Improved bone graft

专利摘要:
The desalted bone matrix (DBM) or other matrix composition of the present invention is mixed with a stabilizer that acts as (1) diffusion barrier, (2) enzyme inhibitor, (3) competition substrate, or (4) masking component. The diffusion barrier acts as a barrier to prevent proteolysis and degradation by the enzyme in the bone graft-inducing factor found in the DBM. Stabilizers can be any biodegradable material such as starch, modified starch, cellulose, dextran, polymers, proteins, collagen. When the stabilizer is degraded in vivo, bone induction factors such as TGF-β, BMP, and IGF are activated or exposed, and the activated factors recruit cells to the site of injury in the perivascular space and into bone-forming cells. Differentiate The present invention also provides a method of making, testing and using the improved osteoinductive matrix composition according to the present invention.
公开号:KR20040047746A
申请号:KR10-2003-7010638
申请日:2002-10-15
公开日:2004-06-05
发明作者:데이비드 크낙크；캐티 트라이아네데스；마이클 디에그만；나네테 포르시트；존 윈터바텀
申请人:오스테오테크, 인코포레이티드；
IPC主号:

专利说明:

IMPROVED BONE GRAFT}
[3] Rapid and effective recovery of bone defects caused by injuries, diseases, wounds, surgery and the like is a long-standing wish of orthopaedic surgery. To this end, various compositions and materials have been used or suggested for use in repairing bone defects. The biological, physical, and mechanical properties of these compositions and materials are among the major factors affecting fitness and efficacy in various orthopedic applications.
[4] Autologous spongy bone ("ACB") is considered the standard of bone graft. ACB is osteoinductive, non-immunogenic and possesses all the structural and functional properties suitable for a particular recipient. However, ACB is only available in some limited environments. Some individuals lack ACBs of the appropriate size and quality for transplantation. In addition, donor site morbidity can cause serious problems for patients and their attending physicians.
[5] Many efforts are being made to identify or develop alternative bone grafts. Desalted bone matrix (“DBM”) implants have been reported to be particularly useful (US Pat. Nos. 4,394,370; 4,440,750; 4,485,097; 4,678,470; 4,743,259; Mulliken et al., Calcif. Tissue Int. 33:71, 1981; Neigel et al., Opthal.Plast.Reconstr.Surg. 12: 108, 1996; Whiteman et al., J. Hand.Surg. 18B: 487, 1993; Xiaobo et al., Clin. Orthop. 293: 360, 1993). Typically, demineralized bone matrices are obtained from carcasses. Bone removes infectious pathogens by embalming and / or other treatments. The bones are then made into fines by milling or grinding, and the mineral components are extracted (eg, soaking the bones in acid solution). The remaining matrix can be processed further to be malleable or shaped to be suitable for implantation into recipient specific sites. Demineralized bone made in this way contains various components, including proteins, glycoproteins, growth factors, and proteoglycans. After transplantation, the presence of DBM induces cell recruitment at the site of injury. The recruited cells can ultimately differentiate into cell-forming cells. This mobilization of cells increases the rate of wound healing and thus results in a more rapid recovery of the patient.
[6] Current DMB formulations have many disadvantages. First, although the collagen-based matrix of DBM is relatively stable, the active factors in the DBM matrix are rapidly degraded. Osteoblastic activity of DBM is significantly reduced within 24 hours from transplantation, and in some cases, osteogenic activity is inactivated within 6 hours. Thus, factors associated with DBM are available for recruiting cells to the site of injury only for a short time after transplantation. In most healing processes, which take two weeks to several months, these implanted materials provide no help in mobilizing cells.
[7] In addition to the active factors present in the DBM, the overall structure of the DBM implant is also believed to contribute to the bone healing ability of the implant.
[8] Summary of the Invention
[9] The present invention relates to improved demineralized bone matrix ("DBM") compositions, methods of making and using compositions according to the present invention, and kits containing such compositions. The present invention provides a rapid reduction in osteoinductive ability observed in existing available DBM compositions by (1) the induction of osteoinductive agents by proteases, sugar-degrading enzymes, or other enzymes present in the host or DBM itself. decomposition; (2) diffusion of osteoinducing agents in the DBM; Or (3) it is due to the reduced activation of osteoinducing agents in the DBM. Accordingly, the present invention provides a DBM composition that protects the osteoinducing agent from degradation and / or a composition that protects the osteoinducing agent from diffusion in the composition. The present invention is also based on the activation of osteoinductive factors found in DBM. In some embodiments, the present invention provides improved form-maintaining properties that contribute to the overall efficacy of the DBM composition. In some embodiments, an IBM composition according to the present invention can be used as a delivery device to administer a bioactive agent.
[10] The protection of active factors in DBMs can include (1) diffusion barriers (eg, polymers, starches), (2) enzyme inhibitors (eg, protease inhibitors), (3) competitive substrates, and / or (4) masking components. moiety). Certain embodiments of the present invention provide DBM compositions containing stabilizers or other factors such as polymers (eg, protease inhibitors). Preferably, the diffusion barrier polymer is metabolized over time, as a result of which the osteoinducing agent is exposed and / or released from the DBM composition over time, or the rate of degradation is delayed. The diffusion barrier of the present invention may also be used as an alternative means of reducing the diffusion of activating enzymes with factors present in the DBM composition. Suitably, such exposure, release, controlled release, or controlled degradation proceeds for a period of at least several hours, preferably one to several days, more preferably weeks or months. In suitable embodiments, the rate of degradation, release, and activation is balanced to yield a DBM composition having a desired level of osteoinductivity over time. Compositions according to the invention containing stabilizers exhibit osteoinductive activity for longer periods of time than observed in comparative compositions lacking stabilizers.
[11] In some embodiments of the invention, the stabilizer is a biodegradation that inhibits or delays the diffusion of osteoinducing agents in polymers, such as biodegradable polymers, especially DBM compositions, or blocks the access of degradation and / or activating enzymes to osteoinducing agents. It contains a sex polymer. Examples of biodegradable polymers are starch, dextran, cellulose, polyester, protein, polysaccharides, polycarbonate, polyarylate, PLGA and the like. Suitably, the polymer is biocompatible and biodegradable. In another embodiment, the DBM composition of the invention contains or is treated with an agent that inhibits the activity of one or more activating enzymes, proteases or glycosidase. Such inhibitors are expected to interact with osteoinducing agents or other active agents in DBM and thus reduce the activity of certain enzymes (regardless of host or DBM) that reduce osteoinductive or wound healing. Alternatively or in addition, the DBM compositions of the invention contain inhibitors provided in time-release formulations (eg, in encapsulated form in a biodegradable polymer). In the case of activating enzymes (eg, enzymes that induce the release, expression or development of osteoinductive factors), inhibitors that reduce the activity of the activating enzyme result in increased osteoinduction for extended periods of time rather than continuous release immediately after implantation.
[12] Some embodiments of the present invention provide a DBM composition wherein the composition or portion thereof is adapted to modulate or adjust the rate of loss of osteoinduction. As one example, DBM compositions can be made from a variety of separate DBM formulations, each of which has DBM particles of different sizes or contains different amounts or types of stabilizers. For example, DBM formulations or powders can be made to have varying half-lives, for example, by varying the nature or content of the stabilizing polymer, the degree of cross-linking of the polymer, the thickness of the stabilizing coating, the particle size, and the inhibitor content of the activating / degrading enzyme. have. The content or position of individual DBM agents in the overall DBM composition according to the present invention can be adjusted to alter the properties of some or all of the composition according to the present invention. In this way, the formulation can be tailored to the patient, type of injury, site of injury, recovery time, underlying disease, and the like. In another aspect, the present invention provides a method for preparing an improved DBM composition according to the present invention. For example, the present invention provides a method of making an improved DBM composition for a particular site or injury.
[13] The invention also provides systems and reagents for making and using DBM grafts, and systems and reagents for treating bone defects using DBM grafts. For example, the DBM composition is provided as a paste in a delivery device such as a syringe. Suitably, the DBM composition may be packaged aseptically and used under sterile conditions (eg, in an operating room).
[14] The present invention also provides a system for characterizing DBM mixtures and a system for identifying and manufacturing DBM-containing materials with improved properties.
[15] In addition, the present invention provides bioactive agents such as growth factors (eg, bone morphogenic proteins, growth factors, hormones, angiogenic factors, cytokines, interleukins, osteopontin, osteonectin) to host animals. Present the system to pass on. The use of the DBM composition as a means of delivery of the bioactive agent provides unexpected results of an improved healing response to the implant without the need to administer the bioactive agent individually. The problem with the introduction of bioactive agents at these sites is that the bioactive agents are diluted and redistributed during the healing process by the recipient's circulatory system (eg blood, lymph) before complete healing proceeds. One solution to the redistribution problem is to attach bioactive components to bone grafts. Bioactive agents suitable for delivery to the DBM composition include agents that promote natural healing processes, ie resorption, angiogenesis, angiogenesis, new growth, and the like. A list of bioactive agents that can be delivered to the DBM compositions of the present invention is appended as Appendix A. In a suitable embodiment of the invention, there is provided a composition according to the invention, wherein the DBM together with the stabilizer is used to deliver the biologically active agent. Stabilizers are expected to protect the biologically active agent from degradation and thus extend the duration of activity after delivery to the recipient animal. In certain embodiments, the bioactive agent is an osteoinductive agent, and in certain embodiments, the DBM may be used to deliver one or more, preferably two or more, more preferably three or more bioactive agents. The bioactive agent can be linked to the DBM. For example, bioactive agents are linked to DBM by electrostatic interactions, hydrogen bonding, pi stacking, hydrophobic interactions, van der Waals interactions, and the like. In certain embodiments, the bioactive agent is attached to the DBM through specific interactions between the receptor and the ligand or between the antibody and the antigen. In other embodiments, the bioactive agent is attached to the DBM via non-specific interactions (eg, hydrophobic interactions).
[1] Related applications
[2] This application claims priority to divided applications USSN 60 / 392,462 "Improved Bone Gaft" (Knaack et al., June 27, 2002) and USSN 60 / 329,156 "Osteoinductive Composition" (Traianedes et al., October 12, 2001). do.
[16] 1 shows three weeks in vivo radiograph showing evidence of bone formation.
[17] FIG. 2 shows six weeks of x-ray or faxitron images.
[18] 3 shows a qualitative assessment of vascular distribution (A) and residual desalted bone fibers (DBF) (B).
[19] A. Vascular distribution and myeloid cell types increase in active DBF in a dose-dependent manner with increasing concentrations of hrhBMP-2x, but this is not evident in the deactivated group. Wild type rhBMP-2 at 5 μg dose is similar to hybrid BMP.
[20] B. The remaining DBFs remain a significant part of the nodule in the deactivation group. Wild-type rhBMP-2 is not as effective as hrhBMP-2x in remodeling DBF.
[21] FIG. 4 shows a comparison of unactivated or hrhBMP-2x treated deactivated DBF matrix with active DBF matrix.
[22] Deactivated : bone formation factor is not clear and only residual DBF is present
[23] Inactivated + 10 μg hrhBMP-2x : residual bone in new bone; Extensive immature bone marrow with adipocytes throughout the nodule; Extensive bone formation at the outer edge of the nodule, but no bone formation at the rim.
[24] DBF : Extensive chondrocyte, bone, and some bone marrow morphology, with borders of residual DBF.
[25] DBF + 10 μg hrhBMP-2x : slender border of new born bone with few residual DBFs in the center and extensive bone formation throughout the nodule; Well-developed hematopoietic bone marrow is present and widespread angiogenesis.
[26] Figure 5 shows a histological comparison of hrhBMP-2x DBF matrix and wild type rhBMP-2 treated DBF matrix. Significant bone formation is seen in the hrhBMP-2x treated group compared to the rhBMP-2 group, as evidenced by much fewer needles and a wide range of adipose bone marrow in the wild type group. More developed blood marrow is evident in the hybrid rhBMP-2x group.
[27] 6 shows the chemical structure of some matrix metalloproteinase inhibitors.
[28] Justice
[29] By "linked" is meant herein that the stabilizer or other chemical entity is linked with the DBM or osteogenic matrix according to the invention such that it is maintained for a long enough time to significantly improve the bone induction of the implant. Specific examples include: 1) free diffusion from DBM as measured by in vitro diffusion assay in simulated body fluids; 2) has an extended half-life in the DBM compared to the free state in solution. In some embodiments, the link is covalent; In other embodiments, they are non-covalent. Examples of non-covalent interactions include electrostatic interactions, hydrogen bonding, hydrophobic interactions, van der Waals interactions. For example, the bioactive agent is linked with DBM or another matrix of the present invention through a polymerizable stabilizer that inhibits diffusion of the bioactive agent from the matrix. Alternatively or in addition, the bioactive agent can be linked to the DBM through physical interaction with one or more entities that are linked to the DBM. For example, in Example 12, BMP-2 is considered to be connected to the DBM, and BMP-2X is considered to be connected closer to the DBM than to BMP-2.
[30] "Demineralized bone activity" as used herein means osteoinductive activity of demineralized bone.
[31] "Desalted bone matrix" herein means any material made by removing mineral material from living bone tissue. In a suitable embodiment, the DBM composition according to the invention is a formulation containing less than 5 w%, preferably less than 1 w% calcium. Partially demineralized bone (eg, formulations containing less than 100 w% calcium at least 5 w% of the starting content) is also considered to belong to the present invention.
[32] By “diffusion barrier” herein is meant any material, coating, film or material that reduces the rate of diffusion of a material from one side of the barrier to another, more specifically from outside to inside. In certain embodiments the diffusion barrier is a polymer comprising proteins, polysaccharides, celluloses, artificial polymers, PLGA, and the like, which prevent the diffusion of activating agents (water, enzymes, etc.) and / or degrading enzymes into the DBM composition. The diffusion barrier may also prevent the release of osteoinductive factors in the DBM composition. In certain embodiments, the diffusion barrier is biodegradable, inducing degradation, activation or release of osteoinduction factors for extended periods of time.
[33] By “matrix” herein is meant a natural or non-natural substantially solid delivery means that can be linked to at least one growth factor for delivery to the site of implantation. The matrix may be completely insoluble or slowly soluble after implantation. After implantation, the appropriate matrix is reabsorbed or degraded and remains substantially intact for at least 1-7 days, preferably at least 2-4 weeks, more preferably at least 60 days. Growth factors may be present internally in the matrix, as in most desalted bones, or added externally to the matrix. The matrix may be in particulate or fibrous form or may be integral. The matrix may comprise a variety of materials and forms in a mixture such as fibers and particles. In a suitable embodiment, the matrix consists of heat pressurized desalted bone fibers. In another embodiment, the matrix contains the resorbable plastic polymer described below suitable for use as a diffusion barrier. In another suitable embodiment, the granulated anhydrous calcium phosphate is used as a matrix that is linked to absorbed growth factors such as BMP, more specifically BMP-2, BMP-4 or derivatives thereof. Other matrix embodiments requiring external addition of growth factors include, but are not limited to, granulated ceramics, preferably calcium sulfate or calcium phosphate. The most preferred matrix is the calcium phosphate matrix, the preparation of which is known to those skilled in the art (Driessens et al. "Calcium phosphate bone cements" Wise, DL, Ed. Encyclopedic Handbook of Biomaterials and Bioengineering, Part B, Applications New York: Marcel Decker; Elliott Structure and Chemistry of the Apatites and Other calcium Phosphates Elsevier, Amersterdam, 1994). The calcium phosphate matrix contains dibasic calcium phosphate dihydrate, monite, tricalcium phosphate, tetracalcium phosphate, hydroxyapatite, nanocrystalline hydroxyapatite, incomplete crystalline hydroxyapatite, substituted Hydroxyapatites, calcium deficient hydroxyapatites are included, but are not limited to these.
[34] Osteoinduction herein refers to properties that can promote bone formation. Substances that can induce the formation of ectopic bones in soft tissues of animals are considered osteoinductive. For example, maximal osteoinductive agents are found in athymic rats analyzed according to the method of Edwards et al. ("Osteroinduction of Human Demineralized Bone: Characterization in a at Model" Clinical Orthopedics & Rel. Res., 35: 219-228, December 1998). Induces bone formation In some cases osteoinduction is considered to proceed through cell recruitment and induction of recruited cells into the osteogenic phenotype. Osteoinduction can also be identified by the ability to induce an osteogenic phenotype of cultured cells (primary, secondary or explants) in tissue culture. Zhang et al. "A quantitative assessment of osteoinductivity of human demineralized bone matrix" J. Periodontal. 68 (11): 1076-84, November 1997 It is desirable to modify the method of tissue culture by in vivo ectopic bone formation assay. Modifications of in vitro assays for proven in vivo ectopic bone formation models are important in that the ability of a compound to induce a clear “bone formation” phenotype in tissue culture is not always associated with the induction of new bone formation in vivo. . BMPs, IGFs, TGF-β, parathyroid hormones, and angiogenesis factors have been found to recruit cells from the bone marrow or perivascular space to the site of injury and then induce differentiation into these cell lines responsible for bone formation of these recruited cells. It is only part of the osteoinductive factor. DBMs isolated from bone or dentin have been found to be osteoinductive agents (Ray et al., "Bone implants" J. Bone Joint Surgery 39A: 1119, 1957; Urist, "Bone: formtin by autoinduction" Science 150: 893, 1965).
[35] Osteoinduction scores indicate a score in the range of 0 to 4 measured according to the method of Edwards et al. (1998) or an equivalent modified test. In the method of Edwards et al., The "0" score represents new bone formation; "1" represents 1% -25% of the implants involved in new bone formation; "2" represents 26-50% of the implant involved in new bone formation; "3" represents 51% -75% of the implant involved in new bone formation; "4" represents> 75% of the implants involved in new bone formation. In most cases, scores are assessed 28 days after transplantation. However, in the improved formulations of the invention, especially those with osteoinductive properties as compared to BMP, the osteoinduction scores can be obtained at an initial time point such as 7 days, 14 days or 21 days after transplantation. In these cases, it is important to include a normal DBM control, such as a DBM powder without carrier, possibly a positive control such as BMP. In particular cases, osteoinduction may record scores at later time points, such as 40 days, 60 days, or 100 days after transplantation. Osteoinduction ratios indicate an osteoinduction score at any point in time expressed as the activity ratio of a particular reference score.
[36] Particles or fibers refer to the formulation of a DBM, DBM composition, or bone sample that has been ground into powder or broken down into particulate form. The dimensions of the particles or fibers are typically 50 microns, preferably 75 microns, more preferably 100 microns and most preferably 150 microns. These dimensions indicate average particle diameters for more spherical particles and particles of other shapes except the smallest cross-sectional dimension of the particles. In certain embodiments, the composition may comprise particles having a larger dimension, preferably at least 1 mm, more preferably at least 1.5 mm, most preferably at least 2 mm. The particles or fibers can have any shape, including wedges, rods, spheres, cubes, disks, ellipses, irregularities, and the like. For example, in certain embodiments the particles are wedge-shaped and have a different dimension of about 2 mm maximum and less than 100 microns. The particles or fibers may be sieved or sorted to collect particles of a particular size. These particles or fibers can be mixed with solutions, slurries, deformable solids or liquids to make the pastes used to administer DBM grafts, DBM compositions of the invention or bone samples. Preferred methods for producing the particles or fibers are described in US Pat. No. 5,607,269; 5,236,456; 5,284,655; 5,314,476; 5,507,813.
