Bone graft

Abstract

The present invention provides bone grafts, such as allografts, and methods of releasing growth factors from tissues with native living cells, such as bone marrow, bone matrix, adventitia and / or inventitia. Also provided are compositions comprising growth factors and uses of the compositions to improve osteoinductivity of implants, such as bone grafts.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 416,756, filed on October 17, 2022, and U.S. Provisional Application No. 63 / 444,120, filed on February 8, 2023, and incorporates the content of each of them herein by reference in its entirety for all purposes. Field of the Invention

[0003] The present invention generally relates to bone grafts (such as allografts), including bone grafts impregnated with growth factors from living tissues. Background of the Invention

[0005] Patients with underlying diseases (such as osteopenia, diabetes, smokers, obesity) suffer from slower bone regeneration and poor graft healing outcomes. Bone grafts supplemented with growth factors can promote bone healing after implantation. There is still a need for bone grafts, such as bone grafts impregnated with growth factors, to improve bone regeneration after implantation. Developing techniques for releasing growth factors from tissues while maintaining their biological activities (including bone formation and angiogenesis) remains a challenge. Summary of the Invention

[0006] The present invention relates to bone grafts, such as allografts, optionally impregnated with growth factors released from tissues having native living cells. The present invention is based on the unexpected discovery by the inventors that cortical bone, cancellous bone, or cortico - cancellous bone from a donor can be machined, minimally processed, and / or assembled into a structural bone graft containing living bone cells for, for example, dental, foot and ankle, and spinal surgeries of the donor, and that a greater number of growth factors can be recovered from bone marrow, bone matrix, periosteum, and / or endosteum having native living cells compared to those from cryopreserved tissues without native living cells.

[0007] An allograft is provided. The allograft includes bone from a donor and living bone cells. The living bone cells are native to the bone and are present on the bone surface. The allograft does not include demineralized matrix (DBM) and has a size greater than 3×3×3 mm 3 3 The allograft may have a content of natural lipids lower than 10 mg of the lipids / cm 3 ​The allograft. The bone can include bone marrow, and at least 80% of the native hematopoietic cells can have been removed from the bone marrow. The allograft can have a pull-out force of at least 1 N against a surgical instrument in the bone. The allograft can be assembled with a bone portion, a metal implant, or a polymer implant. The allograft can be load-sharing.

[0008] A container including the allograft of the present invention and a liquid is provided. The liquid coats the allograft at a liquid-to-bone volume ratio of less than 1. The liquid can contain the composition of the present invention.

[0009] A method for preparing a composition is provided. The preparation method includes: (a) obtaining bone marrow from a first donor, wherein the bone marrow contains first native living cells and is not frozen; (b) incubating the bone marrow in a first aqueous solution to release a first growth factor from the bone marrow into the first aqueous solution, thereby producing a first mixture; (c) collecting a first liquid portion from the first mixture, wherein the first liquid portion contains the first growth factor; (d) obtaining one or more other tissues from a second donor, wherein the one or more other tissues are selected from bone matrix, periosteum, endosteum, and combinations thereof, and the one or more other tissues contain second native living cells; (e) incubating the one or more other tissues in a second aqueous solution to release a second growth factor from the one or more other tissues into the second aqueous solution, thereby producing a second mixture; (f) collecting a second liquid portion from the second mixture, wherein the second liquid portion contains the second growth factor; and (g) combining the first liquid portion and the second liquid portion, thereby preparing the composition, wherein the composition contains the first growth factor and the second growth factor. The preparation method can further include adjusting the pH of the composition to 6.5 to 7.5. The first donor and the second donor can be the same. The preparation method can further include measuring the protein concentration of the composition. The preparation method can further include increasing the protein concentration of the composition. The preparation method can further include removing salts from the composition.

[0010] According to the preparation method, step (a) can include removing red blood cells from the bone marrow. The first aqueous solution can have a pH of 0 to 6.9. The second aqueous solution can have a pH of 0 to 6.9. Step (b) can include disrupting the first living cells. Step (d) can include disrupting the second living cells.

[0011] According to the preparation method, the first living cells can be selected from osteocytes, stem cells, multipotent mesenchymal stromal cells, and combinations thereof. The second living cells can be selected from osteocytes, stem cells, multipotent mesenchymal stromal cells, and combinations thereof. Osteocytes can be selected from osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, bone lining cells, and combinations thereof.

[0012] According to the preparation method, the growth factors released from bone marrow can be selected from insulin-like growth factor (IGF), transforming growth factor (TGF), bone morphogenetic protein (BMP), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), stromal cell-derived factor-1 (SDF-1), and combinations of the foregoing. The growth factors released from one or more other tissues can be selected from insulin-like growth factor (IGF), transforming growth factor (TGF), bone morphogenetic protein (BMP), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), stromal cell-derived factor-1 (SDF-1), and combinations of the foregoing.

[0013] According to the preparation method, the bone matrix can include cellularized cortical fragments, cortico-cancellous fragments, cellularized cortical bone fibers, cellularized cancellous blocks, cellularized cortical blocks, or cortico-cancellous blocks. One or more other tissues can include a bone matrix prepared from bone, and the bone is selected from cancellous bone, cortico-cancellous bone, cortical bone, and combinations of the foregoing.

[0014] The preparation method can further include storing the composition.

[0015] The preparation method can further include lyophilizing or freeze-drying the composition.

[0016] The preparation method can further include sterilizing the composition.

[0017] The preparation method can further include packaging the composition.

[0018] A composition prepared according to the preparation method is provided. The composition can contain growth factors at a concentration of 0.001 ng / g to 42,000 ng / g based on the total weight of the composition.

[0019] A method for improving the osteoinductivity of an implant is provided. The improvement method includes incubating the implant with an effective amount of the composition of the present invention to produce an impregnated implant. The implant includes a bone graft, a metallic material, a synthetic material, or combinations of the foregoing. The impregnated implant has greater osteoinductivity than the implant before incubation. The implant can be incubated with the composition in the presence of a reagent. The incubation can include agitating and / or sonication of the implant and the composition. The improvement method can further include storing the impregnated implant in a container.

[0020] According to the improved method, the implant may include a bone graft. The improved method may further include obtaining bone from a donor and cryopreserving the obtained bone to fabricate the bone graft. The improved method may further include lyophilizing the impregnated bone graft. The improved method may further include demineralizing the impregnated bone graft prior to lyophilization. The improved method may further include sterilizing the impregnated bone graft. The bone graft may be demineralized. The impregnated bone graft may include demineralized bone matrix (DBM) fibers, a first growth factor from bone marrow, and a second growth factor from one or more other tissues, where the one or more other tissues may include osteocytes on the surface of the one or more other tissues. The bone graft may not be demineralized. The bone graft may be selected from cortical pins, blocks or plates, cancellous blocks or strips, cortico-cancellous blocks or strips, cortical bone rings, and combinations of the foregoing. The bone graft may be an allograft of the present invention. The impregnated bone graft may have a pull-out force of at least 1 N against a surgical instrument in bone. The impregnated bone graft may be load-sharing. The impregnated bone graft may be assembled with one or more other bone grafts.

