Bone graft compositions and methods

Abstract

Disclosed herein are various bone allografts that maintain a high percentage of native growth factors and / or cytokines and methods of making them. For example, disclosed is a method of producing a bone graft material that involves harvesting bone having bone marrow from a donor; separating the harvested bone into cancellous and cortical bone; optionally shaping the cortical bone and / or cancellous bone; demineralizing the cortical bone and optionally the cancellous bone with a demineralizing agent to produce a cortical bone with 0 to 99.9% mineral content compared to the original cortical bone and optionally a cancellous bone with 0 to 99.9% mineral content compared to the original cancellous bone; and washing the demineralized cortical bone and / or cancellous bone.

Description

BONE GRAFT COMPOSITIONS AND METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 638,672, filed April 25, 2025, which is hereby incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Demineralized bone allografts have been used to encourage bone growth in various surgical settings including spinal fusion and dental applications. Though they provide an osteoconductive environment to encourage bone forming cell binding, they are generally known to have only low levels of osteoinductive cell signals, i.e. , growth factors and I or cytokines, due to the harsh conditions associated with tissue demineralization. SUMMARY OF THE INVENTION

[0003] Disclosed herein are various bone allografts that maintain a high percentage of native growth factors and / or cytokines and methods of making them.

[0004] For example, disclosed is a method of producing a bone graft material, the method involving harvesting bone having bone marrow from a donor; separating the harvested bone into cancellous and cortical bone; optionally shaping the cortical bone and / or cancellous bone; demineralizing the cortical bone and optionally the cancellous bone with a demineralizing agent to produce a cortical bone with 0 to 99.9% mineral content compared to the original cortical bone and optionally a cancellous bone with 0 to 99.9% mineral content compared to the original cancellous bone; and washing the demineralized cortical bone and / or cancellous bone.

[0005] As can be appreciated by the evidence disclosed herein, the percentage of demineralization affects the amount of growth factors retained in the bone graft material. Therefore, in some embodiments, the methods involve demineralizing the cortical bone and with a demineralizing agent to produce a cortical bone with 0.1 to 99.9% mineral content compared to the original cortical bone, including 1 to 99%, 10 to 95%, 20 to 90%, 30 to 90%, 40 to 90%, 50 to 60%, 50 to 65%, 50 to 70%, 50 to 75%, 50 to 80%, 50 to 85%, 50 to 90%, or 50 to 95%. Therefore, in some embodiments, the methods involve demineralizing the cortical bone and with a demineralizing agent to produce a cortical bone with at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% mineral content and no more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% mineral content. In particular embodiments, the methods involve demineralizing the cortical bone and with a demineralizing agent to produce a cortical bone with at least 50% mineral content for optimal handling properties.

[0006] In some embodiments the demineralizing agent is any combination of the agents disclosed herein. For example, in some embodiments the demineralizing agent is 0.1 to 100% acetic acid, including 0.1 to 15%, 0.1 to 64%, 1 to 15, 1 to 64% , 2.7 to 15%, or 2.7 to 64% acetic acid, such as, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 61 , 62, 64, 65, -% acetic acid, assuming a 14:1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0007] In some embodiments the demineralizing agent is 0.01 to 5% nitric acid, such as-0.01 to 1.5%, 0.01 to 3%, 0.05 to 1.5%, 0.05 to 3%. 0.4 to 1.5%, and 0.4 to 3% nitric acid, such as 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, -% nitric acid, assuming a 14: 1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0008] In some embodiments the demineralizing agent is 0.05 to 45% propanoic acid, including 0.05 to 7%, 0.05 to 15%, 0.2 to 7%, 0.2 to 15%, 0.5 to 7%, and 0.5 to 15% propanoic acid such as 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2,3 ,4, 5, 6, 7, 8, 9,10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 , assuming a 14:1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0009] In some embodiments the demineralizing agent is 0.03 to 30% formic acid, including 0.03 to 2%, 0.03 to 9%, 0.3 to 2%, 0.3 to 9%, 0.6 to 2%, and 0.6 to 9%formic acid, such as 0.03, 0.04, 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30% formic acid, assuming a 14: 1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0010] In some embodiments the demineralizing agent is 0.1 to 40% glycolic acid, including 0.1 to 7%, 0.1 to 25%, 0.4 to 7%, 0.4 to 25%, 1.1 to 7%, 1.1 to 25%, glycolic acid, such as, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, % glycolic acid, assuming a 14:1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0011] In some embodiments the demineralizing agent is 0.6 to 15% ethylenediaminetetraacetic Acid (EDTA), including 0 0.6 to 3%, 0.6 to 9%, 1.3 to 3%, 1 .3 to 9%, 2.3 to 3%, 2.3 to 9%EDTA, including 00.6, 0.7, 0.8, 0.9,1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12, 13, 14, 15%, assuming a 14:1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0012] In some embodiments the demineralizing agent is 0.2 to 25% Hydroxyethylethylenediaminetriacetic acid (HEDTA), including 0 0.6 to 3%, 0.6 to 9%, 1 .3 to 3%, 1.3 to 9%, 2.3 to 3%, 2.3 to 9% HEDTA, such as 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25% HEDTA, assuming a 14:1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0013] In some embodiments the demineralizing agent is 0.03 to 40% ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), including 0.03 to 19%, 0.03 to 38%, 0.2 to 19%, 0.2 to 38%, 0.4 to 19%, 0.4 to 38% EGTA, including 0.03, 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38 or 1M EGTA, assuming a 14: 1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0014] In some embodiments the demineralizing agent is 0.3 to 40% 1 ,3-Diamino-2- hydroxypropane-N,N,N',N'-tetraacetic acid (DPT A), including 0.3 to 20%, 0.3 to 40%, 2.5 to 20%, 2.5 to 40%,DPTA, including 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38 DPTA, assuming a 14:1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0015] In some embodiments the bone is shaped into a particle, strut, fiber, wedge, block, or disc. For example, the particle can be 0.03 to 0.3mm in diameter, including 0.1 to 1 , 1 to 5, 2 to 5, or 3 to 5 mm in diameter, such as , 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, mm in diameter, assuming a 14:1 RT ratio. One of ordinary skill in the art can adjust these concentrations to adapt to alternative ratios.