[37] Polysaccharide herein refers to any polymer or oligomer of carbohydrate moieties. The polymer consists of 2 to hundreds of thousands of sugar units. Polysaccharides can be purified from natural sources such as plants or synthesized de novo in the laboratory. Polysaccharides isolated from natural sources can be chemically modified (eg, ignited, cross-linked) to change chemical or physical properties. Polysaccharides are straight or branched chains. These may include natural and / or artificial carbohydrate moieties. Residual bonds can be typical ether bonds or synthetically possible bonds found in nature. Examples of polysaccharides are cellulose, maltin, maltose, starch, modified starch, dextran, fructose and the like. Glycosaminoglycans are also considered polysaccharides.
[38] Protease inhibitors herein are chemical compounds that can inhibit the enzymatic activity of protein cleavage enzymes (ie, proteases). Proteases inhibited by these compounds include serine proteases, acid proteases, metalloproteases (some examples of matrix metalloprotease inhibitors are shown in FIG. 6), carboxypeptidase, aminopeptidase, cysteine proteases, and the like. do. Protease inhibitors can specifically inhibit only specific proteases or lineages of proteases, or more broadly inhibit most, but not all, proteases. Preferred protease inhibitors are protein or peptide based and commercially available from chemical companies such as Aldrich-Sigma. Proteins or peptide-based inhibitors that attach to DBM (or calcium phosphate or ceramic carriers) are particularly preferred because they remain bound to the matrix and provide stabilizing effects for longer periods of time than inhibitors that freely diffuse. Because. Examples of protease inhibitors include aprotinin, 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF), amastatin-HCl, alpha1-antichymotrypsin, antithrombin III, alpha1-antitrypsin, 4-amino Phenylmethane sulfonyl-fluoride (APMSF), Arfamenin A, Arfamenin B, E-64, Vestatin, CA-074, CA-074-Me, Calpine Inhibitor I, Calpine Inhibitor II, Catephsin Inhibitor, chymostatin, diisopropylfluorophosphate (DFP), dipeptidyl peptidase IV inhibitor, diprotein A, E-64c, E-64d, E-64, eblactone A, eblactone B, EGTA, Elastin, Poroxymitin, Hirudine, Leuhistin, Leupeptin, Alpha2-Macroglobulin, Phenylmethylsulfonyl Fluoride (PMSF), Peststatin A, Feverestin, 1,10-phenanthroline, Phosphora Middon, chymostatin, benzamidine HCl, antipine, f-cylon-aminocaproic acid, N-ethylmaleimide, trypsin inhibitor, 1-chloro-3-tosylamido-7-ami 2-heptanone (TLCK), it is 1-chloro-3-tosyl amido containing the 4-phenyl-2-butanone (TPCK), trypsin inhibitor, sodium EDTA.
[39] Peptides or proteins according to the invention have at least two amino acids linked to each other by peptide bonds. Although artificial amino acids (ie, compounds that do not occur naturally but can be incorporated into the polypeptide chain) and / or amino acid analogs known in the art can be used in the peptides of the invention, only natural amino acids are included wherever possible. In addition, one or more amino acids in the peptides of the present invention may be added to, for example, functionalization or other chemical components such as carbohydrate groups, phosphate groups, farnesyl groups, isophanesyl groups, fatty acid groups, linkers for conjugation. It can be changed by deformation.
[40] Stabilizers are any chemical component that, when included in a composition of the present invention containing DBM and / or growth factors, enhances the osteoinductive properties of the composition in the measurement of certain reference samples. In most cases, the reference sample is the same as the composition which contains no stabilizer but in all other respects contains the stabilizer. Stabilizers do not possess their own osteoinduction and act to increase the half-life of one or more active ingredients or to prolong or delay the release of the active factor in the compositions of the present invention as compared to the same composition lacking stabilizers. In certain embodiments, stabilizers serve to provide a barrier between proteases and sugar-degrading enzymes to protect osteoinductive factors found in the matrix from degradation and / or release. In other embodiments, the stabilizer can be a chemical compound that inhibits the activity of the protease or sugar-degrading enzyme. In suitable embodiments, stabilizers delay the access of enzymes known to release and dissolve active factors. Half-life can be determined by immunological or enzymatic analysis of specific factors attached to or extracted from the matrix. Alternatively, measuring the increase in osteoinductive half-life and measuring the enhanced appearance in the products of the osteoinductive process (e.g., bone, cartilage or osteogenic cells, products or indicators thereof) may have a stabilizing effect on the enhanced osteoinductive matrix composition. Is a useful indicator. In general, the measurement of prolonged or delayed appearance of a strong osteoinductive response indicates increased factor stability with delayed unmasking of factor activity.
[41] A targeting agent is any chemical component that, when included in a composition of the present invention, directs the composition to a specific site or causes the composition of the present invention to remain at a specific site of the recipient's body. Targeting agents can be small molecules, peptides, proteins, biological molecules, polynucleotides, and the like. Typical targeting agents are antibodies, ligands of known receptors, receptors. These targeting agents can be combined with the compositions of the invention in covalent or non-covalent interactions such that the compositions of the invention can be directed to specific tissues, organs, damaged sites or cell types.
[42] Description of Specific Appropriate Embodiments
[43] As disclosed herein, the present invention provides compositions and methods related to compositions containing enhanced DBM or synthetic growth factors. Certain aspects of suitable embodiments in accordance with the present invention are described in more detail below with reference to the drawings. As will be appreciated by those skilled in the art, various embodiments of the invention are included within the scope of the invention as defined in the appended claims, although not specified below.
[44] DBM is basically composed of protein and glycoprotein, collagen is the major protein substituent of DBM. Collagen is relatively stable because it is degraded only by relatively rare collagenase enzymes, while other proteins and active factors present in DBM are rapidly degraded by enzymes present in the host. These host-derived enzymes include proteases and sugar-degrading enzymes (eg, endo- and exo-glycosidases, glycanases, glycolases, amylases, pectinases, galactosidases, etc.). Many of the active growth factors responsible for the osteoinductive activity of DBM exist in protected form in the matrix until activated. Activation may involve alteration of the before and after function of a factor or release of function from a secondary factor or component that binds to a primary growth factor. The present invention is directed to a process which comprises: 1) slowing degradation of active factors present in DBM to prolong their retention time with active ingredients, 2) prolong the release of one or more active factors from the implant, or 3) one or more potentials. By changing the activation kinetics of the factors, it changes the time course in which the active factors present in the DBM can exert osteoinductive activity. The present invention provides for (1) changing the activation kinetics of latent factors, (2) altering the delivery and / or release of active factors from the matrix, or (3) proteolytic degradation of the active factors released from the DBM composition. Decrease to increase the effective osteoinduction of the DBM composition. Increased bone formation is thought to progress through the recruitment of more cells into the osteogenic phenotype.
[45] The present invention provides four methods for the protection of active factors from degradation by host-derived or endogenous enzymes. Factors to be protected may be factors that are endogenous to the DBM or added to the synthetic matrix composition. Protection is provided through a) diffusion barriers, b) enzyme inhibitors, c) competition substrates and / or d) masking components. These four solutions can be used to modulate the activation and / or release of osteoinductive factors in a protected form. For example, diffusion barriers or activating enzyme inhibitors prevent the activating enzyme from reaching or acting on a potential factor. Suitably, the degradation, release, and activation of the active factors in the DBM composition are balanced to yield the desired osteoinduction profile over time.
[46] Desalted Bone Matrix
[47] DBM formulations have been used for years in orthopedic drugs that promote bone formation. For example, DBM has been used to treat bone fractures caused by underlying diseases such as fracture repair, fusion of the vertebrae, joint replacement surgery, and rheumatoid arthritis. DBM is thought to promote bone formation in vivo through bone conduction and osteoinduction. Bone conduction proceeds when the implanted material serves as a backbone to support new bone growth. Bone conduction is particularly important where bone growth is required in "critical size" defects where bone treatment proceeds very slowly or not. In general, it is believed that the bone conduction properties of DBM formulations are provided by the actual shape and adhesion of the implant. DBM compositions comprising intertwined fibers retain superior bone conduction properties compared to formulations with fewer fibers and more granules. Stabilizers that preserve the shape and / or adhesion of DBM substituents can lead to better bone formation properties.
[48] The osteoinductive effect of the implanted DBM composition is believed to be due to the presence of active growth factors present in the isolated collagen-based matrix. These factors include members of the TGF-β, IGF, BMP protein lineage. Specific examples of osteoinductive factors are TGF-β, IGF-1, IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), angiogenic factors and the like. Other osteoinductive factors such as osteocalcin and osteopontin may also be present in the DBM formulation. There may be other osteoinductive factors that have not yet been identified in the DBM.
[49] Various desalted bone matrix formulations can be used in the present invention. Totally or partially desalted, including particulate or fiber-based formulations, mixtures of fiber and particulate formulations, totally or partially desalted formulations, surface desalted formulations as disclosed in Gertzman et al. (US Pat. No. 6,326,018, December 4, 2001). DBM compositions, such as mixtures of formulations, can be made by any method. Preferred DBM compositions are described in Dowd et al., US Pat. No. 5,507,813. DBM formulations also include additives or carriers such as polyhydroxyl compounds, polysaccharides, glycosaminoglycan proteins, nucleic acids, polymers, polaxomers, resins, clays, calcium, salts and / or derivatives thereof. useful.
[50] In certain embodiments, the DBM material used to make the compositions of the present invention is at least 50%, preferably at least 75%, more preferably at least 80%, 85%, 90% or 95%, most preferably 98% The above calcium phosphate is removed. The bone used to make the DBM can be obtained from any source of living or dead tissue. Suitably, the source of bone is suitable for the final recipient of the composition according to the invention. Although heterogeneous sources are possible, it is desirable that the donor and recipient be the same species.
[51] Once the bone sample is obtained, it is made into fines by milling or grinding. In suitable embodiments, the particles have a minimum dimension of at least 75 microns, preferably at least 100 microns, more preferably at least 150 microns. However, the method of the present invention stabilizes implants having particles of less than 100 microns, even less than 75 microns. Particles below 75 microns after desalting are known to have little osteoinductive properties, and the present invention can also be used to enhance the activity of these small size particles. In formulations using these small size DBMs, at least one stabilizer is used that delays the influx of host cells that can remove these small molecules for long enough that the activator in the DBM can induce an osteoinductive response. In addition or alternatively, there is a diffusion barrier that delays the outflow of the factor from the particles. In certain embodiments, the particles have a maximum dimension of at least 200 microns. The particles can be of any shape, including elliptical, circular, cubic, cone, pyramid, wedge, and the like. In certain embodiments, the particles are wedges, pyramids or cones having a maximum dimension of 200 microns. In other embodiments, the DBM composition may contain particle mixtures of various different sizes and / or shapes. After particulateation, the DBM is processed to remove minerals from bone. Hydrochloric acid is an industrially recognized desalting agent, and existing literature contains a number of reports on the preparation of DBM (eg Russel et al. Orthopaedics 22 (5): 524-531, May 1999). In the present invention, DBM is considered to be any substance that provides a backbone with an active osteoinductive factor. DBMs can be made by methods known in the art or by other methods that can be designed by one skilled in the art without undue experimentation. In some cases, large fragments or whole bone may be desalted and become particulate after desalination. DBMs created in this manner are within the scope of the present invention.
[52] In the preparation of the improved DBM composition, the DBM components can be milled before or after desalting or processed into particles of suitable size. In certain embodiments, the particle size is about 75 microns, preferably about 100 to 3000 microns, most preferably about 200 to 2000 microns. After crushing the DBM components to the desired size, the mixture is sieved to screen particles of the desired size. In certain embodiments, the DBM particles are sieved through 50 microns, preferably 75 microns, most preferably 100 microns.
[53] Particularly effective methods for protecting small size particles from cell degradation and / or providing diffusion barriers are embedding them in monolithic bioabsorbable matrices, and then the particle-containing monolithic matrix is at least 70 microns, preferably Is decomposed into particles having a minimum size of at least 100 microns, most preferably at least 150 microns. Suitable matrices for sealing small DBM particles include biocompatible polymers and agglomerated calcium phosphate cements.
[54] In general, the particulate DBM / polymer weight ratio is approximately 1: 5 to 1: 3. In the case of calcium phosphate, the DBM is present up to 75 wt%. Particulation of the monolith can be achieved by conventional milling or grinding, cryomill, or freezing and subsequent grinding. In a suitable embodiment, the frozen DBM is embedded in a resorbable polymer. In another suitable embodiment, the frozen DBM is embedded in one of the condensed calcium phosphates known in the art.
[55] Stabilizer
[56] Diffusion barrier . The diffusion barrier retards the diffusion of degrading enzymes and / or water into the active ingredient in the formulations according to the invention. Enzymes with delayed diffusion into DBM can release the active factor from the matrix or degrade or inactivate the active factor. These barriers may also act to delay the spread of active factors from the site of implantation. In these methods, the barrier provides a longer residence time of the active factor at the site of implantation. This is particularly useful for bone formation in higher animals, such as humans, who require the presence of active factors for longer periods of bone formation.
[57] In general, most materials suitable as diffusion barriers can easily mix with DBM or selective synthetic matrices to form gels, pastes or putty-like consistency, but in some cases the barrier / matrix formulations are relatively unmodified. Made of solid (eg for matrix formulations used for post-external spinal fusion). In a suitable embodiment, the diffusion barrier self-decomposes in a predictable manner, exposing the active factor at a later point in time than normally progresses in the absence of the diffusion barrier. Enzymatically degraded polymers as well as resorbable polymers with known hydrolysis rates are useful as diffusion barriers. Particularly useful are lipase sensitive lipid based carriers such as fatty acids and phospholipids, which are mixed with DBM. In certain DBM embodiments, the composition does not contain phosphatidylcholine. Some formulations that are particularly effective provide extended stability with controlled unmasking of osteoinductive factors. In general, these formulations have two or more diffusion barriers with different degradation times that can expose the same active factor at least two different rates.
[58] Biodegradable polymers used in the stabilizing matrix / growth factor compositions according to the invention include natural polymers such as proteins (eg collagen) and polysaccharides (eg starches, modified starches, maltrins) and poly-or Artificial resorbable polymers, such as sawesters, are included. These polymers, when mixed with the growth factor-containing compositions of the present invention, retard the diffusion of host degrading enzymes and / or water into the active factors contained in the composition, thus delaying the release and / or degradation of the active factors contained therein.
[59] Polymers that may be included in the compositions of the present invention include, for example, natural polymers such as lipids, polysaccharides, proteoglycans, proteins. Preferred polysaccharides include starch, dextran, cellulose, and preferred proteins include collagen. Polysaccharides such as starch, dextran, and cellulose may not be modified or may be physically or chemically modified to affect properties such as properties, solubility, susceptibility to degradation, or in vivo half-life in the hydrated state. Polysaccharides such as starch and cellulose are attractive in that their degradation rates are known. In general, cellulose degrades more slowly in the body and breaks down after weeks or months, while many starch and lipid formulations degrade rapidly within hours or days. In nature, starch is a mixture of two polysaccharides, amylose and amylopectin. The susceptibility of certain starches to starch-degrading enzymes such as amylases, pectinase, β-glucosidase is an important consideration in designing the formulations of the present invention. Those skilled in the art are aware of the various amylase susceptibility of starches derived from various plant sources and can use this knowledge to make formulations with the desired stability time. Preferred starches degrade 10%, preferably 50%, most preferably at least 90% per day. Starches that are less sensitive to degradation by pectinase and / or amylase (amylase-resistant starch; Strach Australasia, Sydney, Australia) are higher levels than enhanced DBM or synthetic growth factor / matrix formulations made from starches that are more sensitive to enzymes. In vivo bone oil can also be used to maximize the half-life. Some modified starches are slightly less sensitive to degradation against amylases; Thus, DBM enhanced with modified starch is expected to have a slightly longer half-life compared to DBM enhanced with unmodified starch. One preferred method of influencing starch amylase sensitivity is the use of starch lipid combination. For a combination of lipids and starch that affect amylase sensitivity, see Crowe et al. "Inhibition of Enzymic Digestion of Amylose by Free Fatty Acids In Vitro Contributes to Resistant Starch Formation" J. Nutr. 130 (8) 2006-2008, August 2000). Lipids and their degrading enzymes, lipases, are similarly considered. Various mono, di-, triglycerides with different grades of sensitivity to lipase degradation are commercially available from commercial sources. Some embodiments include one or more polymerizable materials, preferably tyrosine polycarbonate, polyfumarate, tyrosine polyarylate, poly-orthoesters (e.g. polylactide, polygalactide, co-polymers thereof) It is a biodegradable substance such as. These polymers are biodegradable and their properties can be changed by modifying the chain length or degree of cross-linking of the polymer and / or by modifying the chemical structure of the monomers. In addition, the co-polymer can be made from a combination of resorbable polymers.
