Methods of making osteoinductive bone implants with native growth factors / bone morphogenetic proteins and implants made therefrom

Abstract

The instant disclosure describes bone implants with high levels of native growth factors and native bone morphogenetic proteins as well as methods of preparing such implants.

Description

Attorney Docket No. 82752-429039 Methods of Making Osteoinductive Bone Implants with Native Growth Factors / Bone Morphogenetic Proteins and Implants Made Therefrom RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No.63 / 697,013, filed on September 20, 2024, the contents of which is incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] The present disclosure relates to osteoinductive bone implants and methods of preparation with high levels of native growth factors and bone morphogenetic proteins. BACKGROUND OF THE INVENTION

[0003] Bone grafts, whether an allograft, autograft or xenograft can be employed in patients suffering from painful or otherwise abnormal conditions related to instabilities or abnormalities in the skeletal structure. As a non-limiting example, patients suffering from a spinal instability or excess movement of one or more vertebrae may be treated with a spinal fusion procedure involving removal of a portion of an intervertebral disc located between two vertebrae. A bone graft or spinal implant or a combination of both can then be inserted into or around the area of removed intervertebral disc to facilitate the fusion of two adjacent vertebrae. Such bone grafts or spinal implants can comprise harvested bone fragments made of cortical, cancellous, corticocancellous or a combination of all three aforementioned types of bone material. Patients may also suffer from various degenerative conditions for which implantation of a bone graft can be chosen as a treatment.

[0004] U.S. Patent No.11,491,260 (the ‘260 patent), also assigned to the current applicant, discloses that bone grafts or spinal implants can comprise harvested bone fragments made of cortical, cancellous, corticocancellous or a combination of all three aforementioned types of bone material. By way of example, cancellous bone is recovered from a cadaver, live donor, or harvested autologously from a patient. IfAttorney Docket No. 82752-429039 required by a particular implant application, the harvested cancellous bone may be ground or cut to a desired shape and configuration. Care may be taken to retain as much cellular material, bone marrow, and / or blood within the bone during harvest and cutting operations. The cancellous bone can then be exposed to acetic acid (e.g., 0.1M-17M) as a lysing agent to lyse cells remaining in porous bone structure and on bone surface. The lysing of the cells releases and solubilizes growth factors and bioactive materials contained in the cellular material. Acetic acid also allows the solubilized bioactives to bind to the bone. The cancellous bone may be further demineralized using at least one demineralization wash using any acid, including, but not limited to, acetic, hydrochloric, citric, phosphoric, etc., to alter the mechanical properties of the bone and remove mineral content. The bone may be further rinsed and cleaned to remove excess acid, cell fragments, lipids, or debris. In some embodiments, the bone may be dehydrated by evaporation, vacuum drying, or lyophilization to reduce any residual acetic acid and bring the implant to a more neutral pH. The ‘260 patent also discloses that hydrochloric acid can be employed as a lysing agent that restricts the solubilized growth factors and bioactives from binding to the cancellous bone.

[0005] There continues to be a need for bone implants with even higher levels of native growth factors and bone morphogenetic proteins than those attainable with prior art processes. SUMMARY OF THE INVENTION

[0006] The present disclosure is directed to an improved process for the manufacture of bone implants with high amounts of native growth factors and bone morphogenetic proteins. The inventors have identified a new process that provides for very high levels of native growth factors and bone morphogenetic proteins.

[0007] In an aspect of the composition, a bone implant is prepared by a process comprising the steps of: harvesting bone tissue and marrow from a donor; removing the bone marrow from the bone tissue; exposing the bone marrow, to a lysing agent, theAttorney Docket No. 82752-429039 lysing agent configured to release native growth factors and bone morphogenetic proteins from the harvested bone marrow; demineralizing the harvested bone tissue in the substantial absence of the released native growth factors and bone morphogenetic proteins obtained by lysing the harvested bone marrow; removing or neutralizing the demineralizing acid from the demineralized bone; combining the demineralized bone with the released native growth factors and bone morphogenetic proteins in lysing agent; allowing the released native growth factors and bone morphogenetic proteins to bind to the harvested bone tissue, and rinsing the harvested bone material with growth factors and bone morphogenetic proteins bound thereto.