[0016] In some embodiments the strut, wedge, or block is 0.1 mm to 20cm in any dimension, including 0.1 to 10, 1 to 20, 1 to 10, 5 to 20cm in any dimension, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20cm in any dimension. In some embodiments the fibers are 0.06 to 0.2 mm in diameter, such as 0.06, 0.07, 0.08, 0.09, 0.1 , 0.20.02 to 1 mm in length, including 0.1 to 10, 1 to 20, 1 to 10, 5 to 20cm in length, such as 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, 0.4, 0.5, 1 mm in length. In some embodiments the disc is 0.1 mm to 10mm thick and 0.1 mm to 100mm in diameter, including 0.1 to 1 , 1 to 5, and 5 to 10mm thick and 0.1 to 50, 1 to 100, 10 to 50, and 10 to 100mm in diameter, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 mm thick and 0.1 , 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100mm in diameter.

[0017] In some embodiments the treatment time with the demineralization agent is 1 minute to 3 days, including 1 , 2, 3 days. In some embodiments the number of treatments with the demineralization agent is 1 to 3 times, including 1 , 2, 3 times.

[0018] In some embodiments the demineralization agent volume during treatment is 1 :1 to 1 :50 (weight tissue : volume reagent), including 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1:10, 1 :11 , 1 :12, 1 :13, 1 :14, 1 :15, 1 :16, 1 : 17, 1 :18, 1 :19, 1 :20 1 : 21 , 1:22, 1 :23, 1 : 24, 1 :25, 1 :26, 1 :27,1 :28, 1 :29, 1 :30, 1 : 31 , 1 :32, 1 :33, 1 :34, 1 : 35, 1 :36, 1 :37, 1 : 38, 1 :39, 1 :40, 1 :41 , 1 :42, 1 :43, 1 :44, 1 :45, 1 :46, 1 :47, 1 :48, 1 :49 or 1 :50.

[0019] In some embodiments the temperature during treatment is 1 °C to 60°C, including 1 to 20, 10 to 40, 20 to 60, or 10 to 60°C, such as 4°C or room temperature.

[0020] In some embodiments the donor is a mammal, such as a human donor.

[0021] Also disclosed herein is a bone graft material produced according to a method disclosed herein.

[0022] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0023] FIG. 1 shows a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by Glycolic Acid with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0024] FIG 2 is a summary % of demineralization and Growth Factor sum of cortical powders demineralized by Glycolic Acid with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0025] FIG. 3 is a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by Acetic Acid with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0026] FIG. 4 is a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by Nitric Acid with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times /

[0027] FIG. 5 is a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by Formic Acid with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0028] FIG. 6 is a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by Propionic Acid with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0029] FIG. 7 is a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by EGTA with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0030] FIG. 8 is a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by DTPAwith different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0031] FIG. 9 is a summary % of demineralization and Growth Factor sum of cortical fibers demineralized by HEDTA with different concentrations and reagent (mL) / tissue (g) ratios under different incubation times.

[0032] FIG. 10 shows % of demineralization of cortical fibers demineralized by EDTA with different concentrations under different incubation times.