[0021] According to the improved method, the implant may include a metallic material.

[0022] According to the improved method, the implant may include a synthetic material.

[0023] A product is provided that includes an impregnated implant prepared by the preparation method according to the present invention.

[0024] A container is provided that includes the product of the present invention in a liquid. The liquid may contain the composition of the present invention. The liquid may be a cryopreservation, lyophilization preservation, or radiation protection agent solution. The impregnated implant may be an impregnated bone graft, and the container may be a cannula for injection in minimally invasive surgery (MIS). The cannula may have a particle density of less than 1.2 g / cm 3 The impregnated bone graft may have a maximum extrusion force of 160 N to 200 N. The cannula may be loaded with an impregnated bone graft having a density of at least 0.2 g / cm 3

[0025] A treatment method is provided. The treatment method includes treating cells with an effective amount of the composition of the present invention to reduce the release of pro-inflammatory cytokines from the cells.

[0026] Brief Description of the Drawings

[0027] Figure 1 is a diagram showing treatment steps according to one embodiment of a method for preparing growth factors.

[0028] Figure 2 Shows a comparison of protein concentrations in growth factor extracts prepared from fresh or frozen bone marrow or bone matrix.

[0029] Figure 3 Shows the BMP-2 content in growth factor extracts prepared from fresh or frozen bone marrow or bone matrix.

[0030] Figure 4 Shows the protein concentration in growth factor extracts prepared from fresh bone marrow, O / N frozen bone marrow, or O / N RT bone marrow.

[0031] Figure 5 Shows the expression of BMP2, BMP7, and PDGF growth factors in growth factor extracts prepared from growth factor extracts prepared from fresh bone marrow, O / N frozen bone marrow, or O / N RT bone marrow.

[0032] Figure 6 Shows the BMP-2 concentration in growth factor extracts prepared from fresh bone marrow or fresh bone matrix.

[0033] Figure 7 Shows the concentrations of BMP-4, IGF, TGF-B, BMP-7, VEGF, aFGF, SDF-1, PDFG, and bFGF in growth factor extracts prepared from fresh bone marrow or fresh bone matrix.

[0034] Figure 8 Shows the protein concentration in growth factor extracts prepared from pre-demineralized cortical bone.

[0035] Figure 9 Shows the total protein release over time from coated inactivated square bone grafts in culture.

[0036] Figure 10 Shows the in vitro ALP activity of bone marrow growth factor extract (500 ug / cc), liquid growth factor extract prepared from inactivated mineralized squares (500 ug / cc) (negative control), or BMP-2 solution (200 ng / cc) (positive control) in an in vitro alkaline phosphatase (ALP) assay.

[0037] Figure 11 Shows the increase in alkaline phosphatase activity after the introduction of the following in an in vitro ALP assay: liquid growth factor extracts from representative bone marrow (columns 1 to 3), periosteum (column 4), or bone matrix (columns 5 to 8), each at 500 ug / cc; liquid growth factor extract prepared from inactivated mineralized squares (500 ug / cc) (negative control), or BMP-2 solution (200 ng / cc) (positive control).

[0038] Figure 12showed the following ALK activities: liquid growth factor extracts prepared from bone marrow supernatant (column 4), bone marrow pellet (column 5), undiluted bone marrow (column 9), periosteum (column 8), or bone matrix (column 10), each having a total protein concentration of 500 μg / cc; liquid growth factor extracts (500 μg / cc) prepared from inactivated mineralized blocks (negative control, column 2) or BMP-2 solution (200 ng / cc) (positive control, column 3); high (column 6) and low (column 7) mass amounts of extracts of demineralized bone matrix (DBM) (each having a total protein concentration of 500 μg / cc) used as reference. Column 1 consisted of an untreated cell population.

[0039] Figure 13 showed the ALK activities of demineralized cancellous sponges impregnated with bone marrow growth factor extracts (columns 2, 3, and 4) from three different bone marrow donors, uncoated sponges (column 1), liquid growth factor extracts (500 μg / cc) prepared from inactivated mineralized blocks (negative control, column 5), or BMP-2 solution (200 ng / cc) (positive control, column 6).

[0040] Figure 14 showed images of osteoblast cultures after 21 days of exposure to mineralized bone discs loaded with bone marrow growth factor extracts in an in vitro mineralization assay.

[0041] Figure 15 showed the calcium content in osteoblasts after 21 days of exposure to uncoated bone discs (left column) or mineralized bone discs coated with 500 μg / cc bone marrow growth factor extracts (right column).

[0042] Figure 16 showed the total protein concentration over time of mineralized cancellous blocks coated with growth factor extracts in response to different preservatives.

[0043] Figure 17 showed the BMP-2 concentration over time of mineralized cancellous blocks coated with growth factor extracts in response to different preservatives. Although the performance was the worst without cryoprotectant, sucrose performed as well as or better than trehalose at all observed time points.

[0044] Figure 18 showed live cells in the structural cell graft observed in situ using calcein staining (left panel), and outgrowing cells under an optical microscope after 2 weeks (middle panel) or 4 weeks (right panel).

[0045] Figure 19 showed the cell concentration in the structural cell prototype graft.

[0046] Figure 20Shows the average expression of CD45 and CD166 markers in 30×30×15 mm prototype grafts from three donors before and after processing. 3

[0047] Figure 21 Shows the corrected PBMC proliferation in response to osteocyte lymphocytes (column 1), concanavalin A (column 2), osteocytes from stromal cell grafts (column 4), or osteocytes from bone grafts (column 5).

[0048] Figure 22 Shows cells from outgrowth cultures fixed and stained with alizarin red after 2 weeks (left panel), 3 weeks (middle panel), or 4 weeks (right panel).

[0049] Figure 23 Shows the average BMP-2 or BMP-7 concentration in cells isolated from stromal cell prototype grafts of mineralized donor cells or control donor cells at day 3, day 7, day 14, or day 21.

[0050] Figure 24 Shows a stromal cell graft for posterolateral fusion (PLF) surgery.

[0051] Figure 25 Shows the viability map of a stromal cell prototype graft. DETAILED DESCRIPTION

[0052] The present invention relates to allografts comprising bone from a donor and living cells naturally present in bone, a matrix (such as a bone graft) impregnated with growth factors released from tissue having living cells, and methods for recovering growth factors from tissue having living cells, and methods for impregnating a subject with growth factors. The inventors unexpectedly found that cortical bone, cancellous or cortico-cancellous bone from a donor can be machined, minimally processed, and / or assembled into a structural bone graft comprising living bone cells for use in, for example, dental, foot and ankle, and spinal surgery of the donor. Such structural bone grafts can be allografts in any shape (e.g., blocks or granules) required at the surgical site. The inventors also unexpectedly found that a greater amount of higher activity growth factors can be recovered from tissue having natural living cells (e.g., bone marrow, bone matrix, periosteum, and / or endosteum) compared to those recovered from cryopreserved tissue not having natural living cells. The inventors also unexpectedly found that implants impregnated with growth factors released from bone marrow, bone matrix, periosteum, and / or endosteum comprising natural living cells exhibit improved osteoinductivity.