[0008] In another aspect of the composition, a bone implant is prepared by a process comprising the steps of: harvesting bone tissue and marrow from a donor; exposing the harvested bone tissue and the bone marrow to a lysing agent, the lysing agent configured to release native growth factors and bone morphogenetic proteins from the harvested bone marrow; separating the bone tissue and the lysing agent with released native growth factors and bone morphogenetic proteins from the harvested bone tissue; demineralizing the harvested bone tissue in the substantial absence of the released native growth factors and bone morphogenetic proteins obtained by lysing the harvested bone marrow; removing or neutralizing the demineralizing acid from the demineralized bone; combining the demineralized bone with the released native growth factors and bone morphogenetic proteins in lysing agent; allowing the released native growth factors and bone morphogenetic proteins to bind to the harvested bone tissue, and rinsing the harvested bone material with growth factors and bone morphogenetic proteins bound thereto.

[0009] In another aspect of the composition, the bone implant comprises one or more of native: insulin-like growth factor-1 (IGF-1), transforming growth factor-beta1 (TGF-b1) or bone morphogenetic proteins (BMPs). In other aspects of the composition, the bone implant comprises TGFb1, BMP-2, BMP-4 and / or BMP-7. BRIEF DESCRIPTION OF THE DRAWINGSAttorney Docket No. 82752-429039

[0010] Figure 1 depicts a process in accordance with the present disclosure.

[0011] Figure 2 depicts a process in accordance with the present disclosure.

[0012] Figure 3 depicts a comparative process.

[0013] Figure 4 shows percent change from a process in accordance with the present invention relative to a comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure is related to bone implants with high levels of native growth factors and native bone morphogenetic proteins as well as methods of preparing such implants. The inventors have found implants with high levels of native growth factor and BMPs are obtainable by: harvesting bone tissue and marrow from a donor; separating the bone marrow from the bone tissue; exposing the bone marrow to a gentle lysing agent, the lysing agent configured to release native growth factors and bone morphogenetic proteins from the harvested bone marrow; demineralizing the harvested bone tissue in the substantial absence of the lysing agent with the released native growth factors and bone morphogenetic proteins obtained by lysing the harvested bone marrow; removing or neutralizing the demineralizing acid from the demineralized bone; combining the demineralized bone with the released native growth factors and bone morphogenetic proteins; allowing the released native growth factors and bone morphogenetic proteins to bind to the demineralized harvested bone tissue, and; rinsing the harvested bone material with growth factors and bone morphogenetic proteins bound thereto.

[0015] The inventors have further determined that implants with high levels of native growth factors and BMPs are obtainable by: harvesting bone tissue and marrow from a donor;Attorney Docket No. 82752-429039 exposing the harvested bone tissue and the bone marrow to a lysing agent, the lysing agent configured to release native growth factors and bone morphogenetic proteins from the harvested bone marrow; separating the bone tissue and the lysing agent with released native growth factors and bone morphogenetic proteins from the harvested bone tissue; demineralizing the harvested bone tissue in the substantial absence of the lysing agent with the released native growth factors and bone morphogenetic proteins obtained by lysing the harvested bone marrow; removing or neutralizing the demineralizing acid from the demineralized bone; combining the demineralized bone with the released native growth factors and bone morphogenetic proteins; allowing the released native growth factors and bone morphogenetic proteins to bind to the demineralized harvested bone tissue, and; rinsing the harvested bone material with growth factors and bone morphogenetic proteins bound thereto.