[0033] FIG. 11 shows growth factor remaining for cortical fibers demineralize to a certain level, i.e. 20, 30, 50, 70, 90%, by different reagent, i.e. Glycolic Acid, Nitric Acid, Formic Acid, Propanoic Acid, EGTA, DTPA, HEDTA.DETAILED DESCRIPTION

[0034] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0035] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0037] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which thepublications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0038] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0039] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0040] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0041] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0042] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0043] The term “growth factor” and “cytokine” may refer to the following: Activin A, aFGF, Angiogenin, bFGF, BMP-2, BMP-5, BMP-6, BMP-7, BMP-9, EGF, ENA-78, G-CSF, HB-EGF, HGF, IGF-1 , IGF-2, IL-1b, IL-1 ra, IL-8, OPG, OPN, PDGF-BB, SDF-1a, TGFbl ,TGFb2, TGFb3, TNFa, VEGF, VEGF-C, VEGF-D, IL-10Methods

[0044] As summarized above, methods of producing bone graft materials are provided. An embodiment of the disclosed method is illustrated in Figure 1. Referring to this figure, the method can first involve harvesting bone having bone marrow from a donor and then separating the harvested bone into cancellous and cortical bone. The bone can be demineralized with a demineralizing agent to produce a demineralized bone with 0 to 99.9% mineral content compared to the original bone. The demineralized bone can then be washed. The demineralized bone can then be optionally lyophilized.

[0045] Initial bone sources that may be employed in methods of the invention include autologous, allogeneic, or xenogeneic bone sources. Of interest in certain embodiments are allogeneic bone sources, e.g., mammalian bone sources, including primate bone sources, e.g., human bone sources. In preferred embodiment, the harvested material can be harvested in such a way as to retain as much bone marrow in the harvested sample as possible. The initial bone source may be obtained from the shaft of long bones or from flat bone structures and preferably contain bone marrow. The initial bone source may be raw bone, frozen bone or lyophilized (i.e., freeze dried) bone. In some instances, the initial bone source is cadaver bone, such as human cadaver bone. The harvested bone can then be separated into cancellous and cortical bone.Bone Graft Materials

[0046] Aspects of the invention further include bone graft materials produced by methods of the invention. The bone graft materials may be osteoconductive and / osteoinductive. As used herein “osteoconductive” refers to the ability to provide a scaffold for which bone cells can attach to initiate bone growth. As used herein, “osteoinductive” refers to the ability to induce bone growth. In certain instances, the subject bone graft materials are bioresorbable. Bioresorbable compositions of the invention can be broken down and assimilated in a subject (e.g., a human subject) after a period of time. As such, bioresorbable products allow for a temporary scaffold fortissue growth (e.g., hard tissue) or for the delivery of an agent in a subject (e.g., an agent that promotes bone growth). In certain instances, the bone graft compositions remain in a subject for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 weeks or less following implantation. In certain instances, the bone graft compositions remain in a subject for longer period of time, e.g., 6 months, 1 year, 2 years, 5 years or longer.

[0047] Where desired, the bone graft materials may include one or more osteoinductive factors. Osteoinductive factors that may be present in the compositions include but are not limited to: bone morphogenetic growth factors (BMPs, e.g., BMP-2 and BMP-7), IGF, TGF-p, IGFs (IGF-1 and IGF-2), parathyroid hormone, angiogenic factors (VEGF, aFGF, bFGF), mitogenic factors, osteocalcin and osteopontin.

[0048] In some instances, the bone graft materials include one or more agents that aid in the repair of a bone defect. In certain instances, the agent(s) present in the composition is an agent not found in naturally occurring bone. Exemplary agents include, but are not limited to, antiviral drugs, antibiotics, antimicrobial drugs, growth factors (e.g., osteoinductive or osteogenic factors), and immunosuppresants.

[0049] In certain embodiments, the bone graft materials include are radiographic. As used herein, the term “radiographic” refers to the ability to be visualized by radiography. As such, the compositions may be visualized by X-ray radiography following implantation in the subject, e.g., by standard radiography techniques. In certain embodiments, the radiodensity of the graft composition ranges from +30 to +5000 Hounsfield units (HU), including between +100 to +3,000 HU. As used herein, “radiodensity” refers to the relatively inability of electromagnetic radiation (e.g., X-ray) to pass through a particular material, as measured in HU.