[0053] ​As used herein, the term "bone graft" refers to bone placed within or on the surface of a subject. The bone has been obtained from a donor and may have been processed. In cases where the subject is not the donor, the bone graft is also referred to herein as an allograft.

[0054] As used herein, the term "implant" refers to a device placed within or on the surface of a subject. A metal implant is an implant made of a metallic material. A synthetic implant is an implant made of a synthetic material that does not include metallic materials.

[0055] As used herein, the term "subject" refers to an animal, such as a mammal, which can be a human individual or a non-human individual, such as a cow, pig, dog (e.g., a canine), horse, sheep, or non-human primate (e.g., an ape and a chimpanzee).

[0056] As used herein, the term "donor" refers to the subject from which tissue or cells are obtained.

[0057] The terms "living cells" and "viable cells" are used interchangeably herein and refer to cells that are actively proliferating or metabolically active, as well as cells that are capable of actively proliferating or becoming metabolically active under suitable growth conditions.

[0058] As used herein, the term "native" refers to the origin of a cell. Cells that are native to a source (e.g., a tissue or a donor) are cells that are naturally present in that source. For example, cells that are native to a tissue are naturally present within or on the surface of the tissue. Cells that are native to a source can be isolated from the source and then optionally processed. For example, any method can be used to isolate cells that are native to a tissue, such as a solution containing proteases (e.g., trypsin and collagenase) that does not significantly affect the biological activity of the cells.

[0059] As used herein, the term "tissue" refers to a population of cells having a similar structure and performing a specific function. Tissue can be obtained from a donor. Examples of tissue include bone marrow, bone matrix, periosteum, and endosteum. Tissue obtained from a donor can be processed mechanically, for example, to remove cells or cell debris or to alter the physical or biological properties of the tissue.

[0060] As used herein, the term "fresh tissue" refers to tissue that has not been frozen after being obtained from a donor that has never been frozen or refrigerated. Fresh tissue has native living cells.

[0061] As used herein, the term "frozen tissue" refers to tissue that has been frozen after being obtained from a donor, or tissue obtained from a donor that has been frozen or refrigerated under conditions not suitable for maintaining cell viability. The natural living cells in the tissue before freezing are no longer viable after the tissue has been frozen.

[0062] As used herein, the term "bone marrow" refers to the tissue that is a spongy substance in the center of bone. Bone marrow contains bone marrow progenitor cells, bone marrow stem cells, hematopoietic progenitor cells (HPCs), and red blood cells.

[0063] As used herein, the term "bone matrix" refers to the tissue that is the intercellular substance of bone. The bone matrix contains osteocytes on the surface or within the structure of the bone matrix. The osteocytes in the bone matrix are also referred to as osteocytes. The bone matrix can be mineralized and / or demineralized bone particles and cellularized cortico-cancellous fragments, or mineralized and / or demineralized bone fibers and cellularized cortico-cancellous fragments.

[0064] As used herein, the term "periosteum" refers to the tissue that forms a fibrous sheath covering bone. The periosteum can contain osteocytes on the surface of the periosteum.

[0065] As used herein, the term "endosteum" refers to the tissue that forms a membrane lining the center of bone that has bone marrow. The endosteum can contain osteocytes on the inner bone surface.

[0066] As used herein, the terms "living tissue" and "viable tissue" are used interchangeably herein and refer to tissue that has natural living cells. For example, a bone graft having natural living cells is referred to as a living bone graft or a viable bone graft, while a bone graft that does not have natural living cells is referred to as a non-living bone graft or a non-viable bone graft.

[0067] As used herein, the term "cryopreservation" or "cryopreserved" refers to cooling tissue or cells to preserve the biological activity of the tissue or cells. Cryopreserved tissue (such as a bone graft) or cells can be coated with a cryopreservation solution, or partially or completely immersed in a cryopreservation solution. The cryopreservation solution can contain DMSO, glycerol, trehalose, ethylene glycol (EG), trehalose, HAS, BSA, mannitol, sucrose, glycerol, dextran, sodium alginate, EGCG, glucose, lactose, maltose, NFC, HES, PVP, and / or PEG.

[0068] As used herein, the term "lyopreservation" or "lyopreserved" refers to the lyophilization of frozen tissue or frozen cells, i.e., the removal of water from the frozen tissue or cells under vacuum. Lyopreserved tissue (such as bone grafts) or cells can be coated with or immersed in a lyopreservation solution. The lyopreservation solution can include trehalose, HAS, BSA, mannitol, sucrose, glycerol, dextran, sodium alginate, EGCG, glucose, lactose, maltose, NFC, HES, PVP, and / or PEG.

[0069] As used herein, the term "growth factor" refers to a substance that is naturally present in tissue or cells and has biological activity (such as the ability to stimulate cell proliferation, wound healing, and cell differentiation). Examples of growth factors include insulin-like growth factor (IGF), transforming growth factor (TGF) (such as TGFβ), bone morphogenetic protein (BMP) (such as BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) (such as αFGF and βFGF), stromal cell-derived factor-1 (SDF-1). Growth factors can be released from tissue or cells in which they are naturally present, and the released growth factors can retain biological activity. The released growth factors are also referred to as recovered growth factors. The released growth factors can be named with reference to their source. For example, growth factors released from bone marrow are called bone marrow growth factors (BMGF); growth factors released from bone matrix are called bone matrix growth factors (BGF); growth factors released from the periosteum are called periosteal growth factors (BPGF); growth factors released from the endosteum are called endosteal growth factors (BEGF); and growth factors released from osteocytes are called osteocyte growth factors (BCGF). A composition containing growth factors released from tissue or cells is also referred to as a growth factor extract or growth factor extraction.

[0070] As used herein, the term "cannula" refers to a thin tube used in a surgical procedure to deliver a substance to a treatment site of a subject. The cannula has an outer surface and an inner surface. The outer surface defines the outer diameter of the cannula. The inner surface forms a hole extending along the axis of the cannula and defines the inner diameter. The hole can be cylindrical.

[0071] As used herein, the term "pull-out force" refers to the force required to pull an article out of an object (such as a bone graft or an impregnated bone graft).

[0072] As used herein, the term "load sharing" refers to the distribution of a force applied to an object to one or more other objects. There may be one or more horizontal force distributions between the objects.

[0073] The present invention provides a method for preparing growth factors. According to the method for preparing growth factors, a composition containing growth factors from one or more tissues is prepared. The growth factors are naturally present in one or more tissues. Each of the one or more tissues is from a donor and has natural living cells. The method for preparing growth factors includes incubating the one or more tissues in one or more aqueous solutions to release the growth factors from the one or more tissues into the one or more aqueous solutions to produce one or more mixtures; and collecting one or more liquid portions from the one or more mixtures. The one or more collected liquid portions contain growth factors released from the one or more tissues.