[0016] These methods reduce denaturation of the growth factors and BMPs as a result of: (1) gentle lysing of the bone marrow after it has been removed from the bone, (2) demineralizing bone tissue in the absence of growth factors and BMPs obtained from the bone marrow, and (3) combining the demineralized bone with the released native growth factors and BMPs after the demineralizing acid has been neutralized or removed from the demineralized bone. Separating the bone marrow from the bone tissue before lysing also increases the percentage of cells effectively lysed with a gentle lysing agent.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Attorney Docket No. 82752-429039

[0018] Although various features of the disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0019] “Demineralized”, as used herein, refers to any material generated by removing mineral material from tissue, e.g., bone tissue. In certain embodiments, the demineralized compositions described herein include preparations containing less than 5% calcium and preferably less than 1% calcium by weight. Partially demineralized bone (e.g., preparations with greater than 5% calcium by weight but containing less than 100% of the original starting amount of calcium) is also considered within the scope of the invention. In some embodiments, demineralized bone has less than 95% of its original mineral content.

[0020] “Osteogenic” is used herein to refer to the ability of an agent, material, implant, etc. to enhance or accelerate the growth of new bone tissue by one or more mechanisms such as osteogenesis, osteoconduction, and / or osteoinduction. Osteoinductive, as used herein, refers to the quality of being able to recruit cells from the host that have the potential to stimulate new bone formation.

[0021] The term “BMP(s)” as used herein refers to bone morphogenetic proteins that have been associated with bone growth through the mechanism of providing signals to osteogenic cells to differentiate, proliferate and regenerate new tissue. The term “growthAttorney Docket No. 82752-429039 factors” as used herein refers to known proteins and hormones that promote neovascularization and growth of new tissue. BMPs and growth factors in accordance with the present disclosure include but are not limited to: SDF-1, TNF-α, TGF-β, Activin A, αFGF, bFGF, PDGF-BB, TGF- β2, TGF- β3, IGF-2, VEGF, IGF-1, EGF, HB-EGF, ANG, ANGPT1, ANGPT2, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-9, TNF-α, and TGF- β1.

[0022] The term “bone marrow” as used herein refers to the soft spongy tissue that is in the medullary cavities of bones and includes both red bone marrow (myeloid tissue) and yellow bone marrow (fatty tissue) and is inclusive of hematopoietic cells, red blood cells, white blood cells and platelets as well as all growth factors and bone morphogenetic proteins present in marrow, blood, and bone tissue.

[0023] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value may also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0024] Figure 1 depicts a process in accordance with the present invention. First, bone and marrow are harvested from a patient. After bone and marrow are harvested, they are separated for separate treatment. Marrow is soaked in a gentle lysing agent separate from harvested bone thereby releasing growth factors and BMPs in the lysing agent. Separately, harvested bone is soaked in demineralizing acid and the acid is removed from the demineralized bone or neutralized. After the foregoing separateAttorney Docket No. 82752-429039 treatments, the demineralized bone is then soaked in the gentle lysing agent with growth factors / BMPs allowing the growth factors / BMPs to bind to the demineralized bone. The demineralized bone with growth factors / BMPs bound thereto is then washed one or more times before drying, packaging and gamma sterilization.

[0025] Figure 2 depicts another process in accordance with the present invention. First, bone and marrow are harvested from a patient. After bone and marrow are harvested, they are soaked together in a gentle lysing agent releasing growth factors and BMPs in the lysing agent. The bone is then separated from the lysing agent for demineralization of the bone. The separated bone is soaked in demineralizing acid. After the bone is demineralized separate from the lysing agent, the demineralizing acid is removed or neutralized. After removal or neutralization of the demineralizing acid, the demineralized bone is then soaked in the lysing agent with released growth factors and BMPs allowing the growth factors and BMPs to bind to the demineralized bone. The demineralized bone with growth factors / BMPs bound thereto is then washed one or more times before drying, packaging and gamma sterilization.

[0026] Figure 3 depicts a comparative process wherein bone and marrow are harvested, the bone and marrow are soaked together with a strong lysing agent and then a demineralizing acid is added to the bone after the growth factors / BMPs are bound to the bone.