[60] Enzyme inhibitor . Alternatively or additionally, the compositions of the present invention may be stabilized by the addition of one or more degradation inhibitors that antagonize growth factor active degradation agents found in host organisms and / or transplant compositions. These inhibitors may inhibit the activity of enzymes that activate osteoinductive factors of the DBM composition. Degradation or activation inhibitors useful in the practice of the present invention include, for example, acid protease inhibitors, serine protease inhibitors, metalloprotease inhibitors (FIG. 6; Whittaker et al. "Matrix Metalloproteinase and their Inhibitors-Current Status and Future Challenges"). Celltransmission 17 (1): 3-14), cysteine protease inhibitors, glyconase inhibitors, glycosidase inhibitors. Certain protease inhibitors useful in the practice of the present invention include, for example, aprotinin, 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF), amastatin-HCl, alpha1-antichymotrypsin, antithrombin III, alpha1 Antitrypsin, 4-aminophenylmethane sulfonyl-fluoride (APMSF), arfamenin A, arfamenin B, E-64, bestatin, CA-074, CA-074-Me, calpine inhibitor I, calpine Inhibitor II, catephin inhibitor, chymostatin, diisopropylfluorophosphate (DFP), dipeptidyl peptidase IV inhibitor, diprotein A, E-64c, E-64d, E-64, eblactone A , Eblactone B, EGTA, elastatin, poroxymitin, hirudin, reuhistin, leupeptin, alpha2-macroglobulin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A, fevestin, 1,10 -Phenanthroline, phosphoramidone, chymostatin, benzamidine, HCl, antipine, epsylone-aminocaproic acid, N-ethylmaleimide, trypsin Hazardous Substances, 1-Chloro-3-tosylamido-7-amino-2-heptanone (TLCK), 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin inhibitor , Sodium EDTA, TIMP family metalloproteinase inhibitors. Particularly useful protease inhibitors are those that are stable and effective under acidic conditions. As one of ordinary skill in the art will recognize, the fewer osteoinductive factors that are lost or degraded during the process of bone formation of DBM, the more osteoinductive factors are available for recruitment after implantation of the DMB composition.
[61] Competitive temperament . The use of competing substrates for degradation or activating enzymes in the host can also stabilize the osteoinductive factors or exogenously added growth factors of DBM. Examples of competitive substrates are di- and poly-lysine and the like. Di- and poly-saccharides can be used as competition substrates for glycosidase, amylase and / or pectinase. Stereoisomers of competing substrates are particularly useful.
[62] Shielding component . Certain masking components are commonly used to specifically block enzymatic destruction in a single entity or group of entities. The cleavage or activating enzyme that is blocked is endogenous or exogenous to the matrix. In general, the masking component itself binds to a ligand present in the matrix that is or is not an active factor. Once bound, the masking component spatially interferes with the destruction and / or release of one or more active factors. Over time, the masking component is released from the bond or degrades itself, exposing ligands and / or growth factors that are sensitive to degradation. Diffusion barriers are typical of masking components that prevent access of degradation or activating enzymes to almost all growth factors associated with the matrix.
[63] Growth Factor Binding Proteins : Almost all extracellular matrix growth factors are known to bind with binding proteins that regulate growth factor activity. Purified formulations of these binding proteins can be added to the DBM formulation as a masking component. Typical growth factor binding proteins include, but are not limited to, noggin, chordin, follistatin, TGF-β binding protein, insulin-like growth factor binding protein. Agents that induce release of growth factors from the binding protein may also be added to the DBM composition. In certain embodiments, agents known to induce release of growth factors may be encapsulated in biodegradable polymers such that the agents are released for extended periods of time to induce release of growth factors for extended periods of time.
[64] Lectin . Lectins are proteins that can bind to the sugar component of glycoproteins. Because growth factors are primarily glycoproteins, lectins can be used to bind to growth factors and potentially delay or inhibit the access of proteases or growth factor releasing hormones to active growth factors. Ideally, the lectins are chosen according to the identity of the terminal sugar in the active glycoprotein of interest. Lectins include, but are not limited to, membrane-bound lectins, I-type lectins, and P-type lectins. Specific lectins include galectins, calcium-dependent lectins, selectins, carlectins, and annexin.
[65] Antibody . Monoclonal or polyclonal antibodies specific for the active factor or proteins known to bind to such active factor may be added to the compositions according to the invention to protect certain growth factors from degradation or release enzymes.
[66] The DBM compositions of the present invention may alternatively or additionally be stabilized through exposure to conditions (eg, pH, temperature, etc.) in which the enzyme does not function optimally or the enzyme does not function effectively.
[67] Addition of enzyme inhibitors, competition substrates, masking agents . Incorporation of these in the formulations according to the invention is accomplished by suspending the molecule of interest in a suitable buffer, as is known to those skilled in the art. The buffer is mixed with the frozen matrix at a relatively low liquid-solid volume ratio to form a slurry. The slurry is then frozen and used to make the desired DBM formulation.
[68] One of the unexpected features of the invention is that the integration of one of the enzyme inhibitors, competition substrates or masking agents according to the invention prevents the access of endogenously present degrading enzymes to the active factors present in the matrix, thereby half-life of DBM formulations. Is to further improve. This is particularly true of DBM formulations containing water (eg, hydrogel carriers such as hyaluronic acid or collagen, or DBM formulations containing hydrated starch carriers).
[69] Many osteoinductive factors found in DBM are protected forms and must be "activated" or "released" for osteoinduction. Activation of osteoinductive factors may involve conformational changes, post-translational formulas, peptide cleavage, tertiary or quaternary structural changes, release from DBM, release from binding proteins, and the like. For example, the factor is present in pre- or pro-form, which requires proteolytic cleavage for activity. In addition, osteoinduction factors may bind to binding proteins or matrix proteins of DBM. The same process as proteolysis involved in the degradation of active factors may also be involved in the activation of these factors. Thus, the method that can be used to delay degradation can also affect the rate of activation. Those skilled in the art of preparing DBM compositions can balance the rate of degradation and activation to achieve the desired level of osteoinduction from the implant over time. In addition, pH, ion concentration, or other factors that affect protein function and / or folding may affect the activation of osteoinductive factors found in DBM. These factors may also affect the release of the factor from the binding protein. In certain embodiments, where, for example, the pH plays a role in the activation of the factor, the DBM composition contains chemical compounds such as polymers that break down over time and release acid byproducts to activate the factor in the DBM composition. . In other embodiments, the biodegradable polymer may be a release ion or protease capable of activating osteoinductive factors of the DBM composition.
[70] Release of osteoinduction factors from the delivery matrix is also important for osteoinduction. Many factors have been found to bind DBM through specific binding proteins as described above or through non-specific interactions. Some of these factors must be released from the matrix to be activated, while others are only activated in the bound state. For example, cells are recruited to a matrix by certain factors, and then interact with other factors bound to the matrix. Cells may require interaction with both matrix and factors to induce bone production. The rate of release of osteoinductive factors can be controlled by diffusion barriers or agents that affect the binding of these factors to the matrix or their binding proteins. As noted above, in certain embodiments it is desirable for the diffusion barrier to degrade over time so that cells with released or recruited factors can interact with the matrix. In addition, degradation of the diffusion barrier may allow proteases to enter the DBM implant, activating and / or releasing osteoinductive factors.
[71] As will be appreciated by those skilled in the art, the DBM composition can be balanced with the degradation, activation, and release of osteoinductive factors into a composition with the desired osteoinductive activity. Osteoinduction of a DBM composition may be suitable for a particular use, site of implantation or patient. For example, certain applications require extended bone induction for weeks to months, while other applications require osteoinduction for days to weeks. One skilled in the art can make a DBM composition having a desired osteoinductive time profile.
[72] Reinforcement inspection
[73] The present invention provides a simple in vitro test for the screening of suitable stabilizers. DBMs made with or without biodegradable stabilizers are exposed to enzymes or mixtures of enzymes known to degrade some or all protein components of the DBM under simulated physiological conditions (eg pH 7.4, physiological saline). . In most cases, enzymes are proteases such as trypsin, papain, peptidase and the like. Evidence of matrix or matrix component breakdown is compared between the two formulations. Substances that delay the destruction process are considered promising candidates for further inspection. Preferred indicators of disruption include immunological detection of TGF-β and / or IGF destruction. In addition to the enzymes described above, other enzymes such as collagenase or enzyme mixtures such as glycosidase may be used. In this regard, the natural degrading activity of serum or tissue extracts is particularly useful. Under these conditions, certain marker proteins present in the DBM can be detected by immunological methods such as radioimmunoassay, gel electrophoresis using Western blot, or other methods known in the art.
[74] After identification of the candidate stabilizer in the assay, DBM formulations containing the candidate stabilizer are examined by the osteoinduction assays disclosed herein.
[75] Osteoinductive substances
[76] Other osteoinducers can be added to the enhanced DBM. These agents can be added in activated or non-activated form. These agents can be added at any time during the preparation of the material according to the invention. For example, osteodermal agents are added after the desalting step and prior to the addition of stabilizers so that the added osteoderive agents are protected from exogenous degrading enzymes after transplantation. In some embodiments, the DBM is frozen in a solution containing an osteoinducer. In another suitable embodiment, the osteoinduct is attached to the hydrated demineralized bone matrix and does not freely dissolve. In other cases, the osteoinduct is added to the enhanced DBM after the addition of the stabilizer to make the osteoinducer available immediately after implantation of the DBM.
[77] Osteoinducing agents include any agent that induces or enhances bone formation. Osteoinducing agents can achieve this in any way, for example the agent induces the recruitment of cells responsible for bone formation, induces secretion of the matrix where mineralization proceeds subsequently, and reduction of bone Induced resorption can be induced. Particularly preferred osteoinducing agents include angiogenic factors such as bone forming protein (BMP), converting growth factor (TGF-β), insulin-like growth factor (IGF-1), parathyroid hormone (PTH) and VEGF. In suitable embodiments (Example 12), the inducer is genetically engineered to include an amino acid sequence that facilitates binding of the inducer to the DBM or carrier. Sebald et al. (PCT / EP00 / 00637) describe the production of typical engineered growth factors suitable for use in DBM.
[78] Formulation
[79] The improved osteogenic composition of the present invention can be made to suit a particular application. Such agents can be used to alter the physical, biochemical or chemical properties of DBM formulations. The physician can determine the formulation required for a particular application by considering factors such as type of injury, site of injury, patient's health, risk of infection and the like.
[80] Thus, the compositions of the present invention can be made to have selected resorption / loss ratios of bone induction, or different ratios of one another in various parts of the implant. For example, the formulation process involves the selection of specific stabilizers, the selection of DBM particles of a particular size or composition, and the selection of such agent content. As one example, it is desirable to provide a composition in which the osteoinductive factor is active at a relatively constant content for a defined period of time. DBM compositions containing factors with longer half-lives can be made of less biodegradable polymers or polymers of higher content (eg, thicker coatings). Alternatively or additionally, particle size may be important for determining the half life of a DBM composition according to the present invention. In suitable embodiments, the formulations of the present invention may comprise a mixture of particles, each having a different half-life. Such mixtures can provide stable or possible unmasking of osteoinductive factors for extended periods of days to weeks or months, depending on the extent of the injury. Such compositions can be made to promote bone growth in human patients comparable to bone growth induced by 10 μg, preferably 100 μg, most preferably 1-10 mg rhBMP treatment in collagen sponges.
[81] Physical properties such as the deformability and viscosity of the DBM also depend on the particular clinical application. Improved DBM particles can be mixed with other materials and factors to improve other features of the implant. For example, improved DBM materials can be mixed with other agents to improve wound healing. These agents include drugs, proteins, peptides, polynucleotides, solvents, chemical compounds, biomolecules.
[82] In addition, the particles of DBM (or DBM materials of the present invention) can be made in a variety of shapes and forms. The particles can be made of rods, threads, sheets, fabrics, solids, cones, disks, fibers, wedges, and the like. In certain embodiments, the shape and size of the DBM composition particles affect the time course of osteoinduction. For example, in the cone or wedge shape, the pointed end results in osteoinduction immediately after implantation of the DBM composition, while the thicker end results in osteoinduction later in the treatment process (eg, hours to days or weeks later). In certain embodiments, the particles have a length of at least 2 mm, preferably at least 1.5 mm, more preferably at least 500 microns and most preferably at least 200 microns in the largest dimension. In addition, larger particle sizes lead to bone formation over a longer period of time than small particles. Particles of different properties (eg, composition, size, shape) can be used to form these different shapes and conformations. For example, layers of long half-life particles in a DBM sheet can be placed alternately between layers of shorter half-life particles. In fabrics, strands consisting of short half-life particles can interweave with longer half-life strands.
[83] In a suitable embodiment of the present invention, the fibrous DBM is made in matrix form as disclosed in US Pat. No. 5,507,813 and Examples 13 & 14 (embedded matrix assembly) below. The implemented DBM is then enclosed in a diffusion barrier matrix so that a portion of the matrix is exposed from the matrix material. Particularly preferred blocking matrices are starch, phosphatidyl choline, tyrosine polycarbonate, tyrosine polyarylate, polylactide, polygalactide, or other resorbable polymers or copolymers. Devices made in this way from these matrices have both immediate and long lasting osteoinduction and are particularly useful for promoting bone mass formation in human posterior pole union signs.
[84] In another embodiment of the present invention, a DBM composition according to the present invention having a pre-selected three-dimensional shape can be used for example by Cima et al. U.S. Patents 5,490,962 and 5,518,680; Sachs et al. U.S. It is made with 3-D printing disclosed in patent 5,807,437. The individual layers may consist of a stabilized DBM formulation or may consist of a DBM layer treated with a stabilizer after deposition of multiple layers.
[85] In the course of preparing the improved DBM material of the present invention, the material may be completely aseptically prepared or sterilized to remove infectious pathogens such as HIV, hepatitis B, or hepatitis C. Sterilization can be accomplished by antibiotics, irradiation, chemical sterilization (eg ethylene oxide) or heat sterilization. Other methods known in the art of making DBMs, such as defatting, sonication, lyophilization, can also be used to make improved DBMs. Care should be taken when sterilizing the compositions of the present invention, since the biological activity of demineralized bone is known to be adversely affected by most final sterilization processes. In a suitable embodiment, the DBM composition disclosed herein is aseptically prepared or sterilized as disclosed in Example 11.
[86] Usage
[87] The improved osteogenic composition of the present invention can be used to promote the treatment of bone damage. The composition can be used for bone in the body damaged in any type. The improved DBM composition is designed to produce bone in human patients at similar levels and at levels similar to rhBMPs of 10 μg to 100 μg, preferably 200 μg to 1 mg at collagen sponges. For example, specific bones that can be recovered using the materials of the present invention include bones, foreheads, nose bones, laryngeal bones, parietal bones, temple bones, lower jaw, jawbone, cheekbones, bones, spines, vertebrae, cervical vertebrae, thoracic vertebrae, lumbar spines, Sacrum, sternum, ribs, clavicle, scapula, humerus, ulna, radial, sternum, palmar bone, phalanx, ileum, sciatic, pubis, pelvis, femur, patella, tibia, calf bone, calcaneus, talus, ulna (metatarsal) bone). Types of injury that can be treated with improved DBM include bone defects due to surgical procedures, infections, malignancies, or damage caused by developmental malformations. Substances according to the present invention may include orthopedic, neurosurgery, cosmetic oral and maxillofacial surgical procedures, such as repair of single / compound fractures and non-union; External and internal fixation; Joint reconstruction, such as joint fixation, total arthroplasty, cup arthroplasty of the hip joint, femoral head and humeral head replacement, femoral head surface replacement, total arthroplasty; Recovery of the spinal column, including spinal fusion and internal fixation; Tumor surgery (eg, defect filling); Nucleotomy; Spinal hysterectomy; Extraction of spinal cord tumors; Anterior neck and rib cage surgery; Repair of spinal cord injury, scoliosis, scoliosis, scoliosis; Maxillary fixation of fractures; Jaw surgery; Temporomandibular joint replacement; Alveolar enlargement and reconstruction; Inlay bone grafts; Implant placement and correction; This is useful for maxillary sinus elevation.
[88] The DBM composition of the present invention can also be used as a drug delivery device. In suitable embodiments, the binding with the DBM composition according to the present invention increases the half-life of the associated bioactive agent. Particularly preferred drug delivery devices according to the invention are used to deliver osteoinductive growth factors. Other preferred agents delivered include agents or agents that promote wound healing. However, the compositions of the present invention may alternatively or additionally be used to deliver other pharmaceutical agents, including antibiotics, anti-neoplastic agents, growth factors, hematopoietic factors, nutrients and the like. Bioactive agents that can be delivered to the DBM compositions of the invention include non-collagen proteins such as osteopontin, osteonectin, bone sialo protein, fibronectin, laminin, fibrinogen, vitronectin, thrombospondin, proteoglycans, deco Lean, proteoglycan, beta-glycine, aglycan, aggrecan, veriscan, tenascin, matrix gla protein hyaluronan; cell; amino acid; Peptides; Inorganic elements; Inorganic compounds; Organometallic compounds; Cofactors of protein synthesis; Cofactors of enzymes; Vitamins, hormones; Soluble and insoluble components of the immune system; Soluble and insoluble receptors, including truncated forms; Soluble and insoluble cell surface bound ligands, including truncated forms; Chemokines, interleukins; antigen; Endocytotic bioactive compounds; Tissue or tissue fragments; Endocrine tissues; Enzymes such as collagen, peptidase, oxidase and the like; Multifactor cytoskeleton bearing parenchymal cells; Multifactor carriers containing angiogenic drugs, bioactive agents; Encapsulated bioactive agents; Bioactive agent in the form of a specification; Collagen lattice; Antigenic agents; Cytoskeletal agents; Cartilage fragments; Living cells such as chondrocytes, osteoblasts, osteoblasts, fibroblasts, bone marrow cells, mesenchymal stem cells, and the like; Tissue transplants; Bioadhesives; Bone forming protein (BMP); Converting growth factor (TGF-beta), insulin-like growth factor (IGF-1, IGF-2), platelet derived growth factor (PDGF); Fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), growth factor binding proteins such as insulin-like growth factor binding proteins (IGFBP-2, IGFBP-3, IGFBP-4 , IGFBP-5, IGFBP-6); Angiogenesis agents; Bone promoters; Cytokines; Interleukin; Genetic material; Genes encoding bone promoting action; A cell comprising a gene encoding a bone promoting action; Artificially engineered cells; Exogenously expanded allograft or xenograft cells; Growth hormones such as somatotropin; Bone lysates; Antitumor agents; Fibronectin; Cell attractants and adhesives; Immunosuppressants; Bone resorption inhibitors and promoters; Mitotic factor; Bioactive factors that inhibit and promote secondary messenger molecules; Cell adhesion molecules such as cell-matrix and cell-cell adhesion molecules; Secondary messenger; Monoclonal antibodies specific for cell surface determinants of mesenchymal stem cells; A portion of a monoclonal antibody specific for the cell surface determinant of mesenchymal stem cells; Coagulation factor; Polynucleotides; These compounds are included. The amount of bioactive agent contained in the DBM composition may vary and depends on factors such as the delivered agent, the site of administration, the physical condition of the patient, and the like. In certain cases the optimal level depends on the use of the implant.