[0074] When the one or more tissues consist of one tissue, one liquid portion collected according to the method for preparing growth factors is a composition containing growth factors released from the one tissue.

[0075] When the one or more tissues include two or more tissues, the method for preparing growth factors may include incubating the two or more tissues in an aqueous solution to produce a mixture, and collecting a liquid portion from the mixture. The liquid portion is a composition containing growth factors released from the two or more tissues.

[0076] When the one or more tissues include two or more tissues, the method for preparing growth factors may include incubating the two or more tissues in two or more aqueous solutions to produce two or more mixtures, wherein each or at least one of the two or more tissues may be incubated in one of the two or more aqueous solutions to produce a mixture; and collecting two or more liquid portions from one of the two or more mixtures respectively. The method for preparing growth factors may further include combining the two or more liquid portions into a composition. The composition contains growth factors released from the two or more tissues.

[0077] According to the method for preparing growth factors, the one or more tissues may be selected from bone marrow, bone matrix, periosteum, endosteum, and combinations thereof. The natural living cells may be osteocytes. When the tissue is bone marrow, the natural living bone cells may be in the bone marrow. When the tissue is bone matrix, periosteum, or endosteum, the natural living bone cells may be on the surface of or inside the bone matrix, periosteum, or endosteum. When the one or more tissues contain bone marrow, the method for preparing growth factors may further include removing red blood cells from the bone marrow, for example, by treating the bone marrow with ammonium chloride.

[0078] According to the method for preparing growth factors, one or more tissues can be obtained from one or more donors. Each donor can be an animal, such as a human individual. When the one or more tissues comprise two or more tissues, the two or more tissues can be from the same donor or two or more different donors. Before obtaining the one or more tissues from the one or more donors, the one or more donors can be unrefrigerated or unfrozen. Before obtaining tissue from at least one of the one or more donors, at least one of the one or more donors can be unrefrigerated or unfrozen.

[0079] According to the method for preparing growth factors, the one or more tissues can be unfrozen, cryopreserved, or lyophilized. At least one of the one or more tissues can be unfrozen, cryopreserved, and / or lyophilized.

[0080] The method for preparing growth factors can include obtaining bone marrow from a donor, where the bone marrow includes natural live cells; incubating the bone marrow in an aqueous solution to release growth factors from the bone marrow into the aqueous solution, thereby producing a mixture; and collecting the liquid portion from the mixture, where the liquid portion contains the growth factors released from the bone marrow. The method for preparing growth factors can further include removing red blood cells from the bone marrow, for example, by treating the bone marrow with ammonium chloride. The bone marrow can be unfrozen, cryopreserved, or lyophilized. Before obtaining the bone marrow from the donor, the donor can be unrefrigerated or unfrozen. The live cells can be osteocytes in the bone marrow.

[0081] The method for preparing growth factors can include obtaining a bone matrix from a donor, where the bone matrix contains natural live cells; incubating the bone matrix in an aqueous solution to release growth factors from the bone matrix into the aqueous solution, thereby producing a mixture; and collecting the liquid portion from the mixture, where the liquid portion contains the growth factors released from the bone matrix. The bone matrix can be unfrozen, cryopreserved, or lyophilized. Before obtaining the bone matrix from the donor, the donor can be unrefrigerated or unfrozen. The live cells can be osteocytes, such as osteocytes on the surface of or within the bone matrix.

[0082] The method for preparing growth factors can include obtaining a periosteum from a donor, where the periosteum contains natural live cells; incubating the periosteum in an aqueous solution to release growth factors from the periosteum into the aqueous solution, thereby producing a mixture; and collecting the liquid portion from the mixture, where the liquid portion contains the growth factors released from the periosteum. The periosteum can be unfrozen, cryopreserved, or lyophilized. Before obtaining the periosteum from the donor, the donor can be unrefrigerated or unfrozen. The live cells can be osteocytes, such as osteocytes on the surface of the periosteum.

[0083] A method for preparing a growth factor can include obtaining an endosteum from a donor, wherein the endosteum contains natural living cells; incubating the endosteum in an aqueous solution to release the growth factor from the endosteum into the aqueous solution, thereby producing a mixture; and collecting a liquid portion from the mixture, wherein the liquid portion contains the growth factor released from the endosteum. The endosteum may not be cryopreserved, stored at a low temperature, or freeze-dried. Before obtaining the endosteum from the donor, the donor may not be refrigerated or frozen. The living cells may be osteocytes, for example, osteocytes on the surface of the endosteum.

[0084] A method for preparing a growth factor can include obtaining one or more first tissues from a first donor, wherein the one or more first tissues include first natural living cells; incubating the one or more first tissues in a first aqueous solution to release a first growth factor from the one or more first tissues into the first aqueous solution, thereby producing a first mixture; collecting a first liquid portion from the first mixture, wherein the first liquid portion contains the first growth factor released from the one or more first tissues; obtaining one or more second tissues from a second donor, wherein the one or more second tissues include second natural living cells; incubating the one or more second tissues in a second aqueous solution to release a second growth factor from the one or more second tissues into the second aqueous solution, thereby producing a second mixture; collecting a second liquid portion from the second mixture, wherein the second liquid portion contains the second growth factor released from the one or more second tissues; and combining the first liquid portion and the second liquid portion to produce a composition that contains the first growth factor and the second growth factor, i.e., the growth factor from the one or more first tissues and the growth factor from the one or more second tissues. The one or more first tissues may be selected from bone marrow, bone matrix, periosteum, endosteum, and combinations thereof. The one or more second tissues may be selected from bone marrow, bone matrix, periosteum, endosteum, and combinations thereof. The one or more first tissues and the one or more second tissues may be different. For example, the one or more first tissues may be bone marrow, while the one or more second tissues may be bone matrix, periosteum, endosteum, or combinations thereof. The one or more first tissues may not have been cryopreserved, stored at a low temperature, or freeze-dried. The one or more second tissues may not have been cryopreserved, stored at a low temperature, or freeze-dried. Before obtaining the one or more first tissues from the first donor, the first donor may not have been refrigerated or frozen. Before obtaining the one or more second tissues from the second donor, the second donor may not have been refrigerated or frozen. The first donor and the second donor may be the same or different. The first natural living cells may be osteocytes. The second natural living cells may be osteocytes. The first aqueous solution and the second aqueous solution may be the same or different.