[0027] Harvesting Bone Tissue and Bone Marrow from a Donor

[0028] Cancellous and / or cortical bone is recovered from a cadaver, live donor, or harvested autologously from a patient. The harvested bone can be ground or cut to a desired shape and configuration as can be appreciated. Grinding or cutting to a desired shape may be conducted later in the process as well, for example, after bone and marrow are separated. Cellular material, bone marrow, and / or blood within the bone during harvest and cutting operations should be retained. In some embodiments, depending on the needs of a particular application, the cancellous bone may haveAttorney Docket No. 82752-429039 cortical portions removed prior to further processing. In other embodiments, the cortical bone may have cancellous portions removed prior to processing. As those of ordinary skill in the art will appreciate, harvested bone may be cut or ground in a manner consistent with the manufacture of fibers, granules, putty or sponge implants.

[0029] Separating the Bone Marrow from the Bone Tissue

[0030] Various methods may be used to separate marrow from the bone tissue. In some embodiments, segments of bone are reamed to remove the bone marrow from the bone tissue. In other embodiments, removal of marrow may be achieved through grinding the bone tissue followed by extraction of the marrow. In yet further embodiments, marrow can be removed by other physical or chemical means such as sonication or liquid extraction. It is noted that, while it is desirable to remove as much marrow from the bone as possible, it is not required that all marrow be removed and that in accordance with the present disclosure, some marrow may continue to reside in the bone tissue during subsequent processing steps including demineralization.

[0031] Exposing the Bone Marrow to the Lysing Agent

[0032] The bone marrow is exposed to a gentle lysing agent to facilitate lysis of the cells therein to release growth factors and BMPs. A “gentle” lysing agent is one with a low rate of denaturation of native growth factors and BMPs present in bone marrow. When bone marrow is lysed, it releases growth factors and / or bioactive materials, such as cytokines and nutrients, to stimulate growth, differentiation, and repair. These growth agents can be cytokines such as proteins, hormones, or glycoproteins including members of the TGF-β family (including bone morphogenetic proteins), interleukins, interferons, insulin-like growth factor, lymphokines, BMPs, chemokines, platelet derived growth factors, VEGF, and other stimulative agents that promote growth, repair or regenerate tissues. In embodiments, the native growth factors and BMPs are released into the lysing agent.Attorney Docket No. 82752-429039

[0033] A gentle lysing agent is configured to release native growth factors and BMPs from the harvested bone marrow with a low rate of denaturation and can include a volatile acid or base, such as acetic acid or ammonia at low concentrations. In yet other embodiments, a gentle lysing agent can include detergents that can disrupt cell walls and remove any lipid barriers that may surround the cell. Enzymes, viruses, solvents, surfactants, and hemolysins can also help cleave or remove outer cell membranes releasing the bioactive growth agents contained within.

[0034] For example, the bone marrow may be exposed to acetic acid which may act as a gentle lysing agent as described above. The acetic acid lysing agent may be employed at concentrations sufficient to lyse cells in the bone marrow, but ameliorate denaturation. For example, the acetic acid concentration may be between about 1% to about 6%, between about 2% to about 5%, about 1% to about 3%, or about 5% to about 6%. In other embodiments the acetic acid concentration is about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.0%.

[0035] Although it may be desirable to expose both the removed bone marrow and the bone tissue to a lysing agent since there are growth factors and BMPs in and on the bone tissue, the acetic acid lysing agent also allows the solubilized bioactives from the bone marrow to bind to the bone. To the extent growth factors and bone morphogenetic proteins from the bone marrow are bound to the bone tissue, they are susceptible to denaturation during the demineralization step. Accordingly, it is preferred to expose the bone marrow to the lysing agent in the absence of bone tissue. If bone tissue and bone marrow is lysed together, the bone tissue may be removed from the growth factors and bone morphogenetic proteins prior to demineralizing the bone tissue. In embodiments, this may be accomplished by removing the bone from the lysing agent with growth factors and BMPs residing therein.Attorney Docket No. 82752-429039

[0036] Demineralizing the Bone Tissue in the Substantial Absence of the Released Native Growth Factors and Bone Morphogenetic Proteins Obtained by Lysing the Harvested Bone Marrow

[0037] There are many known methods for demineralizing bone that involve the use of strong acids. See, e.g., Pang, S., Su, F.Y., Green, A. et al. Comparison of different protocols for demineralization of cortical bone. Sci Rep 11, 7012 (2021). Demineralization may be accomplished with hydrochloric acid, formic acid, acetic acid, citric acid, formic acid, phosphoric acid, ethylene-diamine tetraacetic acid, etc. or mixtures thereof.