[89] For example, the DBM compositions of the present invention are prepared to contain one or more compounds selected from: drugs that act on synaptic and neuroeffector junctions (e.g., acetylcholine, methacholine, pilocaprine, atropine, scopolamine, Physostigmine, succinylcholine, epinephrine, norepinephrine, dopamine, dobutamine, isoproterenol, albuterol, propranolol, serotonin); Drugs acting on the central nervous system (e.g., clonazepam, diazepam, lorazepam, benzocaine, bupivacaine, lidocaine, tetracaine, lopivacaine, amitrifthilin, fluoxetine, paroxetine, valproic acid, carbamase Pin, bromocriptine, morphine, fentanyl, naltrexone, naloxone); Drugs that modulate the inflammatory response (eg, aspirin, indomethacin, ibuprofen, naproxen, steroids, chromoline sodium, theophylline); Drugs affecting renal and / or cardiovascular function (e.g. furosemide, thiazide, amylolide, spironolactone, captopril, enalapril, ricinopril, diltiazem, nifedipine, verapamil, digoxin Isordil, dobutamine, lidocaine, quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin, mevalolate); Drugs that affect gastrointestinal function (eg omeprazole, sucralate); Antibiotics (e.g. tetracycline, clindamycin, amphotericin B, quinine, methicillin, vancomycin, penicillin G, amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline, acylclovir, zidobudine (AZT), ddC, ddI , Ribavirin, cefachlor, cephalexin, streptomycin, gentamycin, tobramycin, chloramphenicol, isoniazid, fluconazole, amantadine, interferon); Anticancer agents (e.g., cyclosamide, methotrexate, fluorouracil, cytarabine, mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin, mitomycin C, hydroxyurea, predynisone, tamoxifen, cisplatin, dekar Vagin); Immunomodulators (eg interleukin, interferon, GM-CSF, TNFα, TNFβ, cyclosporin, FK506, azathioprine, steroids); Drugs that act on the blood and / or blood-forming organs (eg interleukin, G-CSF, GM-CSF, erythropoietin, vitamins, iron, copper, vitamin B ₁₂ , folic acid, heparin, warfarin, comarin) ; Hormones (e.g., growth hormone (GH), prolactin, progesterone, TSH, ACTH, insulin, FSH, CG, somatostatin, estrogen, androgens, progesterone, gonadotropin-releasing hormone (GnRH), thyroxine, triiodity Ronin); Hormonal antagonists; Agents that affect calcification and bone replacement (e.g. calcium, phosphate, parathyroid hormone (PTH), vitamin D, bisphosphonates, calcitonin, fluoride), vitamins (e.g. riboflavin, nicotinic acid, pyridoxine, pantothenic acid, biotin, choline, inositol, Carnitine, vitamin C, vitamin A, vitamin E, vitamin K), gene therapeutics (eg, viral vectors, nucleic acid-containing liposomes, DNA-protein conjugates, antisense agents); Or other agents such as targeting agents.
[90] In certain embodiments, the delivered agent binds to the matrix to be absorbed or implanted. Such agents include specific or non-specific interactions; Or binds to the matrix of the DBM composition via covalent or non-covalent interactions. Examples of specific interactions include interactions between ligands and receptors, epitopes and antibodies. Examples of non-specific interactions are hydrophobic interactions, electrostatic interactions, magnetic interactions, dipole interactions, van der Waals interactions, hydrogen bonding, and the like. In certain embodiments, the delivered agent is attached to the matrix using a linker to allow the agent to freely bind to a receptor or site of action in vivo. In a preferred embodiment, the delivered agent is attached to a chemical compound, such as a peptide, recognized by the matrix of the DBM composition. In other embodiments, the delivered agent is attached to an antibody or fragment thereof that recognizes an epitope found in the matrix of the DBM composition. In a particularly preferred embodiment, the agent is BMP, TGF-β, IGF, parathyroid hormone (PTH), growth factor or angiogenesis factor. In certain embodiments, at least two bioactive agents are attached to the DBM composition. In other embodiments, at least three bioactive agents are attached to the DBM composition.
[91] The growth factor stabilization strategy disclosed herein can be applied directly to growth factors associated with synthetic matrices such as ceramic, bone cement or polymers. In these embodiments, one, two or more growth factors bind the synthetic matrix. Growth factors bind to a fixation matrix (e.g., amorphous or crystalline calcium phosphate bound to growth factors such as BMP), wherein the composition is in the presence of a diffusion barrier such as amylose, fatty acids or resorbable polymers, or Made with a mixture of two or more stabilizers. In a suitable embodiment, the weak crystalline calcium phosphate binds to growth factors mixed in the starch / lecithin diffusion barrier.
[92] These aspects of the invention are described in more detail in the following examples, which illustrate certain embodiments of the invention but do not limit the scope of the invention as defined by the claims.
[93] Example 1 Preparation of Desalted Bone Matrix (DBM)
[94] DBMs can be made by any method or technique known in the art (Russel et al. Orthopedics 22 (5): 524-531, May 1999). Next, Glowacki et al. "Demineralized Bone Implants" is a typical manufacturing process for demineralized bone derived from Clinics in Plastic Surgery 12 (2): 233-241, April 1985. Bone or bone fragments obtained from the donor are well washed to remove adherent periosteum, muscle, connective tissue, tendons, ligaments and cartilage. The spongy bone is separated from the dense cortical bone and processed into large pieces. Cortical bone may be cut into small pieces to improve the efficiency of subsequent cleaning and extraction. More dense bones from large animals must be frozen and hammered to make chips less than 1 cm. The resulting bone fragments are thoroughly washed with cold deionized water to remove bone marrow and soft tissue.
[95] The well washed bone is then extracted by frequent exchange of absolute ethanol for at least 1 hour. Typically, a total of 4 L ethanol is used per 100 g of bone. The bone is then extracted by frequent exchange of anhydrous diethyl ether in a steam hood for 1 hour. Typically, 2 L ether is used per 100 g of bone. Bone is dehydrated by extraction of this ethanol and ether and stored at room temperature.
[96] The dehydrated bone is then frozen and ground in a liquid nitrogen impact mill. The crushed bone is then classified into fragments of 75 to 250, 250 to 450, and 450 microns or more. The bone particle fragments are then subjected to 3 hours at room temperature or 4 ° C. in an insulated magnetic stirrer to prevent overheating. Desalting with 0.5 M hydrochloric acid (50 mL / g). Large pieces of bone and blocks are extracted completely at 4 ° C. with frequent exchange of 0.5 M hydrochloric acid. The desalting process can be monitored by radiographic methods by ashing or nondecalcified histology techniques (von Kossa staining). Acids and free minerals are washed with cold deionized water until the pH of the wash solution is equivalent to the pH of the water. The water wash is discarded from the bone fragments; However, at finer particles the wash liquor must be removed by centrifugation. The washing step requires approximately 500 ml water per gram of starting bone particles.
[97] Desalted bone powder is extracted by exchange of absolute ethanol for 1 hour using 200 ml ethanol per gram of starting bone particles. The material is extracted in the steam hood with an exchange of anhydrous ethyl for 1 hour using 100 ml ether per gram of starting bone particles. After removing the last exchanged ether, the desalted bone powder is left overnight in the hood until the remaining ether evaporates. The particles should be odorless, white and separated. Demineralized bone material may be treated with cold ethylene oxide or irradiated for sterilization.
[98] To investigate the bioactivity of the resulting DBM, 25 mg of the material was implanted into each of two thoracic subcutaneous pockets in a haired and anesthetized 28 day old male Charles River CD rat. The transplanted sample is then collected and examined a few days after transplantation. The composition of the induced tissue can be quantified by histomorphologic analysis and biochemical techniques.
[99] Example 2 Another Method of Making a DBM
[100] DBMs can be made by any method or technique known in the art (Russel et al. Orthopedics 22 (5): 524-531, May 1999).
[101] Desalted bone matrix is prepared from the long bone. The interosseous area removes the attached soft tissue and grinds in the mill. The ground material is sieved to yield a powder having particles of approximately 100 μm to 500 μm diameter. The particulate bone is desalted with residual calcium at approximately room temperature with a solution of Triton X-100 (Sigma Chemical Company, St Louis, Mo.) and 0.6 N HCl followed by a fresh 0.6 N HCl solution. The powdered material is washed with deionized water until the pH is above 4.0. It is then immersed in 70% ethanol and freeze-dried to less than 5% residual humidity.
[102] Example 3 Preparation of Preferred DBM Compositions According to the Invention
[103] The carrier is prepared by mixing approximately 6.5% (w / w) modified starch B980, approximately 30% (w / w) maltodextrin (M180), approximately 63.5% (w / w) sterile deionized water. The mixture is pre-gelled by heating to 70 ° C. The pre-gelled mixture is then transferred to a steam autoclave and sterilized / gelled at 124 ° C. for 2 hours. The resulting mixture continuously maintains pudding hardness. The cooled carrier mixture is then mixed with DBM (Example 2) and water in a ratio of approximately 27: 14: 9, respectively. The stabilized DBM is then transplanted into athymic rats to assess bone induction.
[104] Alternative embodiments: Other ingredients, such as glycerol, may be added as a solution dissolved in water (approximately 20% w / v) at the time of pre-gelling or during the final composition mixing, in place of water, which is acceptable treatment properties. It was found to have.
[105] Example 4 Stabilized DBM
[106] The table below describes the formulation of various compositions according to the invention containing separate stabilizers. All formulations are prepared aseptically and can be used as DBM particles, fibers or solid form matrices.
[107]
[108] Example 5 In Vitro Evaluation of Protective Agents
[109] DBM samples with carriers in the presence of stabilizers (or stabilizers of various concentrations and / or formulations) are prepared and incubated with serum or individual enzymes (e.g. papain) in pH 7.4 PBS buffer and 0.5, at 37 ° C. Incubate for 1, 2, 4, 8, 24 hours. The sample is then extracted and Ueland et al. Measure the concentration of growth factors and other matrix proteins as described in "Increased cortical bone content of insulin-like growth factors in acromegalic patients" J Clin Endocrinol Metab 1990 Jan; 84 (1): 123-7.
[110] Samples are prepared for intrinsic and denatured SDS gel electrophoresis and subsequent Western blot analysis or Western ligand blotting ("Increased cortical bone content of insulin-like growth factors in acromegalic patients" J Clin Endocrinol Metab 1990 Jan; 84 (1). 123-7; Walker, JM (Ed) The Protein Protocols Handbook, Second Edition 2002, Humana Press Totowa, New Jersey.
[111] Subsequently, stabilizer-containing samples showing less degradation of growth factors or other proteins than samples without stabilizers were examined for osteoinduction at days 7, 14, 21, and 28 in asymptomatic rat assays. Extracted samples can also be rapidly examined for bioactivity by tissue culture assays as described by Zhang et al. (19997).
[112] Example 6 : Time course confirmation for induction of bone growth by intramuscular implant
[113] This example characterizes the time course of bone growth induction at the intramuscular site using the materials of the present invention as compared to the DBM base powder (Example 1) at 7, 14, 28 and 35 days. This example is similar to the rat model assessing the osteoinduction of DBM identified in Edwards et al. "Osteoinduction of Human Demineralized Bone: Characterization in a Rat Model" Clinical Orthopaedics 357: 219-228, December1998.
[114] This study was conducted in athymic (nude) mice to minimize the potential for heterogeneous incompatible reactions on human tissue implants. An intramuscular region of the hind limb was used for the initial measurement of ectopic osteoinductive properties, because it is naturally free of bone.
[115] 50-75 g of female homologous mu / mu mice were obtained from Harlan (Indianapolis, IN). These mice are kept for one week prior to surgery for adaptation purposes. Aseptic microseparation cages are used for the entire survey period, and sterile water and rodent feeds are provided indefinitely.
[116] Implant Placement : A single intramuscular (IM) site was used on each hind limb of 30 rats. To provide a positive control common to all animals, a single 40 mg sample of rat DBM powder is placed in the left sternal (LP) muscle of each rat. Animals are allowed to function normally after surgery.
[117] Implants : DBM and test material are kept at room temperature. Eight 145 mg test samples and eight 40 mg DBM powder samples are examined at the time of implantation at 7, 14 and 28 days. Each six samples are tested at 35 days. 40 mg DBM powder samples are rehydrated with 100 μl sterile ALLOPREP ^™ (Ostetotech, Eatontown, NJ). Each sample is filled in a 1 ml blunt cut syringe. Transplantation is done randomly so that the same implant is not implanted twice in a single animal.
[118] Anesthesia : Rats are anesthetized with a mixture of ketamine (200 mg), xylazine (400 mg) and physiological saline (10 mL). The dose is 3.5 ml per kg body weight upon intraperitoneal administration.
[119] Procedure : Sterile surgical procedures are conducted in a clean air hood. A 1-cm skin incision is made on the top of each hind limb in a lateral approach, and the skin is separated from the muscle by blunt excision. A surface incision is made along the muscle plane to allow insertion of the tip of the scissors. Blunt ablation is performed deep into the muscle from the line to create a pocket in which the implant is held. A single suture is inserted to suture the muscle pocket, and the skin is sutured with a metal clip.
[120] Implantation of the sample into the left pectoral muscle involves a 1-cm skin incision in the chest, blunt ablation of the muscle to make a pocket, and placement of rat DBM powder using blunt syringe. A single suture is inserted to suture the muscle pocket, and the skin is sutured with a metal clip.
[121] Mice are euthanized with CO ₂ after the designated transplant time. The implant is identified by palpitation, recovered by blunt excision, and carefully trimmed to remove surrounding tissue. Observers of unknown implant type perform microscopic evaluation of the implant. Color, vascularity, hardness and completeness are scored according to the outline outlined in the table below (maximum score is 4 for the strongest response and 0 for samples showing no osteoinductive potential). The model showed a high correlation between visual and histological observations of implant performance only at both ends of the scale.
[122] Microscopic Scoring Guidelines
[123] ColorWhite (W)Gray (G)Red (R) VasculatureNone (N)Some (S)Strong (R) HardnessMist (M)Firmness (F)Hardness (H) completenessDiffusion (D)Uniformity (F)Nodule (N) Score00.5One
[124] Histology : The recovered material is fixed in neutral buffered formalin. After fixation in formalin, the sample is demineralized with 10% formic acid, dehydrated in classified alcohol, sealed and cleaved in JB-4 (Glycol Methacrylate, Polysciences, Inc., Warrington, PA). 5-micron fragments are stained with toluidine blue and evaluated by light microscopy.
[125] These explants are evaluated histologically using a semiquantitative method. In short, scores based on 5-point scores are assigned to each fragment: 4 = 75% new bone formation; 3 = 51-75% new bone formation; 2 = 26-50% new bone formation; 1 = 1-25% new bone formation; 0 = no evidence of intrachondral bone formation processes, including the presence of cartilage or chondrocytes, active osteoblasts, osteoids, newly formed and mineralized bone, or bone marrow and associated fat cells.
[126] Scoring Histological Fragments
[127] ScoreNew bone formation 0No new bone formation One1-25% new bone formation 226-50% new bone formation 351-75% new bone formation 4More than 75% new bone formation
[128] After histological analysis, an average score is calculated for each material type. Based on experience in previous animal models, each group is given an assessment of osteoinduction potential based on average histological scores.
[129] Results : The protocol was performed with a starch stabilizer-containing DBM identified in Example 3, which is the test substance, compared to the control GPS1-2 base DBM powder. At 7 days, DBM and GPS1-2 powder containing starch stabilizer achieved the same level of induction, with histological scores of 0.9 ± 0.4 and 1.0 ± 0, respectively. All samples are hypercellular with multiple chondrocytes present. At 14 days, DBM containing starch stabilizer achieved higher levels of induction than GPS1-2 powder, with histological scores of 3.6 ± 0.5 and 2.9 ± 1.0, respectively. Clusters of chondrocytes were present in all DBMs containing starch stabilizer samples. At this point, half of the powder samples contained clusters of chondrocytes, or individually isolated cells. At 28 days, chondrocytes were hardly present in DBM or GPS1-2 powder containing starch stabilizer. Most samples showed mature bone at this stage. Some tissue infiltration was identified in three DBM and two powder samples containing starch stabilizer. The histological score for DBM containing starch stabilizer remained constant for 14 days, while the histological score for powder improved from 2.9 ± 1.0 to 3.9 ± 0.4 at 14 to 35 days, at 35 days. No significant change was observed in these samples.
[130] Average histological score
[131] product7 days14 days28 days35 days DBM with Starch Stabilizer (GPS1-2)0.9 ± 0.43.6 ± 0.53.6 ± 0.53.5 ± 0.8 DBM (GPS1-2) Powder (Control)1.0 ± 02.9 ± 1.03.9 ± 0.43.7 ± 0.5
[132] CONCLUSIONS : The results of this study suggest that the induction rate of DBM containing starch stabilizer increases up to 14 days and remains constant until termination. GPS1-2 powder exhibited a slower induction rate at 14 days, but was identical to DBM samples containing starch stabilizers at 28 days. At this point, the osteoinductive potentials of both products were nearly identical, with a difference of 0.3 from the mean histological mean, and remained the same until the 35 day time point. DBM samples containing starch stabilizer showed faster bone formation compared to powder control. Qualitative assessment of increased cartilage cell number suggests increased bone formation in DBM samples containing starch stabilizers.
[133] Example 7 Evaluation of Efficacy of Compositions According to the Invention in the Treatment of Bone Defects
[134] BACKGROUND : Fractured autologous allogeneic bone (ABG) has long been regarded as the “golden criterion” for bone induction when bone grafts are needed in orthopedic clinical situations. Unfortunately, the amount of ABG available is limited and there is a surgical mortality of more than 5% during the collection process. Desalted bone matrix (DBM) has been found to have excellent therapeutic potential comparable to ABG. One of the major disadvantages of demineralized bone matrices is that they do not maintain the three-dimensional space of defects. Thus, invasion into the defect site from the surrounding muscle tissue proceeds. The DBM containing the test substance, starch stabilizer, provides a semi-solid structure that maintains three-dimensional space.
[135] The rabbit ulna defect model was modified and used as a substitute for autologous bone grafts in various projects examining osteogenicity and osteoconductive growth factors and efficacy of the matrix. The purpose of this study was to evaluate the osteoinductive capacity of new DBM grafts compared to previous formulations and ABGs.
[136] Materials and Methods :
[137] Study Design Summary:
[138] A. Rabbit Bilateral 2-cm Ulnar Defect
[139] Treatment group:
[140] DBM + starch
[141] 2. starch carrier alone
[142] 3. Autografts (histological data used for comparison)
[143] Surgical Procedure : Six month old male New Zealand white rabbits were used. 2.0 cm defects were made surgically on the bilateral ulna of all rabbits. After complete periostectomy, a thorough defect wash, and a partial interosseous lavage, implantation into each defect (depending on the test group) is performed through known surgical techniques. The wound is first sutured in layers. The test groups are listed in the table below. Once anesthesia is achieved, both forelimbs are removed with the hair removed and the foot is lying down with the feet up. A longitudinal incision (3-4 cm) is made in both ulna to expose the interosseous (middle) part of the ulna. Distal fractures are performed at 1 cm from the vertebral joints, and proximal fractures are performed at 3.0 cm from the vertebral joints, resulting in 2 cm defects. Fractures are performed with a high speed reamer. The sparse block of the resulting interosseous bone is excised to prevent damage to the periosteum. Due to the adherent interosseous membrane of the rabbit paw, no internal fixation is necessary. After irrigation with sterile saline to remove blood, bone and bone marrow residue, the implant is placed in the defect. The deep fascial layer is a 3-0 chromium suture that seals the defect around the defect. The skin is closed with a broken nylon suture. Post-operative finishing material / fibula is affixed and removed 4 days post surgery.
[144] Radiographs : Postoperative radiographs are taken immediately after surgery, and additional radiographs are taken at 3, 6, 9, and 12 weeks. At 6 or 12 weeks, high-resolution (Faxitron) radiographs are taken on both resected and soft tissue removed feet. Three observers without knowledge of the process assess bone formation and remodeling at each time point.
[145] Results : At 3 weeks, in vitro radiographs suggest that bone formation is apparent in starch-based formulations. At 6 weeks, trabeculation was observed and the starch-based formulation almost completely filled the defects of significant size (FIG. 2).