[0085] A method for preparing growth factors may include obtaining bone marrow from a first donor, wherein the bone contains first natural living cells; incubating the bone marrow in a first aqueous solution to release a first growth factor from the bone marrow into the first aqueous solution, thereby producing a first mixture; collecting a first liquid portion from the first mixture, wherein the first liquid portion contains the first growth factor released from the bone marrow; obtaining one or more other tissues from a second donor, wherein the one or more other tissues contain second natural living cells and are selected from bone matrix, periosteum, endosteum, and combinations thereof; incubating the one or more other tissues in a second aqueous solution to release a second growth factor from the one or more other tissues into the second aqueous solution, thereby producing a second mixture; collecting a second liquid portion from the second mixture, wherein the second liquid portion contains the second growth factor released from the one or more other tissues; and combining the first liquid portion and the second liquid portion to produce a composition that contains the first growth factor and the second growth factor, i.e., growth factors from bone marrow and one or more other tissues. The method for preparing growth factors may further include removing red blood cells from the bone marrow, for example, by treating the bone marrow with ammonium chloride. The bone marrow may not be cryopreserved, cryopreserved at low temperature, or freeze-dried. The one or more other tissues may not be cryopreserved, cryopreserved at low temperature, or freeze-dried. Before obtaining the bone marrow from the first donor, the first donor may not be refrigerated or frozen. Before obtaining the one or more other tissues from the second donor, the second donor may not be refrigerated or frozen. The first donor and the second donor may be the same or different. The first natural living cells may be bone cells. The second natural living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

[0086] A method for preparing a growth factor may include obtaining a bone matrix from a first donor, wherein the bone contains first natural living cells; incubating the bone matrix in a first aqueous solution to release a first growth factor from the bone matrix into the first aqueous solution, thereby producing a first mixture; collecting a first liquid portion from the first mixture, wherein the first liquid portion contains the first growth factor released from the bone matrix; obtaining one or more other tissues from a second donor, wherein the one or more other tissues contain second natural living cells and are selected from bone marrow, periosteum, endosteum, and combinations thereof; incubating the one or more other tissues in a second aqueous solution to release a second growth factor from the one or more other tissues into the second aqueous solution, thereby producing a second mixture; collecting a second liquid portion from the second mixture, wherein the second liquid portion contains the second growth factor released from the one or more other tissues; and combining the first liquid portion and the second liquid portion to produce a composition that contains the first growth factor and the second growth factor, i.e., growth factors from the bone matrix and the one or more other tissues. The bone matrix may not be cryopreserved, cryostored, or lyophilized. The one or more other tissues may not be cryopreserved, cryostored, or lyophilized. Before obtaining the bone matrix from the first donor, the first donor may not be refrigerated or frozen. Before obtaining the one or more other tissues from the second donor, the second donor may not be refrigerated or frozen. The first donor and the second donor may be the same or different. The first natural living cells may be bone cells. The second natural living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

[0087] A method for preparing growth factors may include obtaining a periosteum from a first donor, wherein the bone comprises first natural living cells; incubating the periosteum in a first aqueous solution to release a first growth factor from the periosteum into the first aqueous solution, thereby producing a first mixture; collecting a first liquid portion from the first mixture, wherein the first liquid portion comprises the first growth factor released from the periosteum; obtaining one or more other tissues from a second donor, wherein the one or more other tissues comprise second natural living cells and are selected from bone marrow, bone matrix, endosteum, and combinations thereof; incubating the one or more other tissues in a second aqueous solution to release a second growth factor from the one or more other tissues into the second aqueous solution, thereby producing a second mixture; collecting a second liquid portion from the second mixture, wherein the second liquid portion comprises the second growth factor released from the one or more other tissues; and combining the first liquid portion and the second liquid portion to produce a composition comprising the first growth factor and the second growth factor, i.e., growth factors from the periosteum and the one or more other tissues. The periosteum may not be cryopreserved, cryopreserved, or lyophilized. The one or more other tissues may not be cryopreserved, cryopreserved, or lyophilized. Before obtaining the periosteum from the first donor, the first donor may not be refrigerated or frozen. Before obtaining the one or more other tissues from the second donor, the second donor may not be refrigerated or frozen. The first donor and the second donor may be the same or different. The first natural living cells may be bone cells. The second natural living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

[0088] A method for preparing growth factors may include obtaining endosteum from a first donor, wherein the bone comprises first natural living cells; incubating the endosteum in a first aqueous solution to release a first growth factor from the endosteum into the first aqueous solution, thereby producing a first mixture; collecting a first liquid portion from the first mixture, wherein the first liquid portion comprises the first growth factor released from the endosteum; obtaining one or more other tissues from a second donor, wherein the one or more other tissues comprise second natural living cells and are selected from bone marrow, bone matrix, periosteum, and combinations thereof; incubating the one or more other tissues in a second aqueous solution to release a second growth factor from the one or more other tissues into the second aqueous solution, thereby producing a second mixture; collecting a second liquid portion from the second mixture, wherein the second liquid portion comprises the second growth factor released from the one or more other tissues; and combining the first liquid portion and the second liquid portion to produce a composition that comprises the first growth factor and the second growth factor, i.e., growth factors from the endosteum and the one or more other tissues. The endosteum may not be cryopreserved, cryopreserved at low temperature, or freeze-dried. The one or more other tissues may not be cryopreserved, cryopreserved at low temperature, or freeze-dried. Before obtaining the endosteum from the first donor, the first donor may not be refrigerated or frozen. Before obtaining the one or more other tissues from the second donor, the second donor may not be refrigerated or frozen. The first donor and the second donor may be the same or different. The first natural living cells may be bone cells. The second natural living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

[0089] The method for preparing growth factors may further include adjusting the pH of the composition to a neutral pH, such as from about 6.5 to 7.5, 6.5 to 7.0, 7.0 to 7.5, 6.8 to 7.2, 6.8 to 7, 7 to 7.2, 6.9 to 7.1, 6.9 to 7.0, or 7.0 to 7.1.

[0090] The method for preparing growth factors may further include measuring the protein concentration of the composition, the first liquid portion, and / or the second liquid portion, and / or increasing the protein concentration of the composition, the first liquid portion, and / or the second liquid portion.

[0091] The method for preparing growth factors may further include removing one or more salts from the composition, the first liquid portion, and / or the second liquid portion.

[0092] According to the method for preparing growth factors, the aqueous solution can have a pH of 0 to 6.9, 0 to 6.5, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1.0 to 6.9, 1.0 to 6.5, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2.0 to 6.9, 2.0 to 6.5, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3.0 to 6.9, 3.0 to 6.5, 3 to 6, 3 to 5, 3 to 4, 4.0 to 6.9, 4.0 to 6.5, 4.0 to 6.4, 4 to 5, 5.0 to 6.9, 5.0 to 6.5, 5 to 6, 6.0 to 6.9, or 6.0 to 6.5. The aqueous solution can contain an acid selected from acetic acid, hydrochloric acid, citric acid, sulfuric acid, and formic acid. The aqueous solution can be a first aqueous solution and / or a second aqueous solution.

[0093] According to the method for preparing growth factors, the incubation step can include disrupting living cells. The disruption can include agitation (e.g., freeze-thaw cycles, stirring, shaking, rotation, oscillation), cryogenic ball milling, centrifugation, and / or sonication of the tissue. The living cells can be first living cells or second living cells.

[0094] According to the method for preparing growth factors, the living cells can be selected from osteocytes, stem cells, multipotent mesenchymal stromal cells, and combinations thereof. The osteocytes can be selected from osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, bone lining cells, and combinations thereof. The living cells can be first living cells and / or second living cells.