[0050] In some instances, the bone graft materials are flowable compositions. The term “flowable” is meant to include putty and paste-like compositions, as well as more liquid compositions. “Flowable” as used herein therefore refers to both injectable and compactable compositions. In certain embodiments, the viscosity time of the flowable compositions, defined as time periods under which the mixed composition injects through a standard Luer- lok fitting after mixing, ranges up to 10 minutes or longer, such as up to about 7 minutes or longer, and including up to about 4 minutes or longer. Of interest in certain embodiments are paste compositions that have an injectable viscosity that injects in a time period ranging up to about 5 minutes, such as about up to about 4 minutes.

[0051] Where desired, the bone graft material produced, e.g., as described above, may be sterilized. In various embodiments, the bone graft material is sterilized by radiation in a sterilization step, e.g., before and / or after it is placed into any desired packaging, such as sterile packaging. In other embodiments, the material is not sterilized by radiation. In various embodiments, gamma radiation is used in the terminal sterilization step, which involves utilizing ionizing energy from gamma rays that penetrates deeply in material. Gamma rays are highly effective in killing microorganisms, they leave no residues nor have sufficient energy to impart radioactivity to the device. Gamma rays can be employed when the material is present a package and gamma sterilization does not require high pressures or vacuum conditions, thus, package seals and other components are not stressed. In addition, gamma radiation eliminates the need for permeable packaging materials. In various embodiments, electron beam (e-beam) radiation may be used to sterilize the material. E- beam radiation comprises a form of ionizing energy, which is generally characterized by low penetration and high-dose rates. E-beam irradiation is similar to gamma processing in that it alters various chemical and molecular bonds on contact, including the reproductive cells ofmicroorganisms. Beams produced for e-beam sterilization are concentrated, highly-charged streams of electrons generated by the acceleration and conversion of electricity. Other methods may also be used to sterilize the device and / or one or more components of the device, including, but not limited to, gas sterilization, such as, for example, with ethylene oxide or steam sterilization.Utility

[0052] The bone graft materials, e.g., as described above, find use in a variety of applications. The bone graft materials, e.g., as described above, are useful, for example, in instances where bone growth is desired, for example, for repair of a bone defect, for bone replacement, for bone augmentation, etc. The bone graft materials, e.g., as described above, find use in applications where it is desired to introduce an implantable composition having osteoconductive, osteoinductive and / or osteogenic properties into a physiological site of interest, such as in orthopedic applications.

[0053] In orthopedic applications, the bone grafter material may be prepared, as described above, and introduced to a site where bone growth is desired (e.g., bone repair site), including sites containing cancellous and / or cortical bone. In certain embodiments, the bone graft materials are used for the treatment of a subject having a bone disease or disorder. The bone graft materials can be applied at a desired physiological site using any suitable applicator, e.g., syringe, cannula, catheter, spatula, etc., depending on the viscosity of the composition.

[0054] Subjects that can be treated using the subject composition include mammalian subjects, e.g., primates, including humans. In some embodiments, the bone graft materials are used for the treatment of a bone fracture, bone cancer, bone metastases, an autoimmune disease, a metabolic disease, rheumatoid arthritis, or a degenerative bone disease. In some instances, the bone graft materials are used for the repair of a fracture. In other instances, the bone graft materials are used for implant augmentation. In yet other instances, the bone graft materials are used for the replacement of a bone.

[0055] Fractures that can be treated with the subject bone graft materials include simple fractures, compound fractures and non-unions. In such fracture treatment methodologies, the fracture is first reduced. Following fracture reduction, a subject composition is introduced into the cancellous tissue in the fracture region using an application (e.g., a spatula or cannula). Bones that are suitable for repair and replacement using the subject compositions include, but are not limited to: calcaneus, carpal, cervical vertebra, clavicle, ethmoid, femur, fibula, frontal, humerus, ilium, ischium, lumbar vertebra, mandible, maxilla, metacarpal, metatarsal, nasal, occipital, parietal, patella, phalanges, pubis, radius, rib, sacrum, scapula, sternum, tarsal, temporal, thoracic vertebra, tibia, ulna, and zygomatic bones.

[0056] Bone graft materials of the invention are useful in spinal applications including restoration of column support. The bone graft materials are useful for implantation inpatients suffering from defects caused by congenital anomaly, disease, or trauma, including for example, spine fractures; deformity, e.g., kyphotic deformities, e.g., posttraumatic kyphosis; postlaminectomy kyphosis, junctional kyphosis, and Scheuermann's kyphosis; scoliosis, e.g., neuromuscular scoliosis, adult scoliosis, paralytic scoliosis, congenital and syndromic scoliosis; and cervical neck pain. Surgical methods for correcting degenerative conditions, for example in the lumbar spine, include decompression (excision of disc material, hypertrophied bone, or ligament) along with fusion, or fusion alone.