[146] Conclusions : Starch-based formulations have been shown to improve the rate of progression of bone formation.
[147] Example 8 The following table summarizes the results of biocompatibility and safety studies of the starch-based diffusion barrier DBM formulation of Example 3.
[148] All studies listed in the table below were conducted at North American Science Association. Inc. (NAMSA), ISO 9001 certified, with the exception of Study # 12, and approved by the Association for Assesment and Accreditation of Laboratory Animal Care International (AAALAC). . NAMSA is registered with the USDA and complies with FDA guidelines. All samples submitted to NAMSA were inspected according to the laboratory quality guidelines needed to verify valid data.
[149]
[150]
[151] Biocompatibility of DBM Containing Starch Stabilizer . Clearance studies confirmed the removal of DBM containing starch stabilizer carrier from the implant site within 30 days and categorized it into the Class B tissue / bone implant category for ISO 10993 biocompatibility studies. Four assessment tests that consider the Class B tissue / bone graft category are listed in the ISO guidelines. These are: cytotoxicity, sensitization, transplantation, genotoxicity. Acute systemic toxicity can also be applied in certain cases. In addition to the four proposed tests, a total of nine additional safety, biocompatibility, and efficacy studies were performed (including Example 7). These studies are summarized in the table above.
[152] Local reaction
[153] A. Acute intradermal injection and acute muscle transplantation study were conducted. Starch stabilizer-containing DBM made of rabbit DBM was used for muscle transplantation. Starch stabilizer-containing DBM induces minimal irritation in both studies, which is non-irritant in comparison to positive controls in muscle transplantation and has shown minor primary index characterization when administered as an intradermal extract. DBM used intramuscularly without starch carriers has been identified as a common irritant.
[154] B. Cytotoxicity and Genotoxicity . The extract of starch stabilizer showed no ability to induce cell lysis or bacterial mutagenicity. In lysis studies, saline extracts of starch stabilizer-containing DBM were used. In the genotoxicity study, saline and DMSO test extracts were used for two bacterial species: S. typhimurium and E. coli .
[155] C. Hemolysis and Fever . Saline extract of starch stabilizer was judged to be non-pyrogenic and non-hemolytic. In rabbits injected with saline extracts of starch stabilizer-containing DBM body temperature showed no signs of exothermicity, and the extract showed a hemolytic index of zero when added to anticoagulant rabbit blood.
[156] D. Sensitization . The extract of the carrier showed no evidence of delayed skin contact sensitization. Saline and cottonseed oil extracts of the carrier were used in this study. Guinea pigs were injected intradermal with these extracts and subsequently attacked with extract patches after the recovery period.
[157] E. Starch Stabilizer-Containing DBM Exhibit Safety / Toxicity . No evidence of toxicity was observed in a study where starch stabilizer-containing DBM (rabbit DBM) was intramuscularly implanted into the spinal muscle (high dose subcutaneously implanted) and the animals were monitored for 60 days. In these studies, rabbits were implanted with 3.5 cc (low dose) or 17.5 cc (high dose) DBM containing starch stabilizer (˜1.1 gm / cc). Doses (˜1.3 gm / kg; ˜6.41 gm / kg) correspond to 5.6 × and 28 × average human transplant doses (15 cc / 70 kg or 0.23 gm / kg), respectively. For high doses, due to space limitations at the spinal graft site, only 3.5 cc of DBM containing starch stabilizer was implanted into the spinal column and the remaining 14 cc were implanted subcutaneously into the dorsal chest.
[158] Autopsy results on the test animals showed no therapeutic effect. Blood chemistry and urinary levels were all normal except for serum alkaline phosphatase, which is expected to increase by induction of ectopic bone formation due to response to stabilizer-containing DBM.
[159] Example 9 Osteoinduction Analysis of Test Substance
[160] Purpose : The purpose of this example is to evaluate the properties of various potential protective agents and to identify those which do not particularly adversely affect osteoinduction. Suitably, the protective agent is easy to handle, irrigation, non-toxic, degradable and moldable (hardness is similar to a plumber's putty).
[161] METHODS AND MATERIALS : This study is conducted in athymic rat model. Suitably, a single DBM formulation is used for all formulations. Potential protective agent materials are sterilized by irradiation. Various test compositions and control DBMs are implanted in 6-8 sites of animals, respectively. Each animal received bilateral intramuscular transplantation on its hind legs. Each test composition contains 40 mg DBM per bone site. The volume can vary depending on the type of carrier.
[162] Results : The protocol was applied to four separate test compositions plus control DBM. The test composition was implanted in 30 animals; DBM was transplanted into 8 individual animals. The protective agent was sterilized by autoclaving. The following protective agent solution was prepared.
[163]
[164] The following test formulations were prepared:
[165]
[166] In addition, RDBM was placed in the left pectoral muscle as a positive control in all animals.
[167] Result :
[168] ImplantAverageSD HDBM- Contrast Person PullKF-135-040501-102.91.0 Sample E3.40.9 Sample F3.80.5 Sample G3.60.8 Sample H3.31.2
[169] Conclusion : The test composition had no negative effect on bone induction.
[170] Example 10 Osteoinduction in Rabbit Model
[171] Introduction and Methods : 55 male New Zealand white rabbits were assigned to three treatment groups. Test substances were prepared as identified in Example 3. Animals assigned to the low dose treatment group (n = 20) are implanted with 3.5 ml test substance into the right vertebral muscle following the protocol specified procedure. Animals assigned to the high dose treatment group (n = 20) implant 3.5 ml test substance into the right spinal muscle and 7.0 ml test substance into the subcutaneous tissue on each side of the dorsal thoracic region. Animals assigned to the control treatment group (n = 15) implant 3.5 ml control (rehydrated DBM powder) into the right spinal muscle. On days 7, 14 and 28 post-transplantation, 4 animals in the low and high dose treatment groups and 3 in the control group were killed. At 60 days post-transplant, the remaining animals are killed (eight in the low and high dose treatment groups and six in the control treatment group). The transplant site is harvested from each rabbit and fixed in 100% neutral buffered formalin (NBF). Tests and control sites obtained during the 60-day study period after implantation are placed in desalting solution for 3 days after appropriate formalin fixation. All tissue samples are processed by standard histological procedures, cut to 5 μm and stained with hematoxylin and eosin.
[172] RESULTS : Bone induction was observed in the subcutaneous and intramuscular grafts and in the control DBM (no subcutaneous graft 28 days after transplantation). The new bone was histologically characterized as strong, eosinophilic, than the desalted bone component of the test and control material. The new six weeks were filled with bulging (active) osteoblasts, osteogenic precursors, bone sources, and slightly mineralized bone sources. In many cases, bone cells were present and some bone remodeling was confirmed. In some cases cartilage was present. At 60 days post-transplantation, the new bone is similarly characterized, but the coarse fibrovascular stroma (morphologically identical to that found in the bone marrow), which is associated with increased thickness and remodeling and that contains hematopoietic tissues, occurs over six weeks Was observed. Subjectively, the test agent showed a higher level of bone formation at the site of muscle transplantation than at the control site. The amount of cartilage present varied with the site of transplantation. This variation is most likely due to differences in microenvironment for individual implants. Progenitor pluripotents involved in new bone formation, forming fibrous tissue, cartilage or bone under certain microenvironmental conditions. Cartilage in the graft site undergoes endochondral ossification and becomes bone. Differences in tissue response, bone formation, or cartilage formation between the test substance implanted in the subcutaneous tissue and the test substance implanted in the muscle were due to the anatomical and microenvironmental differences between the two tissues. Bone formation was observed on both test and control site implants 28 days after transplantation. Bone content and maturation (manifested as a result of remodeling and the presence of coarse fibrovascular substrates and hematopoietic tissues) increased significantly at 60 days for the test substance.
[173] Presence of new bone and cartilage by treatment group and post-transplantation time Treatment group7 days after transplantation / cartilage14 days after transplant28 days after transplant60 days after transplant High dose muscle subcutaneous0/0 (n = 4) 0/0 (n = 8)0/0 (n = 4) 0/0 (n = 8)2.0 / 1.5 (n = 4) 1.4 / 1.5 (n = 8)3.5 / 0.0 (n = 8) 2.7 / 0.9 (n = 15) Low volume muscle0/0 (n = 4)0/0 (n = 4)2.3 / 0.8 (n = 4)4.0 / 0.4 (n = 5) Control muscle0/0 (n = 4)0/0 (n = 4)0.7 / 0.7 (n = 4)2.5 / 0.2 (n = 6)
[174] Grades in the table above are based on 0-4 scale, with new (survival) bone / cartilage at 0 = 0% transplant area; 1 = new (survival) bone / cartilage in the 1-25% transplant area; 2 = new (survival) bone / cartilage in the transplant area of 26-50%; 3 = new (survival) bone / cartilage in the transplant area of 51-75%; 4 = new (survival) bone / cartilage in the transplant area of 76-100%.
[175] Example 11 : Terminal Sterilization
[176] This example describes a terminal sterilization method that minimizes bone induction loss in a formulation according to the present invention.
[177] DBM formulations according to the invention are made from human tissue in a clean indoor environment. The final implant is placed in individual tray packaging.
[178] Each tray is placed in an Audionvac sealing unit (Audion Electro B.V., Weesp-Holland) equipped with a cylinder filled with 50/50 hydrogen / argon gas. Prior to sealing the tray package, the air is evacuated twice and refilled with the gas mixture. After sealing, the gas mixture remains in each tray package.
[179] The packaged implant is then sealed and treated with 15 KGy gamma radiation from a cobalt 60 light source to reduce the bioburden of the implant to the desired level.
[180] Example 12 Comparing Osteoinduction of DBM Formulations with BMP and / or Other Growth Factors
[181] In a series of studies presented herein, hybrid recombinant human BMPs (hybrid rhBMP, hrhBMP) were created that possess stronger heparin-binding epitopes at the N-terminus compared to wild type BMP. Heparin-binding sites enhance binding to ECM, which increases the local retention time of BMP, maximizing the potential for interaction with suitable cells in vivo (Kubler et al. "EHBMP-2, Initial BMP analog with osteoinductive properties"). Mund Kiefer Gesichtschir. 3 Suppl 1: S134-9, 1999).
[182] The purpose of this study was to compare the osteoinductive potential of hybrid rhBMP-2x (hrhBMP-2x) and wild type BMP-2 (rhBMP-2) to bind hrhBMP-2x to desalted or deactivated (inactivated) bone matrix. Is to check for synergistic potential.
[183] Way
[184] To assess the osteoinductive activity of hybrid rhBMP-2x, 1, 5, 10 μg hrhBMP-2x was placed in a 200 mg osteoinductive human desalted bone fiber (DBF) matrix and placed in athymic rats (6 per group) for 21 days. Displacement was implanted. In order to clarify the difference between the treated and untreated DBMs, a DBF matrix was created in which the osteoinductive potential is approximately 50% of the commonly observed osteoinductive potential. Controls were osteoinduced human DBF matrix alone; Inactivated human DBF matrix alone (“deactivation”, GuHCl extraction); 1, 5, 10 μg hrhBMP-2x containing 5 μg wild type BMP-2, inactivated human DBF bound to the active and inactive matrix. All samples were evaluated histologically with a 5-point scoring system (4 => 75% bone formation in the cross-sectional area of the transplant, 3 = 51-75%, 2 = 26-50%, 1 = 1-25%, 0 = No bone formation) (Edwarss JT, Diegmann MH, Scaborough NL. Osteoinduction of human demineralized bone: characterization in a rat model. Clin. Orthop. 357: 219-28, 1998).
[185] result
[186] Histological scoring revealed in the method section was inadequate for recording scores in most samples including morphogenic factors. Only deactivated samples (inactive DBF matrix) are zero; Deactivation sample + 1 μg hrhBMP-2x is 0.8 ± 0.4; DBF matrix is 2.5 ± 0.8; All other samples were four.
[187] To further distinguish the degree of development of the nodule, a qualitative scoring system was developed that measures the vasculature of the sample and the residual DBF remaining in the sample. The following scale was used:
[188] Vasculature (blood): none = 0; Insignificant = 1; Few vessels, small vessels = 2; Moderate cytoplasm and vessel size = 3, broad cytoplasm, large vessels = 4.
[189] Residual DBF: none = 0; Insignificant = 1; Low = 2; Medium = 3; Wide = 4.
[190] Active DBF matrices treated with hrhBMP-2x include more differentiated nodules with little residual DBM; Extensive new bone formation; The deactivated group induced highly developed vasculature, which was not seen even at the maximum concentration of morphogenesis factors. Deactivated carriers can be likened to collagen sponges—in particular, inert tertiary structures that support bone growth. Although wild-type rhBMP-2 induced well-developed vasculature and bone marrow, the residual bone content was much higher than the active DBF counterpart.
[191] Conclusion : The results show that the modified hrhBMP-2x has stronger osteoinduction than the corresponding wild type. Osteoporosis accelerated and induced bone tissue showed a more compact structure. Synergistic results were achieved when hrhBMP-2x combined with active DBF matrix and non-deactivated DBF. The most likely explanation for this finding is the longer half-life of hrhBMP-2x at the site of transplantation. The persistence of growth factors at this site prolongs interaction time with local cells rather than lysis into surrounding tissues, leading to ectopic bone formation sites. The active matrix appears to significantly increase the bone induction of externally added growth factors due to cooperative interactions with many growth factors already present in demineralized bone (Kubler et al. "Allogenic bone and cartilage morphogenesis. Rat BMP in vivo and in vitro ", J. Craniomaxillofac. Surg. 197): 283-8, 1991; Kubler et al, "Effect of different factors on the bone forming properties of recombinant BMP" Mund Kiefer Gesichtschir. 4 Suppl 2: S465-9, 2000).
[192] Example 13 Process for Making Species-Specific Bone Grafts with Defined Sizes
[193] Long bones from humans, rhesus monkeys, dogs, and rabbits were used to create a species-specific solid phase implant matrix. Bones are sterile trimmed. Cortical bone is processed in a bone milling device disclosed in US Pat. No. 5,607,269 to yield 65 g of elongated bone fibers. The long bone fibers are placed in a reactor and immersed in 0.6 N HCl + 20-2000 ppm non-ionic surfactant solution for 5-10 minutes. After draining the HCl / surfactant, 15 mL of 0.6N HCl per gram of total bone is introduced into the reactor along with the elongated bone fibers. The reaction proceeds for approximately 40-50 minutes. After draining through a sieve, the desalted elongated bone fibers are washed three times with 15 ml of sterile deionized water per g of total bone and repeated at 15 minute intervals. After draining the water, the bone fibers are placed in alcohol and soaked for at least 30 minutes. The alcohol is then drained and the bone fibers are washed with sterile deionized water. The bone fibers are then contacted for at least 60 minutes with a mixture of approximately 4.5 ml glycerol per gram of dry bone fiber and approximately 10.5 ml sterile deionized water per gram of dry bone fiber. The resulting liquid composition, draining excess liquid and retaining approximately 11% (w / v) desalted elongated bone fibers, has a lid of a plurality of protruding teeth (1.5 cm x 3.5 cm wide and length, 4 mm deep). Transfer to a retaining 11 cm x 11 cm mold, with the lid gently placed in the mold to allow the teeth to be buried in the fibers to reduce the pressure on the composition as little as possible. The size of the protrusions can be tailored to the size of the bone graft needed for the animal model of interest. The resulting cut pieces were 4.5 cm long, 2.5 cm wide, 8 mm high (thick), and troughs were 3.5 cm long, 1 cm wide, 4 mm high (thick). The mold is then placed in an oven at 46 ° C. for 4 hours. The composition is then frozen at −70 ° C. overnight and then frozen for 48 h. After freezing, the mold disintegrates and the sponge-like form composition is cut into individual pieces with troughs.
[194] The resulting composition is a sponge-like tertiary structure with viscous, flexible, visible pores, has a defined shape, including teeth created by lid protrusion, and does not require hydration prior to use, but rapidly It is hydrated and, once wetted with fluid, remains circular and does not require freezing for storage.
[195] Example 14 Methods of Making Partly Enclosed DBM / Polymer Composites
[196] The following method is used to make a desalted bone matrix partially encapsulated in a resorbable polymer. This partially enclosed DBM provides an initial level of bone induction from the non-sealed DBM portion and provides a continuous source of active DBM that does not degrade as the polymer degrades over time. The method is particularly suitable for sealing DBM in tyrosine polycarbonate Integra Life Sciences (DT) and poly (L-lactide-D, L-lactide 70/30) (Boehringer Ingelheim). The device is particularly useful for posterior spinal fusion, where it can be located in the lateral trough to promote bone formation. The method can be used to half seal a matrix of suitable shape made from the method disclosed in Example 10, or a collection of desalted cortical fibers, wherein the fibers have a long axis of fibers that are cut one inch long and are substantially parallel to each other. The fibers arranged in a cylindrical bundle can be partially enclosed by the method.
[197] An approximately 1/2 inch high stainless steel adjustable diameter circular clamp is used to hold the ground polymer, with the lower portion of the desalted bone. The fiber bundle or matrix sample is centered on the clamp and leaves the outer space of the clamp to receive the ground polymeric material. Thereafter, heat is applied under the clamp until the polymer melts. The clamp is rigid (reduced in diameter) and the polymer is still fluid, causing the polymer to flow into the bottom of the fiber bundle. Thereafter, the polymerizable material is cooled and the clamp is removed to seal the lower portion of the fiber in the solid polymer.
[198] In suitable embodiments, resorbable polymers are used. The temperature is used to melt the polymer to an appropriate viscosity, allowing the dissolved polymer to flow into and out of the desalted bone. Most of the temperatures used are 0-15 ° C. above the glass transition temperature of the polymer. The polymer preferably has a glass transition temperature of less than 100 ° C., more preferably less than 80 ° C., most preferably less than 60 ° C., since the DBM's bioactivity becomes poor if the temperature is maintained above 60 ° C. for a significant period of time. . In the case of tyrosine polycarbonate, a DT temperature of 115 ° C. is used for 10 minutes. 70 ° C. is suitable for poly (L-lactide-D, L-lactide 70/30). The method is also applicable where a suitable polymer solvent is used in place of heat to facilitate polymer flow.
[199] Example 15 DBM Formulations Containing Stabilized DBM Mixtures with Extended Half-Life Diffusion Barrier
[200] Two desalted bone preparations are prepared:
[201] Desalted Bone Formulation # 1 . DBM is made from approximately 150-1000 micron bone particles desalted, lyophilized and pre-expanded with 100% glycerol, and excess glycerol is removed by filtration. Lactomer 9-1, Caprolactone Glycolide & Calcium Stearoyl Lactylate (Tyco Inc. North Haven, CT) is homogeneously mixed with DBM at 10: 1 per weight. The mixture is dissolved at 70 ° C. in the mold. After cooling, the polymer DBM monolith is ground in a cold mill and filtered to a particle size of approximately 130-1200 microns.
[202] Desalted Bone Formulation # 2. Lecithin-based DBM formulations are prepared according to the method of Han et al. (“Synergistic Effects of Lecithin and Human DBM on Bone Induction in Nude Rats” Abstract from the 28th Annual Meeting of the Society for Biomaterials (2002)). In short, Pospholipon 90G (American Lecithin Company) mixes with desalted bone in a weight ratio of 40% lecithin: 60% DBM to 60% lecithin: 40% DBM.
[203] A third starch based demineralized bone is prepared according to Example 2 except that only one third of the total demineralized bone is added to the starch carrier. In place of the remaining two-thirds of desalted bone, desalted bone derived from formulations # 1 &# 2 of this example is added in equal amounts. The composition is then mixed to make an implant formulation.
[204] Example 16 : Competitive Substrates
[205] Poly-L-lysine can be used as a competitive inhibitor for serine protease enzymes. This example describes the formulation of desalted bone incorporating poly-L-lysine. Poly-lysine (10-300 kD) is prepared in 1 mM HCl in a concentration range of approximately 1-10 mg / ml. Prepare demineralized bone. After the final wash, it is mixed with the poly-L-lysine solution at a 1/5 concentration ratio to make a thick slurry (˜0.33 gm / ml). The desalted bone / substrate mixture is lyophilized. The demineralized bone thus produced is either used directly or formulated into a carrier.
[206] Example 17 Fatty Acid / Starch Diffusion Barrier Matrix
[207] Desalted bone is prepared as shown in Example 14 except that the polymer / DBM formulation is replaced with an equivalent weight of lecithin formulation.