[0095] According to the method for preparing growth factors, the growth factors released from one or more tissues can be selected from insulin-like growth factor (IGF), transforming growth factor (TGF) (e.g., TGFβ), bone morphogenetic protein (BMP) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) (e.g., αFGF and βFGF), stromal cell-derived factor-1 (SDF-1), and combinations thereof.

[0096] According to the method for preparing growth factors, the growth factors released from bone marrow can be selected from insulin-like growth factor (IGF), transforming growth factor (TGF) (e.g., TGFβ), bone morphogenetic protein (BMP) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) (e.g., αFGF and βFGF), stromal cell-derived factor-1 (SDF-1), and combinations thereof.

[0097] According to the method for preparing growth factors, the growth factors released from the bone matrix can be selected from insulin-like growth factors (IGFs), transforming growth factors (TGFs) (e.g., TGFβ), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factors (PDGFs), vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs) (e.g., αFGF and βFGF), stromal cell-derived factor-1 (SDF-1), and combinations of the foregoing. In one embodiment, the composition or extract comprising the growth factors released from the bone matrix can further comprise calcium ions (Ca ++ ) from the bone matrix, and the released growth factors and Ca ++ can be impregnated onto a bone graft, a metal implant, or a polymer implant.

[0098] According to the method for preparing growth factors, the growth factors released from the periosteum can be selected from insulin-like growth factors (IGFs), transforming growth factors (TGFs) (e.g., TGFβ), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factors (PDGFs), vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs) (e.g., αFGF and βFGF), stromal cell-derived factor-1 (SDF-1), and combinations of the foregoing.

[0099] According to the method for preparing growth factors, the growth factors released from the endosteum can be selected from insulin-like growth factors (IGFs), transforming growth factors (TGFs) (e.g., TGFβ), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factors (PDGFs), vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs) (e.g., αFGF and βFGF), stromal cell-derived factor-1 (SDF-1), and combinations of the foregoing.

[0100] According to the method for preparing growth factors, the bone matrix can include cellularized cortical fragments, cortico-cancellous fragments, cellularized cortical bone fibers, cellularized cancellous blocks, cellularized cortical blocks, or cortico-cancellous blocks. For example, the bone matrix can include (i) mineralized and / or demineralized bone particles and cellularized cortico-cancellous fragments, or (ii) mineralized and / or demineralized bone fibers and cellularized cortico-cancellous fragments. The bone matrix can be prepared from bone selected from cancellous bone, cortico-cancellous bone, cortical bone, and combinations of the foregoing.

[0101] The method for preparing growth factors may further include storing the composition; lyophilizing or freeze-drying the composition; sterilizing the composition; and / or packaging the composition. The composition may be sterilized by gamma radiation, electron beam (e-beam), x-rays, or ethylene oxide (EtO).

[0102] In one embodiment, the method for preparing growth factors includes Figure 1 the processing steps shown: obtaining tissue from a donor, such as bone marrow; treating the tissue with ammonium chloride, for example, to remove red blood cells from the bone marrow; treating the tissue with an acid to release growth factors from the tissue to produce a sample; centrifuging the sample and neutralizing the pH of the sample; desalting the pH-neutralized sample; concentrating the desalted sample using a stirred cell concentrator; and measuring the protein content in the concentrated sample. Thus, a growth factor extract is recovered from the donor tissue.

[0103] For each method for preparing growth factors of the present invention, a composition prepared according to the method for preparing growth factors is provided. The growth factors in the composition may include BMGF, BGF, BPGF, BEGF, and / or BCGF. For example, the composition may include BCGF, and osteocytes may be in the bone marrow, and / or on the surface of the bone matrix, periosteum, and / or endosteum. The composition may be dried. The dried composition may be in the form of a powder. The dried composition may be rehydrated.

[0104] The composition may contain growth factors at a concentration of about 0.001 ng / g to 100,000 ng / g, 0.001 ng / g to 50,000 ng / g, 0.001 ng / g to 42,000 ng / g, 0.001 ng / g to 40,000 ng / g, 0.001 ng / g to 30,000 ng / g, 0.001 ng / g to 20,000 ng / g, 0.001 ng / g to 10,000 ng / g, 0.001 ng / g to 5,000 ng / g, 0.001 ng / g to 1,000 ng / g, 0.001 ng / g to 500 ng / g, 0.001 ng / g to 100 ng / g, 0.001 ng / g to 50 ng / g, 0.001 ng / g to 10 ng / g, 0.001 ng / g to 1 ng / g, 0.001 ng / g to 0.01 ng / g, or 0.001 ng / g to 0.1 ng / g, based on the total weight of the composition.

[0105] The composition may be packaged, lyophilized, and / or sterilized until used in a surgical procedure, such as applied to a surgical site to improve bone formation. For example, the composition may be applied to the surface of a metal or polymer implant and then a surgical procedure is performed.

[0106] The present invention also provides a method for improving the osteoinductivity of an implant. The improvement method includes incubating the implant with an effective amount of the composition of the present invention to produce an impregnated implant. The implant includes a bone graft, a metallic material, a synthetic material, or a combination of the foregoing. The impregnated implant has greater osteoinductivity than the implant before incubation.

[0107] According to the improvement method, the implant can be incubated with the composition in the presence of a reagent. The reagent can be a solvent, a preservative, or a combination of the foregoing. Incubation can include agitating (e.g., stirring, shaking, rotating, oscillating) and / or sonication of the implant and the composition. The improvement method can further include storing the impregnated implant in a container.

[0108] In the case where the implant includes a bone graft, the improvement method can further include obtaining bone from a donor and cryopreserving the obtained bone to fabricate the bone graft. The improvement method can further include lyophilizing the impregnated bone graft. The improvement method can further include demineralizing the impregnated bone graft before lyophilization. The improvement method can further include sterilizing the impregnated bone graft. The bone graft can be an allograft of the present invention.

[0109] The bone graft can be demineralized. The bone graft can include demineralized bone matrix (DBM) particles, DBM fibers, DBM blocks from, for example, cortical bone plates, DBM sponges from, for example, cancellous bone blocks, and / or DBM putties, optionally with a carrier.

[0110] The impregnated bone graft can include impregnated demineralized bone matrix (DBM) particles, DBM fibers, DBM blocks from, for example, cortical bone plates, DBM sponges from, for example, cancellous bone blocks, and / or DBM putties, optionally with a carrier. The impregnated DBM putty can be flowable and delivered through an injection needle into deep defects. The carrier can be derived from bone. For example, the impregnated bone graft can include impregnated DBM fibers.

[0111] The impregnated bone graft can contain growth factors from one or more tissues selected from bone marrow, bone matrix, periosteum, endosteum, and combinations of the foregoing. For example, the impregnated bone graft can contain growth factors from bone marrow (BMGF) and growth factors from one or more other tissues selected from bone matrix, periosteum, endosteum, and combinations of the foregoing. The bone marrow can contain natural live bone cells in the bone marrow. The one or more other tissues can contain natural live bone cells on the surface of the one or more other tissues, the one or more other tissues selected from bone matrix, periosteum, endosteum, and combinations of the foregoing. The one or more other tissues can include the periosteum.