[0038] In accordance with the present disclosure, it is desirable to demineralize the bone tissue in the substantial absence of the released native growth factors and BMPs. It is noted that bone tissue will continue to retain native growth factors and BMPs as naturally present in and on bone tissue. It is therefore consistent with the present disclosure for naturally present growth factors and BMPs to be present during demineralization of bone tissue. It is the “released” native growth factors and BMPs lysed from the bone marrow that are preferably not present during demineralization. The released growth factors are present in the lysing agent or may be transferred to another fluid that is later combined with the demineralized bone. The released native growth factors and BMPs obtained from the bone marrow are later recombined with the bone tissue in lysing agent after demineralization and removal or neutralization of the demineralizing acids.

[0039] Removing or Neutralizing the Demineralizing Acid from the Demineralized Bone

[0040] After demineralization of the bone tissue, it is desirable to neutralize or remove the demineralizing acid from the demineralized bone. This can be achieved by either simply removing the demineralized bone from a vessel containing the demineralizing acid and the demineralized bone or by removing the acid from the vessel, e.g., draining the demineralizing acid from the vessel. It may be desirable to wash the demineralizedAttorney Docket No. 82752-429039 bone before combining it with the released native growth factors and BMPs. Although not all demineralizing acid must be neutralized or removed, it is desirable to neutralize or remove as much as possible to avoid risk of denaturation of the native released growth factors and BMPs when combined.

[0041] Combining the Demineralized Harvested Bone With the Native Released Growth Factors / BMPs

[0042] After the demineralizing acid is removed from the demineralized bone, or the demineralizing acid is neutralized, the demineralized bone is combined with the native growth factors and BMPs released from the marrow. This may be accomplished by immersing demineralized bone in lysing agent containing the native proteins and BMPs released from the bone marrow. The native proteins and BMPs may also be transferred to another liquid or medium for subsequent combination with demineralized bone. Such other liquids or media may be chosen that promote or at least do not inhibit binding of the native growth proteins and BMPs to bone.

[0043] Marrow that has been previously lysed with the gentle lysing agent may be optionally present during demineralization provided that substantially all of the lysing agent with released growth factors / BMPs is separated from the bone when the demineralizing acid is introduced to the bone.

[0044] Allowing the Released Native Growth Factors / BMPs to Bind to the Harvested Demineralized Bone Material

[0045] After the demineralized harvested bone is combined with the native growth factors / BMP’s the combination should soak together for a period of time sufficient to permit binding of the released native growth factors / BMPs to bind to bone. Depending on the lysing agent, other liquid or media used to include the native growth proteins / BMPs, a time of a few minutes to a few hours may be required. Those of ordinary skill in the art will be able to determine the optimal residence time byAttorney Docket No. 82752-429039 measuring the concentration of one or more desired proteins in an extraction analyte after soaking with a BMP Responsive Report Osteoblast Cell assay, Pierce BCA protein assay kit, ELISA, or the like.

[0046] Rinsing

[0047] The bone may be further rinsed and cleaned by a rinsing agent after exposure to the acetic lysing agent and after growth factors and / or bioactive materials bind to the bone. Rinsing can be conducted in order to remove excess acetic acid, cell fragments, lipids, and / or debris. Additionally, pH of the harvested bone may be substantially neutralized. In some embodiments, the pH of the harvested bone can be neutralized by the rinsing agent and rinsing step. In other embodiments, pH neutralization may not be required. Further pH neutralization of the harvested bone may be accomplished by dehydrating by evaporation, vacuum drying, or lyophilization to reduce the acetic acid lysing agent to a residue and bring the implant to a more neutral pH.