[0057] Where desired, a posterior surgical approach may be employed. The choice of approach is dictated by the site of primary pathology. Pathology that involves vertebral bodies may be approached anteriorly through the thorax, abdomen or flank. Pathology involving posterior elements are best approached posteriorly for example, through a vertical midline approach or posterior lateral muscle spinning approach.

[0058] Those of ordinary skill in the art to which the present invention pertain, including for example an orthopedic surgeon and a spinal surgeon, can readily select and employ a particular bone graft material of the invention, without undue experimentation. An ideal graft, for example for use in lumbar interbody fusion, should be: osteoinductive, non- immunogenic, provide immediate mechanical stability, and be appropriately sized and shaped for the particular application / patient. Indications, diagnostic criteria, graft selection and surgical technique, are factors that can be readily selected, optimized and employed by those of ordinary skill in the art without undue experimentation, and are discussed in: Master Techniques in Orthopaedic Surgery, The Spine, edited by Bradford, David S., Lippincott- Raven, ISBN 0-7817-0033-7, Philadelphia, Pa., (1997), hereby incorporated herein by reference in its entirety. When implanting a cervical fusion graft, an anterior cervical approach may be employed.

[0059] Where desired, compositions that are implanted in a subject may be imaged, e.g., using any suitable radiography technique to obtain an image of the implanted composition. In certain instances, images of the composition are produced by passing a radiation source (e.g., an X-ray generator) over a subject at the site where the composition has been implanted and capturing the radiation that passes through the subject onto a recording medium. In certain embodiments, the recording medium is film. In other embodiments, the recording medium is a digital recording medium (e.g. a digital detector). In some embodiments, the X-ray generator and recording medium is capable of measuring bone mineral density at the site of implantation of the subject composition. Exemplary systems for obtaining X-ray images are described in U.S. Pat. Nos. 5,204,888; 6,285,740;6,320,931 ; 6,325,537; 6,666,579; and 7,672,432, the disclosure of which is herein incorporated by reference.Kits

[0060] Also provided are kits that include a bone graft material, e.g., as described above. In addition to the bone graft materials, the subject kits may further include a number of additional components, e.g., applicators, such as described above. The component(s) of the kit may be present in sterile packaging, e.g., a sterile pouch, as desired. The various components may be present in the same or different containers, e.g., sterile packaging.

[0061] In addition to above-mentioned components, the subject kits may include instructions for using the components of the kit to practice the subject methods. The instructional material may also be instructional material for using the bone graft materials, e.g., it may provide surgical techniques and protocols for a particular application in which the bone graft material is to be employed. The instructions for practicing the subject methods may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0062] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1:MethodDemineralization of Tissue Samples

[0063] Weigh 0.5-1 gram of dry bone fibers, or powders which were milled by Motors mill with 250um sieve (S55PZE-7831 , US Motors), in 50 mL tube and record the masses of both tube and tissue. Add DI water (mL,10 x mass of dry tissue (g)) into the tube, wait for 10 minutes, vortex, and then centrifuge each tube at 4700 rpm for 5 minutes at room temperature (RT). Aspirate and discard the supernatant.

[0064] Calculate the volume of demineralization reagents, e.g. DI Water, 0.6 N HCL, Glycolic Acid, Formic Acid, Nitric Acid, Propanoic Acid, EDTA, EGTA, DTPA, HEGTA, to be added to each tissue in 14 or 42 hydrated tissue (g) / reagent (mL) ratios, where mass of hydrated tissue = 1 .2* mass of dry tissue. Table 1 shows demineralized reagents and their concentrations. All the chelators were dissolved at neutral pH.

[0065] Add the calculated volume of demineralization reagents, seal the cap and place each tube on a shaker at 100 rpm under 4 °C for certain periods of times: 5 minutes, 20 minutes, 2 hours, and 16 hours. Then centrifuge each tube at 4700 rpm for 5 minutes at RT. Decant the supernatant, i.e. demineralized solution (DMS 1).

[0066] The demineralized bone tissue will be washed by adding 10 to 15 mL of DI water into each tube. Vortex and invert each tube occasionally and then centrifuge each tube at 4700 rpm for 5 minutes at RT. Decant and discard the supernatant. The tissue wash process will be repeated for 5 times. The washed tissue will be stored at -80 °C for at least 1 hour before being lyophilized.

[0067] Weigh the tissue + tube and get the weight of the tissue by subtracting the weight of tube. Then the lyophilized demineralized tissue will be hydrated by adding DI water (mL, 10 x mass of dry tissue (g)) into the tube. Vortex and then centrifuge each tube at 4700 rpm for 5 minutes at RT. Aspirate and discard the supernatant.