[0112] The bone graft can be non - demineralized. The bone graft can include cortical, cancellous, or cortico - cancellous granules.

[0113] The bone graft can be selected from cortical pins, blocks or plates, cancellous blocks or strips, cortico-cancellous blocks or strips, cortical bone rings, and combinations of the foregoing.

[0114] The impregnated bone graft can have a pull-out force of at least about 0.1 N, 0.5 N, 1 N, 5 N, 10 N, 20 N, or 50 N for a surgical instrument in the impregnated bone graft. The impregnated bone graft can be load-sharing. The implanted bone graft can share forces with one or more load-bearing constructs (such as plates and / or screws). Different levels of force distribution can exist between the implanted bone graft and the load-bearing constructs. The impregnated bone graft can have machining properties, e.g., be capable of being further machined, cut, drilled, or shaped. The impregnated bone graft can be assembled with one or more additional bone grafts.

[0115] According to the improved method, the implant can include a metallic material. Such metallic implants can be joint implants, e.g., joint implants in the knee, shoulder, elbow, ankle, or finger; spinal cages, which can be expandable and / or 3D printed; or metallic plates, screws, or dental implants.

[0116] According to the improved method, the implant can include a synthetic material. The synthetic material can be a synthetic bone void filler, such as tricalcium phosphate (TCP) or bioglass. The synthetic implant can be a polymer implant, such as a PEEK implant.

[0117] For each improved method of the present invention, a product is provided that includes an impregnated implant prepared according to the method.

[0118] For each product of the present invention, a container is provided. The container includes the product in a liquid. The liquid can contain the composition of the present invention. The liquid can be a cryopreservation, lyophilization preservation, or radiation protection agent solution. The impregnated implant can be an impregnated bone graft, and the container can be a cannula for injection in minimally invasive surgery (MIS). The cannula can have a density less than about 1 g / cm 3 、1.1 g / cm 3 、1.2 g / cm 3 、1.3 g / cm 3 、1.4 g / cm 3 、1.5 g / cm 3 、1.6 g / cm 3 、1.7 g / cm 3 、1.8 g / cm 3 、1.9 g / cm 3 or 2.0 g / cm 3The particle density. The impregnated bone graft may have a maximum extrusion force of about 10 N to 500 N, 10 N to 400 N, 10 N to 300 N, 10 N to 200 N, 100 N to 500 N, 100 N to 400 N, 100 N to 300 N, 100 N to 200 N, 150 N to 500 N, 150 N to 400 N, 150 N to 300 N, 150 N to 250 N, 150 N to 200 N, or 160 N to 200 N (such as about 180 N). The cannula may be loaded with an impregnated bone graft having a density of at least about 0.01 g / cm 3 ³, 0.05 g / cm 3 ³, 0.1 g / cm 3 ³, 0.15 g / cm 3 ³, 0.2 g / cm 3 ³, 0.3 g / cm 3 ³, 0.4 g / cm 3 ³, 0.5 g / cm 3 ³ or 1.0 g / cm 3 ³ of the impregnated bone graft.

[0119] The present invention also provides a treatment method. The treatment method includes treating cells with an effective amount of the composition of the present invention to reduce the release of pro-inflammatory cytokines by the cells.

[0120] The present invention also provides an allograft. The allograft includes bone from a donor and living bone cells. The living bone cells are naturally present in the bone and on the surface of the bone. The allograft does not include demineralized bone matrix (DBM). The size of the allograft is greater than about 1×1×1 mm 3 ³, 2×2×2 mm 3 ³, 3×3×3 mm 3 ³, 4×4×4 mm 3 ³, 5×5×5 mm 3 ³, or 10×10×10 mm 3The bone cells can be selected from osteocytes, osteoblasts, bone lining cells, progenitor cells, and combinations of the foregoing. The bone can be selected from cancellous bone, cortico-cancellous bone, cortical bone, and combinations of the foregoing. The allograft can have at least about 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, one million, two million, or three million viable bone cells per cubic centimeter of the allograft. The allograft can have a native lipid content of less than about 1 mg, 5 mg, 10 mg, 50 mg, or 100 mg per cubic centimeter of the allograft. The bone can include bone marrow, and at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the native hematopoietic cells may have been removed from the bone marrow. The allograft can have a pull-out force of at least about 0.1 N, 0.5 N, 1 N, 5 N, 10 N, 20 N, or 50 N for a surgical instrument in the bone. The allograft can be assembled with a bone portion, a metal implant, or a polymer implant (e.g., a cage, plate, or screw). The bone portion can contain native viable bone cells. The allograft can be load-sharing. The allograft can share force with one or more load-bearing constructs (e.g., plates and / or screws). There can be different levels of force distribution between the allograft and the load-bearing constructs. The allograft can have machining properties, e.g., be capable of being further machined, cut, drilled, or shaped.

[0121] For each allograft of the present invention, a container is provided. The container includes the allograft in a liquid, the liquid coating the allograft. The liquid contains the composition of the present invention. The volume ratio of the liquid to the bone is less than about 0.1, 0.5, 1, 5, or 10. The liquid can be a cryopreservation, lyophilization preservation, or radioprotectant solution.

[0122] As used herein, the term "about" modifies, for example, dimensions, volume, amount, concentration, processing temperature, processing time, yield, flow rate, pressure, and the like and ranges thereof of components in a composition, and refers to variations in the numerical amounts that can be produced by, for example: typical measurement and processing procedures used in preparing compounds, compositions, concentrates, or use formulations; inadvertent errors in these procedures; differences in the manufacture, source, or purity of starting materials or components used to implement the methods; and similar considerations. The term "about" also includes amounts that differ due to, for example, the aging of a composition, formulation, or cell culture or mixture having a particular initial concentration, and amounts that differ due to mixing or processing a composition or formulation or mixture having a particular initial concentration. Whether or not modified by the term "about", the appended claims include equivalents of these amounts. The term "about" further may refer to a range of values similar to the reference value. In certain embodiments, the term "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0123] Example 1. Preparation of Growth Factor Extract from Tissue

[0124] Methods for preparing growth factor extracts and subsequent reapplication are provided. The procedure is applicable to tissues including bone matrix (such as osteocyte matrix), bone marrow, periosteum, and / or endosteum, whereby growth factors are extracted from the tissue, optionally combined, and subsequently reapplied to a target implant, which includes, for example, a bone graft such as DBM fibers.

[0125] Growth factors are extracted from osteocyte matrix. The osteocyte matrix can include demineralized bone particles and cellularized cortico-cancellous bone fragments, or demineralized bone fibers and cellularized cortico-cancellous bone fragments. These products are then lyophilized, after which growth factor extraction is further facilitated. Growth factors extracted from other bone matrices can be prepared using the same procedure. In one aspect, the following steps can be carried out in a clean room or laminar flow hood to maintain aseptic conditions:

[0126] 1. Prepare a 10% acetic acid solution by diluting with deionized water.

[0127] 2. Generate a volume equivalent to twice the volume of the extracted osteocyte matrix product.