[0048] Rinsing solutions can be water, saline (NaCl, PBS, etc.), peroxides, alcohol (isopropyl, ethanol, etc.), crystalloids, sterilizing fluids (antibiotics such as gentamicin, vancomycin, bacitracin, polymixin, amphotericin, ampicillin, amikacin, teicoplanin, etc.), preserving fluids (DMEM, DMSO, mannitol, sucrose, glucose, etc.), storage agents, and / or other fluids used in processing of allografts. The resulting product yields a cancellous bone implant with increased bioactivity. In some embodiments, ground particulate filler implants as well as structural cancellous bone implants with increased bioactivity may be formed.

[0049] Drying, sterilization and packaging of resultant bone implant materials may be accomplished by any methods known to those of ordinary skill in the art. Resultant bone implants may also be lyophilized and reconstituted with water prior to use.

[0050] One embodiment of the present disclosure relates to osteoinductive implants made from cellular bone tissue as well as methods of making osteoinductive implants.Attorney Docket No. 82752-429039 Implants made from cellular bone tissue can include osteoinductive and / or osteoconductive materials to facilitate fusion and / or new bone growth in or around an area of implant insertion. Accordingly, in accordance with one embodiment, a portion of cancellous, corticocancellous and / or cortical bone or any combination thereof can be harvested from a donor. In one embodiment, the harvested material can be harvested in such a way as to retain as much bone marrow in the harvested sample as possible.

[0051] It is noted that the particular order of steps could be altered and still be within the scope of the present disclosure and claims. For example, the demineralizing step could be completed after removing the marrow from the bone tissue but before lysing is conducted. The same result is achievable as long as the released native growth factors and BMPs are not present with bone tissue during demineralizing. Examples

[0052] EXAMPLE 1 - Example in Accordance with the Present Disclosure

[0053] Thawed bone shaft with blood / bone marrow was obtained from a human donor. Segments were reamed to remove marrow and blood which was collected in a first basin including acetic acid as a gentle lysing agent allowing the growth factors / BMPs to be released in the lysing agent. The bone segments were then separated from the marrow and blood by removing them from the first basin, patted dry, and machined into fibers. The fibers were then demineralized with HCl in a second basin separate from the first basin. The HCl was then removed by washing the bone fibers. After the demineralized fibers were washed, they were subsequently soaked together with the lysing agent and blood / bone marrow allowing growth factors / BMPs to bind to the bone fibers. This was followed by successive washes, packaging and lyophilization.

[0054] EXAMPLE 2 – Example in Accordance with the Present Disclosure (Prophetic)Attorney Docket No. 82752-429039

[0055] Thawed bone shaft with blood / bone marrow was obtained from a human donor. Segments were reamed to remove marrow and blood which was collected in a first basin including acetic acid as a gentle lysing agent. The bones segments were then soaked in the first basin with the lysing agent allowing the growth factors / BMPs to be released in the lysing agent. The bone segments were then removed from the first basin, patted dry and machined into fibers. The fibers were then demineralized with HCl in a second basin separate from the first basin until sufficiently demineralized. The HCl was then removed by washing the bone fibers. After the demineralized fibers were washed, they were subsequently soaked together with the lysing agent and blood / bone marrow allowing growth factors / BMPs to bind to the bone fibers. This was followed by successive washes, packaging and lyophilization.

[0056] EXAMPLE 3 – Comparative Example

[0057] Thawed bone shaft with blood / bone marrow was obtained from a human donor. Segments were reamed to remove marrow and blood which was collected in a basin including acetic acid as a strong lysing agent. The bone segments with were then machined into fibers and subsequently soaked together with the lysing agent and blood / bone marrow allowing the released growth factors / BMPs to bind to the bone fibers. Demineralizing HCl was then added directly to the mixture of bone fibers with bound growth factors / BMPs, lysing agent, blood and bone marrow. The bone fibers were soaked in this mixture until sufficiently demineralized followed by successive washes, packaging and lyophilization.

