture of 4.5 ml glycerol per gram of dry bone particles and 10.5 ml sterile, deionized water per gram of dry bone particles for at least 60 minutes. Excess liquid was drained and the resulting liquid composition containing approximately 11 (w/v) demineralized elongate bone particles was transferred to a 11 cm11 cm mold containing a lid having a plurality of protruding indentations such as those depicted in FIG. 4. The dimensions of the protrusions were specific for the size of the osteoimplant required for the Rhesus monkey. The lid was gently placed on the mold such that the indentations became immersed into the liquid composition to exert as little pressure on the composition as possible. The mold was then placed in an oven at 46 C. for 4 hours. The composition was then frozen overnight at 70 C. and then lyophilized for 48 hours. Following lyophilization, the mold was disassembled and the formed composition was cut into individual pieces that contained troughs corresponding to the dimensions of the lid protrusions. The resulting pieces had dimensions of 4.5 cm in length, 2.5 cm in width and about 8 mm in height with trough dimensions of 3.5 cm in length, 1 cm in width and 4 mm of depth.

(44) The resulting composition was cohesive, flexible, and sponge-like with an obvious continuous three-dimensional structure possessing visible open pores. The implant had a defined shape including the indentations made by the lid protrusions, did not require rehydration before use, and was more rapidly hydratable in comparison to Grafton Flex. The material retained its shape once wetted with fluids and freezing was not required for storage.

(45) The density of bone is based on calculation of the defined mold volume used and the amount of demineralized bone particles used to fill the volume of the mold. In making the composition described in this example, 12 g demineralized fibers occupied a volume of 105 cm.sup.3. Therefore, the density was approximately 0.114 g of bone/cm.sup.3. These calculations are approximate as there can be a range in weights (about 10-20 g) and a range in volumes of about 100-120 cm.sup.3 (which can be defined by the dimensions of the mold used).

Example 2

(46) Evidence of Osteoinduction by Grafton DBM in Non-Human Primate Spine Fusion

(47) While autogenous iliac crest bone graft remains the gold standard, much work continues to identify viable bone graft extenders, enhancers, and substitutes. While several demineralized bone matrix formulations have been shown to be variably osteoinductive in rodent ectopic bone assays, few have demonstrated efficacy in higher species and more challenging applications such as posterolateral spine fusion. To date, none have been tested in a non-human primate posterolateral spine fusion model which has been previously determined to be extremely challenging with less than 40% of animals achieving successful fusion with autogenous iliac crest bone graft. The purpose of this example was to test the osteoimplant described in Example 1 for evidence of osteoinduction and its use as an extender/enhancer for autogenous bone graft in a non-human primate.

(48) Four skeletally mature rhesus macaques underwent single level lumbar posterolateral arthrodesis through a Wiltse muscle-splitting approach under general anesthesia. The transverse processes were decorticated with an electric burr. Autogenous iliac crest bone graft was harvested bilaterally through separate fascial incisions. In these four animals, rhesus-specific osteoimplant material (described in Example 1) was implanted with the usual autograft (4 g) on one side of the spine and one half the usual autograft (2 g) on the opposite side. Radiographs were taken at intervals until euthanasia at 24 weeks. The lumbar spines were excised and palpated manually to determine fusion status as fused or not fused and then underwent CT scanning to visualize the amount of bone formation. Radiographs and CT scans were evaluated blindly and assessed semi-quantitatively for the area of the fusion mass (3=good, 2=fair, l=poor) and the amount of bridging between the transverse processes on each side (0=<25%, 1=25%, 2=50%, 3=75%, 4=100%). Points were added for each site in each animal. Three of four monkeys receiving the osteoimplant plus autograft were graded as fused. Six of eight sites in the were rated as good for area of fusion mass on CT (computer tomography) scans. Six of eight sites had at least 50% bridging. The quality and amount of bone was better in the osteoimplant group and best with the 4 g of autograft. Although the assessment of bone formation was semi-quantitative, given the spectrum of fusions previously obtained in this model with autograft alone, these data support evidence of osteoinduction of the osteoimplant in a challenging model. These data support the role of this osteoimplant as an osteoinductive graft extender and graft enhancer in rhesus posterolateral spine fusion.

Example 3

(49) Implantation of Osteoimplant in a Human Patient to Promote Spinal Fusion

(50) Human-specific osteoimplant was made in the same manner described in Example 1. However, the mold dimensions and final dimensions of the osteoimplant were altered to adjust to the approximate size required for human posterolateral spinal fusion procedure (known by those skilled in the art). The dimensions of the osteoimplant pieces were approximately 5.0 cm in length, 2.5 cm in width and approximately 1 cm in height with trough dimensions 4 cm in length, 1.5 cm in width and depth approximately 0.7 cm. The trough design specifically allowed for the surgeon to fill the center of the osteoimplant with autograft or allograft or both. Autograft is usually obtained from local bone at the site of the procedure, or marrow, or iliac crest or a combination. The fluids rapidly dispersed within the osteoimplant hydrating the osteoimplant. The osteoimplant is placed either trough down facing the decorticated transverse processes or trough facing away from the decorticated transverse processes to allow blood to be absorbed by the sponge-like nature of the osteoimplant. The osteoimplant remains as a three-dimensional cohesive structure retaining the autograft or allograft or both at the implant site. The surgery then follows usual closure procedure known to those skilled in the art.