[0068] Calculate the volume of 0.6 N HCL to be added to each tissue in 14 hydrated tissue (g) / reagent (mL) ratio, where mass of hydrated tissue = 1 ,2x mass of dry tissue. Then add the calculated volume of 0.6 N HCL, seal the cap and place each tube on a shaker at 100 rpm under 4 °C for 30 minutes. Then centrifuge each tube at 4700 rpm for 5 minutes at RT. Collect the supernatant, i.e. demineralized solution (DMS 2). The second demineralization process will be repeated 3 times to ensure the demineralization is complete. Take 200 pL of DMS2 aliquot from each sample for later Calcium measurement using QuantiChrom™ Calcium Assay Kit.

[0069] The tissue after the second demineralization will be washed by adding 10 mL of DI water into each tube. Vortex and invert each tube occasionally for 5 minutes and thencentrifuge each tube at 4700 rpm for 5 minutes at RT. Decant and discard the supernatant. The tissue wash process will be repeated for 5 times. The washed tissue would be stored at -80 °C for at least 1 hour before being lyophilized.Dialysis of DMS2

[0070] Label 30 mL 2K MWCO (Molecular weight cut-off) Dialysis cassettes with the information of DMS 2. Rehydrate the dialysis cassettes in DI water for at least 4 minutes before use. Add DI water (100x volume of each solution in the cassette) into a pitcher. Load each of the DMS samples into a labeled 30 mL 2K MWCO Dialysis cassette. Dialyze the cassettes for 2 hours twice and then overnight under 4 °C.

[0071] After dialysis, transfer DSM2 samples into serum, and place the after-dialyzed DMS samples at -80°C for at least 1 hour before transferring to lyophilizer for at least 48 hours.Protein Extraction from tissue samples

[0072] Prepare extraction buffer: 38.2 g Guanidinium Chloride (GuHCL), 4.1 g sodium acetate and 2 tablet / s of Protease inhibitor for 100 mL of extraction buffer.

[0073] Add a certain volume of extraction buffer (40 mL extraction buffer / gram lyophilized tissue after the second demineralization) into each tube and seal the cap. Place each tube on a shaker at 100 rpm at 4°C for 24 hours. Centrifuge each tube at 4700 rpm for 5 minutes at RT. Decant the supernatant through a cell strainer, discard the tissue.Dialysis of Extract buffers

[0074] Label 15 mL 2K MWCO (Molecular weight cut-off) Dialysis cassettes with sample information. Rehydrate the dialysis cassettes in DI water for at least 4 minutes before use.

[0075] Add DI water (100x volume of each solution in the cassette) into a pitcher. Load each of the Extract samples into a labeled 15 mL 2K MWCO Dialysis cassette. Dialyze the cassettes for 2 hours twice and then overnight under 4 °C.

[0076] After dialysis, transfer each sample into a labeled serum vial. Take an aliquot of 200 pL of each sample in 1 .5 mL tube to measure total protein concentration, recorded as Pre-lyo BCA, following the protocol from the BCA kit (Pierce™ BCA Protein Assay Kit). This aliquot will be stored at 4°C if not used directly. Place the remaining samples at -80°C for at least 1 hour. Place the frozen samples into the lyophilizer at least 48 hours. After lyophilization, store at 4°C until ready to be reconstituted.Reconstitute samples.

[0077] Reconstitute each DMS2 sample with 4.2 mL of DI water.

[0078] For Extract samples, calculate each sample's protein concentration from the Pre-Lyo BCA results. Then reconstitute each sample to a 1.5 mg / mL concentration with DI water. Take an aliquot of 200 pL of each sample in 1.5 mL tube to measure total proteinconcentration, recorded as Post-recon BCA. Normalize each sample to 1 mg / mL concentration based on Post-recon BCA with DI water. Aliquot 2 mL of each sample into 5 mL conical tube for protein characterization assay, i.e. instance ELISA.Protein Characterization

[0079] Masses of eight growth factors (GFs), i.e. Bone morphogenetic protein 2 (BMP-2), Bone morphogenetic protein 7 (BMP-7), Hepatocyte growth factor (HGF), Insulinlike growth factor 1 (IGF-1), Osteoprotegerin (OPG), Osteopontin (OPN), Human Transforming Growth Factor beta 1 (TGF-pi), Vascular endothelial growth factor (VEGF), in both DMS2 and Extract for each sample were measured by ELISA (Enzyme-linked Immunosorbent Assay).Calculation - % of Demineralization

[0080] The calcium in DMS2 measured in section 1.6 was used to calculate the % of demineralization. No Ca2+left in DMS2 for 0.6 N HCL demineralized tissue, indicating 100% demineralization. Ca2+measured in the DMS2 for DI Water demineralized tissue was considered as Ca2+in mineralized tissue, given no Ca2+is removed by DI water. Hence, % of demineralization for a sample = (Ca2+(mg) in DI Water - Ca2+(mg) in sample) / (Ca2+(mg) in DI Water - Ca2+(mg) in 0.6 N HCL control) xioo.Calculation - % of Normalized Growth Factor Sum