[208] Example 18 Osteoinduction of DBM Compositions in Athymic Rat Models
[209] The purpose of this example is to evaluate the osteoinductive potential of a DBM composition using an ectopic osteoinduction 28 day transplant model (Edwards et al., Clin Orthop. Rel. Res. 35: 219-228, 1998; Urist, Science 150 : 893-899, 1965. The DBM composition comprises cubic DBM particles with DBM fibers (USSN 60 / 159,774, October 15, 1999; WO0232348). Chondrocytes are the leading cell type in the DBM cube after 28 days of transplantation. In this study, the transplantation time was extended to 49 days to confirm evidence of continuous bone remodeling in the desalted cortical cube.
[210] MATERIALS AND METHODS : Each dose of approximately 600 mg ground sample is packaged in a 2.5 ml blunt syringe. Eighteen female athymic mice were obtained from Harlan Sprague Dawley Inc. (Indianapolis, Ind.). Animal weights at the time of surgery ranged from 186 g to 236 g. Evaluate 28 and 49 days after transplant.
[211] Transplantation sites are evaluated histologically. The fibrous component scores independently of the cube and scores on a 5-point semiquantitative scale based on the percentage of fiber regions participating in new bone formation. The cube portion assigns scores based on the percentage of the Haversian system participating in new bone formation.
[212] Results : New bone, bone marrow and adipocytes were present throughout the fibrous part of the nodules. Chondrocytes were present in the central bone at all time points. At 28 days, the mean bone induction score for the fiber portion was 3.1 ± 0.5 in the 89.8 ± 5.8% fiber portion of the Haversian canal occupied by the cube portion. The cube was surrounded by new bone or bone marrow, and pockets of chondrocytes appeared in and between cubes.
[213] At 49 days, the mean bone induction score for the fiber section was 3.5 ± 0.5 in the 98.1 ± 2.4% fiber section of the Haversian canal occupied by the cube. Notable differences from the 28-day sample include nearly complete remodeling of the fibrous portion, large number of chondrocyte pockets and new bone regions in the cube, and remodeling at the corners of the cube.
[214] CONCLUSIONS : Cortical cube plays an important role in osteoinduction of DBM compositions. The cube is cut from the cortical bone and the central tube provides natural porosity. The cartilage that persists after 28 days coincides with a delay in bone formation due to delayed angiogenesis. On day 49, the cube showed slower than fiber but evidence of remodeling. The bone remodeling was faster on the outer surface than on the inner surface. The cube continues to provide support matrix and osteoinduction signals important for normal bone formation during the treatment response.
[215] Example 19 Establishment of Treatment Characteristics of Compositions According to the Invention
[216] This example describes the addition of desalted bone to a stabilizer and / or diffusion barrier according to the present invention for producing a moldable osteoinductive implant composition. This example describes the establishment of an appropriate carrier viscosity, the mixing of the carrier with the DBM, and the modification of the final treatment characteristics of the finished composition.
[217] Carrier viscosity . The starch based compositions of the present invention were prepared as shown in Example 3 with a starch to water ratio of approximately 5% to 45%. Starch powder was mixed with water and the mixture was made into sterile hydrated starch formulation by autoclaving. Thereafter, autoclaved starch was checked for viscosity. DBM compositions were prepared using starch formulations having a viscosity in the range of 5000 to 20000 sCp.
[218] The viscosity of the starch carrier was measured using a Brookfleld viscometer (HB-DV III +) backed by Rheocalc32 software and equipped with a suitable sample adapter (SSA27 / 13RPY s / n RP66162, spindle # 27).
[219] Mixing of Carrier and DBM . Starch carriers having a viscosity of approximately 5000 sCpi were mixed with various amounts of DBM (approximately 10% to 50% DBM per weight) to make compositions with similar hardness as in model clay or bread dough. Changes with smaller amounts of DBM resulted in the composition in a viscous but nearly flowable form. Formulations with more DBM yielded very hard hardness products, where formulations with high levels of DBM were ground and fragmented during mixing. These formulations were then quantitatively evaluated for treatment as described below.
[220] Analysis of Composition Treatment Properties . The following method was used to establish the hardness in the treatment properties of the composition according to the invention. Compositions using starch-based carriers having a penetration resistance value of 25-120N are acceptable, penetration resistance values of 30-90N are more preferred, and penetration resistance values of 40-65N are considered even more preferred.
[221] A 1 "diameter X9" length seal (14 tpi) push rod was placed on the actuator of the MTS 858Bionix inspection device equipped with a 1 kN force transducer. One piece of 1.5 "diameter X6" length PVC pipe was placed perpendicular to the center of the force transducer and a large weight boat was positioned below it to support the protruding bone formulation. A 1 "ID X0.5" thick spacer is placed on top of the PVC tube, and the 7.00 g bone mixture is weighed into a 5cc syringe and loaded at the end of the syringe using a clean dry 5cc syringe plunger with the tip removed just below the o-ring. (load) to make a flat surface. The loaded syringe was positioned perpendicular to the spacer / PVC pipe assembly with the plunger facing up. The entire assembly (PVC pipe, spacer, syringe) was placed in the center of the load cell just below the push bar. The center of the plunger was located one line with the center of the push rod. The 1 kN load range was used for the first test of each new bone formulation. If the maximum load required to protrude the bone mixture was below 90N, higher accuracy was achieved using the 100N load range during subsequent inspections. The test sample was preloaded to 5N under load control, the displacement was zero, and the test was carried out. The bone formulation protruded at a rate of 5 mm / min up to a displacement of up to 20 mm in the compressed state. Thereafter, the average maximum capacity required to extrude each bone formulation was measured.
[222] Example 20 Detection of Amylase Sensitivity
[223] This example describes the evaluation of starch-based stabilizers and diffusion barriers (carriers). Increasing amylase resistance of starch based carriers increases the effective retention time of the carriers after transplantation, thus increasing the stabilizing effect of the carriers.
[224] Quantification of resistant starch requires the use of pancreatic α amylase and amyloglucosidase to effectively detect degradation of amylase-resistant starch into glucose.
[225] Degradation of the starch and starch / lipid compositions of Examples 3, 9, 15, and 17, including new candidate amylase resistant starches and modified starches, was performed by Megazyme International Ireland Ltd. Resistant Starch Assay Kit (amyloglucosidase α-amylase method AOAC method 996.11, AACC Method 76.13, ICC Standard Method No. 168). In general, the slowest dissolving agents have the longest stabilizing effect in vivo.
[226] Example 21 Starch / Lipid Carrier Composition
[227] The following compositions were prepared in a manner similar to that disclosed in Examples 3, 9, 15, and 17. The carrier is sterilized by autoclaving for 20 minutes prior to mixing with DBM.
[228] Compound # 1-Carrier 1 consists of approximately 8% Penford Maps 281 and 5% lecithin, with the remainder water.
[229] Compound # 2-Carrier 2 consists of approximately 8% Penford Maps 281 and 15% lecithin, with the remainder water.
[230] Compound # 3-Carrier 3 consists of approximately 6% GPC B980 and 5% lecithin, with the remainder water.
[231] Compound # 4-Carrier 4 consists of approximately 6% GPC B980 and 15% lecithin, with the remainder water.
[232] Each of the four carrier mixtures was mixed with human DBM to yield a bone content of approximately 25%. These bone mixtures were then examined for osteoinductance as identified in Example 6.
[233] Another embodiment
[234] The foregoing is a preferred non-limiting embodiment of the invention. As will be appreciated by those skilled in the art, various modifications to these embodiments do not depart from the spirit or scope of the present invention as defined in the claims herein.
[235] Appendix A
[236] In the DBM composition of the present invention, the biologically active agent is capable of biological activity including small molecules, chemical compounds, proteins, polypeptides, polynucleotides, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, synthetic analogues thereof and biologically processed analogues. Having any substance.
[237] Examples of biologically active compounds that can be used in the DBM compositions of the present invention include any hydrophilic or hydrophobic biologically active compound. Appropriately, the drug is one that has been recognized for its safety and effectiveness by the relevant government agency. For example, 21 C.F.R. Drugs approved for human use by the FDA under 330.5, 331-361; 21 C.F.R. Drugs approved for livestock by the FDA under 500-582 are available for use in the novel polymer network of the present invention.
[238] Drugs that are not themselves liquid at room temperature can be mixed with DBM and other polymers. In addition, peptides and proteins normally dissolved by tissue-activated enzymes, such as peptidase, may be manually protected in polymers or DBMs.
[239] "Biologically active compounds" include pharmacologically active substances which exhibit local or systemic effects in animals, plants or viruses. Thus, it is meant any substance used for the diagnosis, treatment, alleviation or prevention of a disease or for the enhancement of physical or mental development and condition in an animal, plant or virus. As used herein, “animal” includes mammals such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, mice; Birds; reptile; Pisces; insect; Arthropods; Protists (eg, protozoa); Means protozoa. "Plant" means higher plants (pizza plants, seedlings), fungi, protozoa (zozobacteria).
[240] A biologically active compound is any substance with biological activity, such as proteins, polypeptides, polynucleotides, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, synthetic analogs and biologically processed analogs thereof. "Protein" is known in the art and includes peptides. A protein or peptide is any biologically active protein or peptide that is naturally occurring or synthesized.
[241] Examples of proteins include antibodies, enzymes, steroids, growth hormones, growth hormone-releasing hormones, gonadotropin-releasing hormones, agonists and antagonists thereof, succinate stimulating hormones such as somatostatin and its analogs, emperor hormones and follicle-stimulating hormones, Peptide-T, Tyrocalcytonin, Parathyroid Hormone, Glucagon, Vasopressin, Oxytocin, Angiotensin I and II, Bradykinin, Calidine, Adrenal Cortical Stimulating Hormone, Thyroid Stimulating Hormone, Insulin, Glucagon and Many Analogs Included.
[242] Biologically active compound lines that can be loaded with crosslinked gels by the methods of the present invention include anti-AIDS substances, anti-cancer substances, antibiotics, immunosuppressants (e.g., cyclosporins), anti-viral substances, enzyme inhibitors, Neurotoxins, opioids, sleeping pills, antihistamines, lubricants, neurostabilizers, anti-convulsants, muscle relaxants and anti-Parkinson drugs, anti-seizure and muscle contractors, mobilizers and anti-choline drugs, anti-glaucoma compounds, anti Parasites and anti-protozoal compounds, anti-hypertensives, analgesics, antipyretics, anti-inflammatory agents such as NSAIDs, local anesthetics, eye drops, prostaglandins, anti-depressants, anti-psychotic drugs, anti-emetic agents, imaging agents ), Including but not limited to certain targeting agents, neurotransmitters, proteins, cellular response modifiers, vaccines.
[243] A more complete list of compound lines suitable for loading into polymers using the methods of the present invention can be found in Pharmazeutische Wirkstoffe (Von Kleemann et al. (Eds) Stuttgart / New York, 1987).
[244] Typical examples of biologically active substances are given below:
[245] Anti-AIDS substances are substances used to treat or prevent autoimmune deficiency syndrome (AIDS). Examples of such substances include CD4, 3'-azido-3'-deoxythymidine (AZT), 9- (2-hydroxyethoxymethyl) -guanine acylclovir, phosphonoformic acid, 1-adamantana Min, peptide T, 2'3 'dideoxycytidine and the like.
[246] Anti-cancer substances are substances used to treat or prevent cancer. Examples of such substances include methotrexate, cisplatin, prednisone, hydroxyprogesterone, hydroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, testosterone propionate, fluoxymesterone, vinblastine, vincristine, vindesine , Daunorubicin, doxorubicin, hydroxyurea, procarbazine, aminoglutetimide, mechloretamine, cyclophosphamide, melphalan, uracil mustard, chlorambucil, busulfan, carmustine, romuslin, Dacarbazine (DTIC: dimethyltriazenomidazolecarboxamide), methotrexate, fluorouracil, 5-fluorouracil, cytarabine, cytosine arabinoxide, mercaptopurine, 6-mercaptopurine, thioguanine and the like.
[247] Antibiotics are substances that inhibit or kill the growth of microorganisms. Antibiotics can be produced synthetically or by microorganisms. Examples of antibiotics are penicillin, tetracycline, chloramphenicol, minocycline, doxycycline, vanomycin, bacitracin, kanamycin, neomycin, gentamycin, erythromycin, cephalosporin and the like.
[248] Anti-viral agents are substances that can destroy viruses or inhibit their replication. Examples of anti-viral agents include methyl-P-adamantane methylamine, 1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9- [2-hydroxy-ethoxy] methyl Guanine, adamantanamin, 5-iodine-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside and the like.
[249] Enzyme inhibitors are substances that inhibit enzymatic reactions. Examples of enzyme inhibitors include edroponium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine HCl, tacrine, 1-hydroxy maleate, iodinetobercidine, p Bromotetramisol, 10- (alpha-diethylaminopropionyl) -phenothiazine hydrochloride, carmidazolium chloride, hemicolinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3-phenylpropargylamine, N ⁶ -monomethyl-L-arginine acetate, carbidopa, 3-hydroxybenzylhydrazine HCl, hydralazine HCl, chlorgiline HCl, deprenyl HCl, L (-)-deprenyl HCl, D (+)-hydroxylamine HCl, eproniazide phosphate, 6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargiline HCl, quina Clean HCl, Semicarbazide HCl, Tranylcipromine HCl, N, N-diethyla Noethyl-2,2-diphenylvalerate hydrochloride, 3-isobutyl-1-methylxanthine, paraberine HCl, indomethacin, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3- Dichloro-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, p-aminoglutetidemide, p-aminoglutetimide tin Acid salts, R (+)-p-aminoglutetimide stannate, S (-)-3-iodtyrosine, alpha-methyltyrosine, L-alpha-methyltyrosine, D, L-acetazolamide, dichlorfena Mead, 6-hydroxy-2-benzothiazolesulfonamide, allopurinol and the like.
[250] Neurotoxins are substances that have a toxic effect on the nervous system, such as nerve cells. Neurotoxins include adarlinergic toxins, cholinergic neurotoxins, dopaminergic toxins, and other neurotoxins. Examples of aderergic neurotoxins are N- (2-chloroethyl) -N-ethyl-2-bromobenzylamine hydrochloride and the like. Examples of cholinergic neurotoxins are acetylethylcholine mustard hydrochloride and the like. Examples of dopaminergic toxins are 6-hydroxydopamine HBr, 1-methyl-4- (2-methylphenyl) -1,2,3,6-tetrahydro-pyridine hydrochloride, 1-methyl-4-phenyl-2 , 3-dihydropyridinium HBr, 1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate, N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine HCl, 1 -Methyl-4-phenylpyridinium iodine and the like.
[251] Opioids are substances with opiate-like effects that do not originate from opium. Opioids include opioid promoters and opioid antagonists. Opioid promoters are codeine sulfate, fentanyl silicate, hydrocodone bistantate, loperamide HCl, morphine sulfate, noscapine, norcodeine, normorphine, thebaine and the like. Opioid antagonists are nor-vinaltorphamine HCl, buprenophrine, chlornaltrexamine 2HCl, funnaltrexamion HCl, nalbuphine HCl, nallopine HCl, naloxone HCl, naloxazine, naltrexone HCl, naltrindol HCl, and the like.
[252] Sleeping pills are substances that induce hypnotic effect. Hypnotics include pentobarbital sodium, phenobarbital, secobarbital, thiopental and mixtures thereof, heterocyclic hypnotics, dioxopyreridine, glutarimide, diethyl isovaleramide, bromoisovaleryl urea, urethanes, disulfane And so on.
[253] Antihistamines are substances that competitively inhibit the effects of histamine. Examples are pyrillamine, chlorpheniramine, tetrahydrazolin and the like.
[254] Lubricants are substances that increase the lubricity of the environment in which they are delivered. Examples of biologically active lubricants are water and saline.
[255] Neurostabilizers are substances that provide neurostable effects. Examples of neurostabilizing agents are chloropromazine, promazine, flufenzaine, reserpin, deserpiddine, meprobamate and the like.
[256] Anti-convulsants are substances that have the effect of preventing, reducing or eliminating spasms. Examples of such agents are primidone, phenytoin, valproate, Chk, ethosuccimid and the like.
[257] Muscle relaxants and anti-Parkinson's are agents that relax muscles or reduce or eliminate symptoms associated with Parkinson's disease. Examples of such agents are mefenesin, metocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa / carbidopa, biferidine and the like.
[258] Anti-seizure agents and muscle contractors are substances that can prevent or alleviate muscle seizures or contractions. Examples of such agents are atropine, scopolamine, oxyphenonium, papaverine and the like.
[259] Motivators and anticholiners are compounds that induce bronchial relaxation. Examples of such compounds are ecothioparts, pilocarpine, physostigmine salicylate, diisopropylfluorophosphate, epinephrine, neostigmine, carbacol, methacholine, betacol.
[260] Anti-glaucoma compounds include betaxalol, pilocarpine, timolol, timolol salts, mixtures of timolol and pilocarpine and / or salts thereof.
[261] Anti-parasites, anti-protozoal agents, and anti-fungal agents include ivermectin, pyrimethamine, trisulfypyrimidine, clindamycin, amphotericin B, nystatin, flucitocin, natamycin, and myconazole do.
[262] Anti-hypertensive agents are substances that can counteract hypertension. Examples of such materials are alpha-methyldopa and pivaloyloxyethyl esters of alpha-methyldopa.
[263] Painkillers are substances that can prevent, reduce or alleviate pain. Examples of analgesics are morphine sulfate, codeine sulfate, meperidine, nallopine and the like.
[264] Antipyretic agents are substances that can relieve or reduce heat, and anti-inflammatory agents are substances that can offset or inhibit inflammation. Examples of such agents are aspirin (salicylic acid), indomethacin, sodium indomethacin trihydrate, salicyamide, naproxen, colchicine, phenopropene, sulindac, diflunisal, diclofenac, indoprofen, sodium salicylate Silamide and the like.
[265] Local anesthetics are substances that have anesthetic effects on the local area. Examples of such anesthetics are procaine, lidocaine, tetracaine, dibucaine and the like.
[266] Eye drops include diagnostic agents such as sodium fluresin, rose bengal, methacholine, adrenaline, cocaine, atropine. Ophthalmic surgical additives include alpha-chymotrypsin and hyaluronidase.
[267] Prostaglandins are a class of long-chain hydroxy fatty acids known in the art that have a variety of biological effects and are known in the art.
[268] Anti- depressants are substances that can prevent or alleviate depression. Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzin, isocaboxazide, etc. to be.
[269] Anti-psychotic drugs are substances that change psychotic behavior. Examples of such agents are phenothiazine, butyrophenone, thioxanthene and the like.
[270] Anti-nausea drugs are substances that can prevent or alleviate nausea or vomiting. An example of such a substance is dramamin.
[271] Imaging agents are agents that can image tumors at desired sites, for example in vivo. Examples of imaging agents include substances with labels that can be detected in vivo, such as antibodies attached to fluorescent labels. Antibodies include whole antibodies and fragments thereof.
[272] Certain targeting agents include agents that can deliver a therapeutic drug to a desired site, such as a tumor, and provide a therapeutic effect. Examples of targeting agents include agents that can carry toxins or other agents that provide a beneficial effect. The targeting agent may be an antibody bound to a toxin, such as lysine A or an antibody bound to a drug.
[273] Neurotransmitters are substances that, when excited, are released and migrated from neurons to inhibit or stimulate target cells. Examples of neurotransmitters are dopamine, serotonin, q-aminobutyl acid, norepinephrine, histamine, acetylcholine, epinephrine and the like.
[274] Cell response modifiers are chemotactic factors such as platelet-derived growth factor (PDNF). Other chemotactic factors include neutrophil-activated protein, monocyte chemotactic protein, macrophage-inflammatory protein, platelet factor, platelet based protein, melanoma growth promoting activity; Epithelial growth factor, converting growth factor (alpha), fibroblast growth factor, platelet-derived endothelial growth factor, insulin-like growth factor, nerve growth factor, bone growth / cartilage-inducing factor (alpha and beta), or other Bone forming proteins are included.
[275] Other cell modifying agents include interleukin, including interleukin 1-10, interleukin inhibitors or interleukin receptors; Interferons including alpha, beta, gamma; Hematopoietic factors including erythropoietin, granulocyte colony promoter, macrophage colony promoter, granulocyte-macrophage colony promoter; Tumor necrosis factors, including alpha and beta; Conversion growth factors (beta), including beta-1, beta-2, beta-3, inhibin, actibin; It is a bone forming protein.