[0058] The results depicted in Figure 4 show about 100%-300% improvement in concentration of IGF-1, TGFb1, BMP-4 and BMP-7 when a process in accordance with the disclosure over the comparative example. The percent biofactor concentrations were calculated using the following formulaAttorney Docket No. 82752-429039 ^^ ^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ ^^^^^^^ 1 ^^^ ^^^^^ ^^^^^^^^^^ ^ × 100^^^^^^^ 3 (%)

[0059] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

Claims

Attorney Docket No. 82752-429039 What is claimed:

1. A bone implant prepared by a process comprising the steps of: harvesting bone tissue and marrow from a donor; separating the bone marrow from the bone tissue; exposing the bone marrow to a gentle lysing agent, the lysing agent configured to release native growth factors and bone morphogenetic proteins from the harvested bone marrow; demineralizing the harvested bone tissue in the substantial absence of the lysing agent with the released native growth factors and bone morphogenetic proteins obtained by lysing the harvested bone marrow; removing or neutralizing the demineralizing acid from the demineralized bone; combining the demineralized bone with the released native growth factors and bone morphogenetic proteins; allowing the released native growth factors and bone morphogenetic proteins to bind to the demineralized harvested bone tissue, and; rinsing the harvested bone material with growth factors and bone morphogenetic proteins bound thereto.

2. A bone implant prepared by a process comprising the steps of: harvesting bone tissue and marrow from a donor; exposing the harvested bone tissue and the bone marrow to a lysing agent, the lysing agent configured to release native growth factors and bone morphogenetic proteins from the harvested bone marrow; separating the bone tissue and the lysing agent with released native growth factors and bone morphogenetic proteins from the harvested bone tissue;Attorney Docket No. 82752-429039 demineralizing the harvested bone tissue in the substantial absence of the lysing agent with the released native growth factors and bone morphogenetic proteins obtained by lysing the harvested bone marrow; removing or neutralizing the demineralizing acid from the demineralized bone; combining the demineralized bone with the released native growth factors and bone morphogenetic proteins; allowing the released native growth factors and bone morphogenetic proteins to bind to the demineralized harvested bone tissue, and; rinsing the harvested bone material with growth factors and bone morphogenetic proteins bound thereto.

2. The bone implant of claims 1 or 2, wherein the bone implant comprises one or more of native: insulin-like growth factor (IGF), transformed growth factor (TGF) or bone morphogenetic proteins (BMP).

3. The bone implant of claims 1 or 2, wherein the bone implant does not include viable stem cells.

4. The bone implant of claims 1 or 2, wherein the rinsed bone comprises a bone implant comprising demineralized bone and bone morphogenetic proteins derived from the bone material.

5. The bone implant of claims 1 or 2, wherein the bone implant comprises bone material and morphogenetic proteins obtained from a single donor.

6. The bone implant of claims 1 or 2, wherein the lysing agent is acetic acid at a concentration between about 1% to about 6%, between about 2% to about 5%, about 1% to about 3%, or about 5% to about 6%.

7. The bone implant of claims 1 or 2, wherein the lysing agent is acetic acid at a concentration of about 1.5 to about 6.0%.

8. The bone implant of claims 1 or 2, wherein the acid is hydrochloric acid.Attorney Docket No. 82752-429039 9. The bone implant of claims 1 or 2, wherein the bone material comprises cortical bone tissue.

10. The bone implant of claims 1 or 2, wherein the bone material comprises cancellous bone tissue.

11. The bone implant of claims 1 or 2, wherein the bone material comprises a mixture of cortical and cancellous bone tissues.

12. The bone implant of claims 1 or 2, wherein the combining the demineralized bone with the released native growth factors and bone morphogenetic proteins is accomplished by combining the demineralized bone with lysing agent that includes the released native growth factors and bone morphogenetic proteins.

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