[0081] For each sample, all GFs measured by ELISA were added up as the remaining GFs for the demineralized tissue. Then the total GF sum for each sample was normalized by that of 0.6N HCL. Cortical tissues demineralized by DI water are considered to maintain all, i.e. 100%, the GFs, and 0.6N HCL are considered to remove all GFs, i.e. 0%. Therefore, % of normalized GF Sum for a sample = (GF sum for DI Water - GF sum for sample) / (GF sum in DI Water - GF sum in 0.6 N HCL) *100.Results

[0082] Figure 1. shows a summary of % of demineralization and Growth Factor (GF) sum of cortical fibers demineralized by Glycolic Acid (GA) with different concentrations and reagent (mL) / tissue (g) (RT) ratios under different incubation times. A reverse relationship between demineralization and GF remaining can be observed. It takes 5-minute incubation in 0.18 M of GA and 3x30-minute incubation in 69 M GA to initiate and finish the cortical fiber demineralization, respectively. GFs in the tissue started to be affected when the demineralization reached to ~30%, which requires 20-minute incubation in 2 M GA. Based on the demineralization vs GF data, certain concentrations of GA with different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 2.

[0083] Figure 2. shows a summary of % of demineralization and GF sum of cortical powders demineralized by GA with different concentrations and RT ratios under different incubation times. A reverse relationship between demineralization and GF sum can be observed. It only takes 5-minute incubation in 0.02 M of GA and 20-minute incubation in 22 M GA to initiate and finish the cortical powder demineralization, respectively, where less GA is required to compared to cortical fibers. Besides, cortical fibers and powders have different measurements, shown in Table 4. Therefore, it suggests that geometry of cortical tissues affects demineralization. GFs in the tissue started to be affected when the demineralization reached ~40%, which requires 20-minute incubation in 2.4 M GA. Based on the demineralization vs GF data, certain concentrations of GA with different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 3.

[0084] Figure 3. shows a summary of % of demineralization and GF sum of cortical fibers demineralized by Acetic Acid (AA) with different concentrations and RT ratios under different incubation times. A reverse relationship between demineralization and GF sum can also be observed. Based on the demineralization vs GF data, certain concentrations of AAwith different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 5.

[0085] Figure 4. shows a summary of % of demineralization and GF sum of cortical fibers demineralized by Nitric Acid (NA) with different concentrations and RT ratios under different incubation times. A reverse relationship between demineralization and GF sum can be observed. It would take 5-minute incubation in 0.01 M of NA and 20-minute incubation in 11 M NA to initiate and finish the cortical fiber demineralization, respectively. GFs in the tissue started to be affected when the demineralization reached ~30%, which requires 20- minute incubation in 0.9 M NA. Based on the demineralization vs GF data, certain concentrations of NA with different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 6.

[0086] Figure 5. shows a summary of % of demineralization and GF sum of cortical fibers demineralized by Formic Acid (FA) with different concentrations and RT ratios under different incubation times. A reverse relationship between demineralization and GF sum can be observed. It would take 5-minute incubation in 0.1 M of FA and 20-minute incubation in 80 M FA to initiate and finish the cortical fiber demineralization, respectively. GFs in the tissue started to be affected when the demineralization reached ~20%, which requires 20- minute incubation in 0.9 M FA. Based on the demineralization vs GF data, certainconcentrations of FA with different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 7.

[0087] Figure 6. shows a summary of % of demineralization and GF sum of cortical fibers demineralized by Propionic Acid (PA) with different concentrations and RT ratios under different incubation times. A reverse relationship between demineralization and GF sum can be observed. It would take 5-minute incubation in 0.1 M of PA and 20-minute incubation in 80 M PA to initiate and finish the cortical fiber demineralization, respectively. GFs in the tissue started to be affected when the demineralization reached ~31%, which requires 20-minute incubation in 0.9 M PA. Based on the demineralization vs GF data, certain concentrations of PA with different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 8.

[0088] Figure 7. shows a summary of % of demineralization and GF sum of cortical fibers demineralized by EGTA with different concentrations and RT ratios under different incubation times. It takes 2-Hour incubation in 0.01 M of EGTA and 72-Hours incubation in 1 M EGTA to initiate and finish the cortical fiber demineralization, respectively. It seems more demineralization led to a very slightly higher GF sum, which is not share the same trend as other reagents. Based on the demineralization vs GF data, certain concentrations of EGTAwith different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 9.