权利要求:
Claims (105)
[1" claim-type="Currently amended] An implantable bone growth inducing composition, wherein the composition is composed of:
matrix;
At least one growth factor;
Stabilizer;
Here, the stabilizer enhances the osteoinductive properties of the composition, leading to improved bone formation ability compared to the composition without the stabilizer.
[2" claim-type="Currently amended] The composition of claim 1, wherein the stabilizer is selected from diffusion barriers, enzyme inhibitors, competition substrates, masking components, compounds thereof.
[3" claim-type="Currently amended] The method of claim 1 wherein the stabilizer is selected from natural polymers, non-natural polymers, modified or derived natural polymers, modified or derived non-natural polymers, compounds thereof, and the matrix is at least partially encapsulated in the stabilizer. The composition characterized in that.
[4" claim-type="Currently amended] 4. The composition of claim 3 wherein the natural polymer is selected from lipids, polysaccharides, compounds thereof.
[5" claim-type="Currently amended] The composition of claim 4 wherein the lipid is a fatty acid.
[6" claim-type="Currently amended] The composition of claim 4 wherein the polysaccharide is starch.
[7" claim-type="Currently amended] 4. The composition of claim 3 wherein the stabilizer is resorbable or biodegradable.
[8" claim-type="Currently amended] 4. The non-natural polymer of claim 3, wherein the non-natural polymer is poly-lactic acid, poly-glycolic acid, copolymers of poly-lactic acid and poly-glycolic acid (PLGA), polydextran, polyester, polyvinyl alcohol, tyrosine polycarbonate, A composition characterized in that it is a resorbable polymer selected from tyrosine polyarylate, poly-orthoester, polylactide, polyglycolide, polyether, poly-fumarate polyester, copolymers thereof.
[9" claim-type="Currently amended] The composition of claim 3, wherein the matrix is at least partially particulate.
[10" claim-type="Currently amended] 10. The composition of claim 9, wherein the matrix is selected from ceramics, polymers, bones, desalted bones, extracellular matrices, compounds thereof.
[11" claim-type="Currently amended] The composition of claim 10 wherein the ceramic contains calcium phosphate or calcium sulfate.
[12" claim-type="Currently amended] 12. The method of claim 11, wherein the calcium phosphate is amorphous calcium phosphate, weakly crystalline hydroxyapatite, nanocrystalline hydroxyapatite, stoichiometric hydroxyapatite, calcium deficient hydroxyapatite, substituted And hydroxyapatite, tricalcium phosphate, tetracalcium phosphate, dicalcium phosphate dihydrate, monocalcium phosphate.
[13" claim-type="Currently amended] 4. The composition of claim 3, wherein the matrix is a non-naturally resorbable polymer or derivative thereof.
[14" claim-type="Currently amended] 4. The polymer of claim 3 wherein the polymer is poly-lactic acid, poly-glycolic acid, a copolymer of poly-lactic acid and poly-glycolic acid (PLGA), polydextran, polyester, polyvinyl alcohol, tyrosine polycarbonate, tyrosine polyarray , Poly-orthoesters, polylactides, polyglycolides, polyethers, poly-fumarate polyesters, copolymers thereof.
[15" claim-type="Currently amended] The composition of claim 1, wherein the growth factor is selected from osteogenic factors, angiogenesis factors, angiogenic factors, and compounds thereof.
[16" claim-type="Currently amended] The composition of claim 15, wherein the osteogenic factor is selected from BMP, TGF, IGF, MCSF, statins, GSF.
[17" claim-type="Currently amended] An implantable bone growth inducing composition, wherein the composition is composed of:
Micronized ceramic;
Growth factors associated with ceramics;
Resorbable or biodegradable polymers;
Here, the micronized ceramic is dispersed in the polymer, and the osteoinductance of the composition is higher than that of the composition consisting of only the micronized ceramic and growth factors bound thereto.
[18" claim-type="Currently amended] 18. The composition of claim 17, wherein the polymer is selected from polysaccharides, lipids, resorbable polymers, resorbable plastics, derivatives thereof, compounds thereof.
[19" claim-type="Currently amended] 18. The composition of claim 17, wherein the ceramic is calcium phosphate ceramic.
[20" claim-type="Currently amended] 18. The composition of claim 17, wherein the polymer is starch.
[21" claim-type="Currently amended] 21. The composition of claim 20, further comprising a lipid.
[22" claim-type="Currently amended] A desalted bone matrix (DBM) composition consisting of desalted bone matrix (DBM) and stabilizers.
[23" claim-type="Currently amended] The composition of claim 22, wherein the DBM comprises particles of at least 1 mm maximum dimension.
[24" claim-type="Currently amended] The composition of claim 22, wherein the DBM comprises particles of at least 1.5 mm maximum dimension.
[25" claim-type="Currently amended] The composition of claim 22, wherein the DBM comprises particles of at least 2 mm maximum dimension.
[26" claim-type="Currently amended] 23. The composition of claim 22, wherein the DBM composition comprises particles, wherein the particles are tapered, wedge or conical in shape and have a maximum dimension of at least 1 mm and another dimension of 100 microns.
[27" claim-type="Currently amended] The composition of claim 22, wherein the stabilizer is selected from diffusion barriers, enzyme inhibitors, competition substrates, masking components, compounds thereof.
[28" claim-type="Currently amended] The method of claim 22, wherein the stabilizer is selected from natural polymers, non-natural polymers, modified or derived natural polymers, modified or derived non-natural polymers, compounds thereof, and the matrix is at least partially encapsulated in the stabilizer. The composition characterized in that.
[29" claim-type="Currently amended] 29. The composition of claim 28, wherein the natural polymer is selected from lipids, polysaccharides, compounds thereof.
[30" claim-type="Currently amended] 30. The composition of claim 29, wherein the lipid is a fatty acid.
[31" claim-type="Currently amended] 30. The composition of claim 29, wherein the polysaccharide is starch.
[32" claim-type="Currently amended] 29. The composition of claim 28, wherein the stabilizer is resorbable or biodegradable.
[33" claim-type="Currently amended] The composition of claim 22 wherein the stabilizing agent is selected from protease inhibitors, glycosidase inhibitors, compounds thereof.
[34" claim-type="Currently amended] The composition of claim 22, wherein the competition substrate is selected from polypeptides, poly-amino acids, polysaccharides, compounds thereof, derivatives thereof.
[35" claim-type="Currently amended] 23. The composition of claim 22 wherein the stabilizing agent is a masking agent selected from lectins, antibodies, growth factor binding proteins, derivatives or compounds thereof.
[36" claim-type="Currently amended] The method of claim 28, wherein the non-natural polymer is selected from the group consisting of poly-lactic acid, poly-glycolic acid, copolymers of poly-lactic acid and poly-glycolic acid (PLGA), polydextran, polyester, polyvinyl alcohol, tyrosine polycarbonate, A composition characterized in that it is a resorbable polymer selected from tyrosine polyarylate, poly-orthoester, polylactide, polyglycolide, polyether, poly-fumarate polyester, copolymers thereof.
[37" claim-type="Currently amended] The method of claim 22, wherein the stabilizing agent is aprotinin, 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF), amastatin-HCl, alpha1-antichymotrypsin, antithrombin III, alpha1-antitrypsin, 4-aminophenylmethane sulfonyl-fluoride (APMSF), Arfamenin A, Arfamenin B, E-64, Vestatin, CA-074, CA-074-Me, Calpine Inhibitor I, Calpine Inhibitor II, Catephsin inhibitors, chymostatin, diisopropylfluorophosphate (DFP), dipeptidyl peptidase IV inhibitors, diproteins A, E-64c, E-64d, E-64, eblactone A, eblactone B , EGTA, elastinal, poroxymitin, hirudin, reuhistin, leupeptin, alpha2-macroglobulin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A, fevestin, 1,10-phenanthroline , Phosphoramidone, chymostatin, benzamidine HCl, antipine, epsilon-aminocaproic acid, N-ethylmaleimide, trypsin inhibitor, 1-chloro-3-tosylamido-7-ami No-2-heptanone (TLCK), 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin inhibitor, sodium EDTA, composition thereof .
[38" claim-type="Currently amended] A desalted bone matrix (DBM) composition consisting of desalted bone matrix (DBM) and a diffusion barrier.
[39" claim-type="Currently amended] The composition of claim 38, wherein the DBM comprises particles of at least 1 mm maximum dimension.
[40" claim-type="Currently amended] The composition of claim 38, wherein the DBM comprises particles of at least 1.5 mm maximum dimension.
[41" claim-type="Currently amended] The composition of claim 38, wherein the DBM comprises particles of at least 2 mm maximum dimension.
[42" claim-type="Currently amended] 39. The composition of claim 38, wherein the DBM composition comprises particles, wherein the particles are tapered, wedge or conical in shape and have a maximum dimension of at least 1 mm and another dimension of 100 microns.
[43" claim-type="Currently amended] 39. The diffusion barrier of claim 38, wherein the diffusion barrier is selected from natural polymers, non-natural polymers, modified or derived natural polymers, modified or derived non-natural polymers, compounds thereof, and the DBM is at least partially embedded in the diffusion barrier. A composition, characterized in that.
[44" claim-type="Currently amended] The composition of claim 43, wherein the natural polymer is selected from lipids, polysaccharides, compounds thereof.
[45" claim-type="Currently amended] 45. The composition of claim 44, wherein the lipid is a fatty acid.
[46" claim-type="Currently amended] 45. The composition of claim 44, wherein the polysaccharide is starch.
[47" claim-type="Currently amended] 45. The composition of claim 44, wherein the stabilizer is resorbable or biodegradable.
[48" claim-type="Currently amended] A demineralized bone matrix composition consisting of demineralized bone matrix (DBM) and related excipients, wherein the demineralized bone matrix composition is at least 10% higher in bone induction than a composition without DBM alone.
[49" claim-type="Currently amended] 49. The composition of claim 48, wherein the DBM comprises particles of at least 1 mm maximum dimension.
[50" claim-type="Currently amended] 49. The composition of claim 48, wherein the DBM comprises particles of at least 1.5 mm maximum dimension.
[51" claim-type="Currently amended] 49. The composition of claim 48, wherein the DBM comprises particles of at least 2 mm maximum dimension.
[52" claim-type="Currently amended] 49. The composition of claim 48, wherein the DBM composition comprises particles, wherein the particles are tapered, wedge or conical in shape and have a maximum dimension of at least 1 mm and another dimension of 100 microns.
[53" claim-type="Currently amended] 49. The composition of claim 48, wherein the composition has at least 20% higher osteoinduction than a composition without DBM alone.
[54" claim-type="Currently amended] 49. The composition of claim 48, wherein the composition has at least 35% higher osteoinduction than a composition without DBM alone.
[55" claim-type="Currently amended] 49. The composition of claim 48, wherein the excipient is selected from diffusion barriers, enzyme inhibitors, competition substrates, masking components, compounds thereof.
[56" claim-type="Currently amended] 49. The excipient according to claim 48, wherein the excipient is selected from natural polymers, non-natural polymers, modified or derived natural polymers, modified or derived non-natural polymers, compounds thereof, and the matrix is at least partially embedded in the stabilizer. A composition, characterized in that.
[57" claim-type="Currently amended] The composition of claim 56 wherein the excipient is selected from lipids, polysaccharides, compounds thereof.
[58" claim-type="Currently amended] 59. The composition of claim 56, wherein the excipient is a lipid / polysaccharide compound.
[59" claim-type="Currently amended] 59. The composition of claim 58, wherein the lipid is phosphatidylcholine and the starch is amylase resistant starch.
[60" claim-type="Currently amended] 49. The composition of claim 48, wherein osteogenicity is measured in the thymus space of the athymic rat hindlimb muscle or rabbit at least 7 days after transplantation.
[61" claim-type="Currently amended] 61. The composition of claim 60, wherein the osteoinductive property is measured at least 14 days after transplantation.
[62" claim-type="Currently amended] 61. The composition of claim 60, wherein the osteoinductance is measured at least 21 days after transplantation.
[63" claim-type="Currently amended] 63. The composition of claim 60, wherein the osteoinductive property is measured at least 28 days after transplantation.
[64" claim-type="Currently amended] 61. The composition of claim 60, wherein the osteoinductive property is measured at least 40 days after transplantation.
[65" claim-type="Currently amended] 61. The composition of claim 60, wherein the osteoinductance is measured at least 60 days after transplantation.
[66" claim-type="Currently amended] A DBM composition consisting of DBM and excipients, wherein the composition retains at least 25% of the osteoinductive properties of the 10 μg BMP-collagen sponge formulation.
[67" claim-type="Currently amended] 67. The composition of claim 66, wherein the DBM comprises particles of at least 1 mm maximum dimension.
[68" claim-type="Currently amended] 67. The composition of claim 66, wherein the DBM comprises particles of at least 1.5 mm maximum dimension.
[69" claim-type="Currently amended] 67. The composition of claim 66, wherein the DBM comprises particles of at least 2 mm maximum dimension.
[70" claim-type="Currently amended] 67. The composition of claim 66, wherein the DBM composition comprises particles, wherein the particles are tapered, wedge or conical in shape and have a maximum dimension of at least 1 mm and another dimension of 100 microns.
[71" claim-type="Currently amended] 67. The composition of claim 66, having at least 50% of osteoinductive properties of the 10 μg BMP-collagen sponge formulation.
[72" claim-type="Currently amended] 67. The composition of claim 66, having at least 75% of osteoinductive properties of the 10 μg BMP-collagen sponge formulation.
[73" claim-type="Currently amended] 67. The composition of claim 66, having at least 90% of osteoinductive properties of the 10 μg BMP-collagen sponge formulation.
[74" claim-type="Currently amended] 67. The composition of claim 66, wherein the excipient is selected from diffusion barriers, enzyme inhibitors, competition substrates, masking components, compounds thereof.
[75" claim-type="Currently amended] 67. The method of claim 66, wherein the excipient is selected from natural polymers, non-natural polymers, modified or derived natural polymers, modified or derived non-natural polymers, compounds thereof, and the matrix is at least partially embedded in the stabilizer. A composition, characterized in that.
[76" claim-type="Currently amended] 76. The composition of claim 75, wherein the excipient is selected from lipids, polysaccharides, compounds thereof.
[77" claim-type="Currently amended] 76. The composition of claim 75, wherein the excipient is a lipid / polysaccharide compound.
[78" claim-type="Currently amended] 78. The composition of claim 77, wherein the lipid is phosphatidylcholine and the starch is amylase resistant starch.
[79" claim-type="Currently amended] 67. The composition of claim 66, wherein the osteoinductance is measured in the thymus space of the athymic rat hindlimb muscle or rabbit at least 7 days after transplantation.
[80" claim-type="Currently amended] 80. The composition of claim 79, wherein the osteoinductance is measured at least 14 days after transplantation.
[81" claim-type="Currently amended] 80. The composition of claim 79, wherein the osteoinductance is measured at least 21 days after transplantation.
[82" claim-type="Currently amended] 80. The composition of claim 79, wherein the osteoinductive property is measured at least 28 days after transplantation.
[83" claim-type="Currently amended] 80. The composition of claim 79, wherein the osteoinductive property is measured at least 40 days after transplantation.
[84" claim-type="Currently amended] 80. The composition of claim 79, wherein the osteoinductance is measured at least 60 days after transplantation.
[85" claim-type="Currently amended] 23. The composition of claim 22, wherein the stabilizer is a tertiary structure disrupting agent.
[86" claim-type="Currently amended] 86. The composition of claim 85, wherein the tertiary structure splitting agent is selected from alkylating agents and sulfidyl modifiers.
[87" claim-type="Currently amended] 86. The composition of claim 85, wherein the tertiary structure splitting agent is selected from guanidine hydrochloride, dithiothreitol, acetic iodide, methyl iodide, alkyl iodide.
[88" claim-type="Currently amended] A process for preparing a desalted bone matrix composition, comprising the following steps:
Providing a DBM;
Providing a stabilizer;
The DBM is contacted with a stabilizer to produce a safer DBM composition in vivo.
[89" claim-type="Currently amended] 89. The method of claim 88, wherein providing the DBM consists of treating the DBM with a protease inhibitor.
[90" claim-type="Currently amended] A drug delivery device comprising: a drug delivery device, comprising:
Desalted bone matrix;
Bioactive agents that are delivered and absorbed into the matrix;
Stabilizer.
[91" claim-type="Currently amended] 93. A drug delivery device according to claim 90, wherein the bioactive agent is an osteoinductive factor.
[92" claim-type="Currently amended] 91. The drug delivery device according to claim 90, wherein the bioactive agent is selected from bone morphogenetic protein, TGF-β, IGF.
[93" claim-type="Currently amended] 91. The drug delivery device according to claim 90, wherein the bioactive agent is a bone forming protein.
[94" claim-type="Currently amended] 91. The drug delivery device of claim 90, wherein the bioactive agent covalently binds to the matrix.
[95" claim-type="Currently amended] 91. The drug delivery device of claim 90, wherein the bioactive agent non-covalently binds to the matrix.
[96" claim-type="Currently amended] 91. The drug delivery device of claim 90, wherein the bioactive agent is selected from small molecules, chemical compounds, cells, polynucleotides, proteins, peptides, drugs, viruses.
[97" claim-type="Currently amended] 93. A drug delivery device according to claim 90, wherein the bioactive agent is selected from antibiotics, anti-neoplastic agents, growth factors, hematopoietic factors, wound healing factors, nutrients.
[98" claim-type="Currently amended] In bone-inducing compositions for transplantation to bone defect sites, bone-induced desalted bone matrices are contained in hydrated polysaccharide carriers, and the type and content of polysaccharides present in the carriers are fluid ) And sufficient to provide at least one osteoinductive ability in athymic rat model analysis.
[99" claim-type="Currently amended] 99. The osteoinductive composition of claim 98, wherein the polysaccharide is selected from starch and cellulose.
[100" claim-type="Currently amended] 99. The osteoinductive composition of claim 98, wherein the starch is selected from corn starch, wheat starch, potato starch, rice starch, compounds thereof.
[101" claim-type="Currently amended] 99. The osteoinductive composition of claim 98, wherein the cellulose is at a level of 0.5% to <3.0% methyl cellulose per weight of the osteoinductive composition.
[102" claim-type="Currently amended] Implants consisting of bone derived particles and starch or starch mixtures.
[103" claim-type="Currently amended] Implants consisting of calcium phosphate particles, starch or starch mixture, and biologically active factors.
[104" claim-type="Currently amended] A composition consisting of desalted bone matrix (DBM) and a stabilizer, wherein the stabilizer is characterized by extending the half-life of DBM activity in vivo.
[105" claim-type="Currently amended] A composition consisting of a DBM and an excipient, wherein the excipient delays the release rate of the DBM or extends the osteoinductive life.