Example 4

(51) Evaluation of the Osteoinductive Potential of Example 1

(52) The osteoinductive potential of Example 3 (human-specific osteoimplant) for posterolateral fusion (PLF) was evaluated using the standard heterotopic osteoinductive implant model (see, Edwards J T, Diegmann M H, Scarborough N L, Osteoinduction of human demineralized bone: Characterization in an animal model, Clin Orthop Rel Res 357:219228 (1998) which is a modification of Urist M R, Bone formation by autoinduction, Science, 150:893-899 (1965)). Implants are placed in the hind limb, intramuscular sites of athymic rats and evaluated histologically after 28 days.

(53) Animal Model

(54) The study was conducted in the athymic (nude) rat to minimize the potential for a cross species incompatibility response to xenograft tissue implants. The hind-limb intramuscular site is ideal for the initial determination of heterotopic bone induction properties of implant materials, as bone is not present in this area.

(55) Implant Placement

(56) The study utilized a singular intramuscular (IM) implantation site in each hind limb of the animals. Different specimen types were placed in the sites in a randomized fashion, such that the same animal did not have the same treatment in both hind limbs. To provide a common positive control over all animals, a single 40 mg sample of rat DBM powder was placed intramuscularly over the left pectoralis (LP) muscle on the left side of each rat. Animals were allowed normal activities following surgical procedures. Four samples of each material were used for analysis.

(57) Procedure

(58) Briefly, rats were anesthetized with a mixture of ketamine (250 mg), xylazine (11 mg), and physiological saline (10 ml). The dosage is 3.6 ml/kg body weight administered intraperitoneally. Aseptic surgical procedures were carried out in a laminar airflow hood. A 1 cm skin incision was made on each upper hind limb using a lateral approach and the skin was separated from the muscle by blunt dissection. A superficial incision aligned with the muscle fiber plane was made to allow for insertion of the tips of the scissors. Blunt dissection of the muscle to create a pocket and positioning of the rat DBM powder or devitalized fibers was made using a blunt syringe. In each case, the skin was closed with metal clips.

(59) Rats were euthanized with CO.sub.2 following 28-day implantation time. Implant materials were located by palpitation, retrieved by blunt dissection and cleaned of the surrounding tissue by careful trimming. An observer blinded to implant type performed a macroscopic evaluation of the implant material. Color, vascularity, hardness and integrity were scored according to the scheme outlined in Table 1; the highest score for the most robust response would be 1, while a specimen showing little or no osteoinductive potential would score 0. Experience with this model has shown a high correlation between visual observations and histological observations of DBM implant performance.

(60) Histology

(61) Retrieved materials were fixed in neutral buffered formalin, dehydrated in a series of graded ethanol solutions, embedded in JB-4 (glycol methacrylate, Polysciences, Inc., Warrington, Pa.) and sectioned. Toluidine blue was used for staining and each material was evaluated using a light microscope at magnifications up to 200.

(62) A numerical score of 0, 1, 2, 3, or 4 was given to grade the extent of new bone formation for each explant when examined under the light microscope. Assignment of scores was according to the descriptions given in Table II below. Histological sections for each explant were scored independently by two individuals blinded to treatment groups.

(63) Following histological analysis, average scores were calculated for each material type or sample group. Based on previous experience with this animal model, each group was assigned an assessment of osteoinductive potential based on the average histological scores. Sample groups scoring 0 show no osteoinductive response; groups scoring up to 2 show a slight osteoinductive response and groups scoring 3 or above show a robust osteoinductive response.

(64) TABLE-US-00001 TABLE I Macroscopic Observation Scoring Guidelines Color White (W) Gray (G) Red (R) Vascularity None (N) Some (S) Robust (R) Hardness Mushy (M) Firm (F) Hard (H) Integrity Diffuse (D) Flat (F) Nodule (N) Score 0 0.5 1

(65) TABLE-US-00002 TABLE II Scoring of Histological Sections Score New Bone Formation 0 No new bone 1 Few areas of new bone formation 2 Numerous areas of new bone formation 3 Greater than 50% of nodule involved in new bone formation 4 Greater than 75% of nodule involved in new bone formation
Results

(66) Histology showed evidence of robust cartilage, bone and marrow formation in the samples. Scores for the individual samples were averaged and the meanSD of the osteoinductive score for 13 individual samples derived from Example 3 was 3.30.7. Historically, demineralized bone powder produces a comparable osteoinductive score of 3.60.8 while guanidine hydrochloride extracted samples routinely display lack of inductivity. The foregoing results demonstrate that the osteoimplant of the invention possesses excellent osteoinductivity with the additional advantage of being a cohesive three-dimensional, lower density, porous matrix.

Example 5

(67) This example illustrates the preparation of a plug in accordance with the invention which is intended for insertion in the cavity of an implant as shown in FIGS. 2, 2a and 3, or in the cavity of a bone defect site.

(68) Mineralized milled and sieved fibers of cortical bone were fully demineralized with a 30 ml volume of 0.6M HCl/0.025% Triton X-100 per gram of bone at room temperature. The acid was decanted and the remaining fibers were washed with deionized water to remove the remaining acid. The demineralized elongate fibers were soaked in a 70% ethanol and deionized water solution for at least 30 minutes. The elongate demineralized fibers were washed with deionized water to remove the ethanol solution. The wet elongate demineralized fibers were added to a glycerol-deionized water solution and mixed well. The fibers and the glycerol-deionized water solution was allowed to sit for at least one hour. The elongate demineralized fibers were lightly packed in a cylindrical vessel to form a mold with a desired configuration which was capped to prevent loss of moisture. The capped vessel was heated in an oven at 46 degrees Celsius for a period of from two to six hours. The capped vessel was then placed in a freezer for from 6 to 24 hours at 70 degrees Celsius. The vessel was then removed from the freezer and the cap was removed. The uncapped vessel was then lyophilized for 48 hours. The lyophilized plug was then removed from the container.