[0089] Figure 8. shows a summary of % of demineralization and GF sum of cortical fibers demineralized by DTPA with different concentrations and RT ratios under different incubation times. A reverse relationship between demineralization and GF sum can be observed. It would take 2-hour incubation in 0.1 M of DTPA and 72-hour incubation in 14 M DTPA to initiate and finish the cortical fiber demineralization, respectively. GFs in the tissue started to be affected when the demineralization reached ~19%, which requires 16-hour incubation in 0.9 M DTPA. Based on the demineralization vs GF data, certain concentrations of DTPA with different event / time / RT ratio combinations were chosen to set ranges where demineralization and GF remaining are affected in different levels, as shown in Table 10.

[0090] Figure 9. shows a summary of % of demineralization and GF sum of cortical fibers demineralized by HEDTA with different concentrations and RT ratios under different incubation times. A reverse relationship between demineralization and GF sum can be observed. It would take 2-hour incubation in 0.1 M of HEDTA and 72-hour incubation in 14 M HEDTA to initiate and finish the cortical fiber demineralization, respectively. GFs in the tissue started to be affected when the demineralization reached ~23%, which requires 16-hour incubation in 0.9 M HEDTA. Based on the demineralization vs GF data, certain concentrations of HEDTA with different event / time / RT ratio combinations were chosen to setranges where demineralization and GF remaining are affected in different levels, as shown in Table 11.

[0091] Figure 10. shows % of demineralization and GF sum of cortical fibers demineralized by HEDTA with different concentrations under different incubation times, in 14:1 RT ratio. It would take 2-hour incubation in 0.3 M of EDTA and 16-hour incubation in 7 M EDTA to initiate and finish the cortical fiber demineralization, respectively. Based on this data, certain concentrations of HEDTA with different event / time / RT ratio combinations were chosen to set ranges where demineralization and GFs remaining are affected in different levels, as shown in Table 12.

[0092] Figure 11. shows GF remaining for cortical fibers demineralize to a certain level by different reagents. The data demonstrates that different amounts of GF remained in tissue demineralized by different reagents even though they were demineralized to a same level, indicating reagents and their specific demineralization mechanisms affect GF remaining for cortical fibers.

[0093] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0094] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the inventiondescribed herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A method of producing a bone graft material, the method comprising:(a) harvesting bone having bone marrow from a donor;(b) separating the harvested bone into cancellous and cortical bone;(c) optionally shaping the cortical bone and / or cancellous bone;(d) demineralizing the cortical bone and optionally the cancellous bone with a demineralizing agent to produce a cortical bone with 10 to 95% mineral content compared to the original cortical bone and optionally a cancellous bone with 10 to 95% mineral content compared to the original cancellous bone,(e) washing the demineralized cortical bone and / or cancellous bone.

2. The method of claim 1 , wherein step (d) comprises demineralizing the cancellous bone with the demineralizing agent to produce cortical bone with 50 to 90% mineral content compared to the original cortical bone.

3. The method of claim 1 , wherein step (d) comprises demineralizing the cancellous bone with the demineralizing agent to produce cortical bone with 60 to 80% mineral content compared to the original cortical bone.

4. The method of any one of claims 1 to 3, wherein the demineralizing agent is glycolic acid, nitric acid, propanoic acid, formic acid, ethylenediaminetetraacetic Acid (EDTA), Hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 ,3-Diamino-2-hydroxypropane-N,N,N',N'- tetraacetic acid (DPT A), or any combination thereof.

5. The method of any one of claims 1 to 4, wherein the cortical bone is shaped into a particle, strut, fiber, wedge, block, or disc.

6. The method of claim 5, wherein the particle is 0.1 to 5mm in diameter.

7. The method of claim 5, wherein the strut, wedge, or block is 0.1 mm to 20cm in any dimension.

8. The method of claim 5, wherein the fibers are 0.1 to 5mm in diameter and 0.1 to 20cm in length.

9. The method of claim 5, wherein the disc is 0.1 mm to 1cm thick and 0.1 mm to 10cm in diameter.

10. The method of any one of claims 1 to 9 wherein the treatment time with the demineralization agent is 1 minute to 3 days.11 . The method of any one of claims 1 to 10, wherein the number of treatments with the demineralization agent is 1 to 3 times.

12. The method of any one of claims 1 to 11 , wherein the demineralization agent volume during treatment is 1 :1 to 1 :50 (weight tissue : volume reagent).

13. The method of any one of claims 1 to 12, wherein the temperature during treatment is 1°C to 60°C.

14. The method of any one of claims 1 to 13, wherein the donor is human.

15. A bone graft material produced according to the method of any one of claims 1 to 14.

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