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同族专利:

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公开号 | 公开日

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WO2003030956A2|2003-04-17|

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EP2295088A1|2011-03-16|

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AU2008255147B2|2011-04-28|

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EP1434608A2|2004-07-07|

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WO2003030956A3|2003-11-06|

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US20080145392A1|2008-06-19|

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US7163691B2|2007-01-16|

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US20030143258A1|2003-07-31|

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JP2005505351A|2005-02-24|

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AU2008255147A1|2009-01-08|

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CA2433038A1|2003-04-17|

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EP2295088B1|2016-12-07|

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US7959941B2|2011-06-14|

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EP1434608B1|2018-08-22|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-10-12|Priority to US32915601P

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2001-10-12|Priority to US60/329,156

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2002-06-27|Priority to US39246202P

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2002-06-27|Priority to US60/392,462

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2002-10-15|Application filed by 오스테오테크, 인코포레이티드

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2002-10-15|Priority to PCT/US2002/032941

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2004-06-05|Publication of KR20040047746A

优先权:

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申请号 | 申请日 | 专利标题

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US32915601P| true| 2001-10-12|2001-10-12|

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US60/329,156|2001-10-12|

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US39246202P| true| 2002-06-27|2002-06-27|

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US60/392,462|2002-06-27|

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PCT/US2002/032941|WO2003030956A2|2001-10-12|2002-10-15|Improved bone graft|