[0128] 3. Example: For 50 cc of osteocyte matrix product, 100 cc of 10% acetic acid is generated by mixing 90 cc acetic acid with 10 cc dH2O.

[0129] 4. Remove the osteocyte matrix product from the sachet and wash it in a sterile basin with HBSS for 30 seconds, twice, as applicable.

[0130] 5. Place the bone cell matrix product in a suitable sterile, sealed container and add an appropriate volume of 10% acetic acid solution.

[0131] 6. Place on a shaker at 4 °C for 20 to 24 hours.

[0132] 7. Transfer the sample to a 50 mL conical tube.

[0133] 8. Equilibrate and centrifuge at 4,000 xg for 15 minutes at 4 °C.

[0134] 9. Collect the supernatant and neutralize with 0.3 x (recorded volume) 5N NaOH.

[0135] 10. Check the pH.

[0136] 11. Select a sufficient number of Zeba TM spin desalting columns for the total volume of the extract.

[0137] 12. For each column: Unscrew the bottom closure of the column and loosen the cap; place the column in a 50 mL conical tube; add 5 mL of PBS to the column; centrifuge at 1,000 xg for 2 minutes, discard the buffer, and repeat twice; place the column in a new 50 mL tube.

[0138] 13. Apply the sample portion to the column and centrifuge at 1,000 xg for 2 minutes.

[0139] 14. Pool the portions.

[0140] Growth factors are extracted from bone marrow. Bone marrow is collected by rasping the bone marrow contents of long bones (e.g., femur, tibia, and / or humerus) into a sterile basin and then transferring it to a sterile 50 mL conical tube to facilitate further extraction. In one aspect, the following steps can be performed in a clean room or laminar flow hood to maintain aseptic conditions:

[0141] 1. Prepare a 10% acetic acid solution by diluting with deionized water.

[0142] 2. Generate a volume equivalent to twice the volume of the extracted bone marrow.

[0143] 3. Example: For 50 cc of bone marrow, prepare 100 cc of 10% acetic acid by mixing 90 cc of acetic acid with 10 cc of dH2O.

[0144] 4. Combine the bone marrow with an appropriate volume of 10% acetic acid solution in a sterile, sealed container.

[0145] 5. Place on a shaker at 4 °C for 20 to 24 hours.

[0146] 6. Transfer the sample to a 50 mL conical tube.

[0147] 7. Balance and centrifuge at 4,000 x g for 15 minutes at 4 °C.

[0148] 8. Collect the supernatant, record the volume, and neutralize with 0.3 × (recorded volume) of 5N NaOH.

[0149] 9. Check the pH.

[0150] 10. Select a sufficient number of Zeba TM spin desalting columns for the total volume of the extract.

[0151] 11. For each column: Unscrew the bottom closure of the column and loosen the cap; place the column in a 50 mL conical tube; add 5 mL of PBS to the column; centrifuge at 1,000 x g for 2 minutes, discard the buffer, and repeat twice; place the column in a new 50 mL tube.

[0152] 12. Apply the sample portion to the column and centrifuge at 1,000 x g for 2 minutes.

[0153] 13. Pool the portions.

[0154] Extract growth factors from the periosteum. The periosteum is recovered by using an osteotome on long bones (e.g., femur, tibia, and / or humerus) to recover the thin tissue on the bone surface. Collect the soft tissue fragments on a sterile basin and transfer them to a sterile 50 mL conical tube to facilitate further extraction. In one aspect, the following steps can be performed in a clean room or laminar flow hood to maintain sterile conditions:

[0155] 1. Prepare a 10% acetic acid solution by diluting with deionized water.

[0156] 2. Generate a volume equivalent to twice the volume of the extracted periosteum.

[0157] 3. Example: For 50 cc of periosteum, prepare 100 cc of 10% acetic acid by mixing 90 cc of acetic acid with 10 cc of dH2O.

[0158] 4. Wash twice in HBSS in a sterile basin for 30 seconds.

[0159] 5. Place the periosteum in a suitable sealed sterile container and add an appropriate volume of 10% acetic acid solution.

[0160] 6. Place on a shaker at 4 °C for 20 to 24 hours; record the start time.

[0161] 7. Transfer the sample to a 50 mL conical tube.

[0162] 8. Balance and centrifuge at 4,000 x g for 15 minutes at 4 °C.

[0163] 9. Collect the supernatant, record the volume, and neutralize it with 0.3×(recorded volume) of 5N NaOH.

[0164] 10. Check the pH.

[0165] 11. Select a sufficient number of Zeba TM spin desalting columns for the total volume of the extract.

[0166] 12. For each column: Unscrew the bottom closure of the column and loosen the cap; Place the column in a 50 mL conical tube; Add 5 mL of PBS to the column; Centrifuge at 1,000 x g for 2 minutes, discard the buffer, and repeat twice; Place the column in a new 50 mL tube.

[0167] 13. Apply the sample portion to the column and centrifuge at 1,000 x g for 2 minutes.

[0168] 14. Pool the portions.

[0169] Extract growth factors from the endosteum. The endosteum is recovered by using an osteotome or reamer on a long bone with marrow (e.g., femur, tibia, and / or humerus) to recover the thin tissue lining the center of the bone. Collect the soft tissue fragments on a sterile basin and transfer them to a sterile 50 mL conical tube to facilitate further extraction. In one aspect, the following steps can be performed in a clean room or laminar flow hood to maintain aseptic conditions:

[0170] 1. Prepare a 10% acetic acid solution by diluting with deionized water.

[0171] 2. Generate a volume equivalent to twice the extracted endosteum.

[0172] 3. Example: For 50 cc of endosteum, prepare 100 cc of 10% acetic acid by mixing 90 cc of acetic acid with 10 cc of dH2O.

[0173] 4. Wash in HBSS in a sterile basin for 30 seconds, twice.

[0174] 5. Place the endosteum in a suitable sealed sterile container and add an appropriate volume of 10% acetic acid solution.

[0175] 6. Place on a shaker at 4°C for 20 to 24 hours and record the start time.

[0176] 7. Transfer the sample to a 50 mL conical tube.

[0177] 8. Equilibrate and centrifuge at 4,000 x g for 15 minutes at 4°C.

[0178] 9. Collect the supernatant, record the volume, and neutralize it with 0.3×(recorded volume) of 5N NaOH.

[0179] 10. Check the pH.

[0180] 11. Select a sufficient number of Zeba TM spin desalting columns for the total volume of the extract.

[0181] 12. For each column: Unscrew the bottom closure of the column and loosen the cap; Place the column in a 50 mL conical tube; Add 5 mL of PBS to the column; Centrifuge at 1,000 x g for 2 minutes, discard the buffer, and repeat two more times; Place the column in a new 50 mL tube.

[0182] 13. Apply the sample portion to the column and centrifuge at 1,000 x g for 2 minutes.

[0183] 14. Pool the portions.

[0184] Growth factor extraction solution. In order to reapply the extracted growth factors to a recipient implant (e.g., a bone graft such as DBM fibers), the final growth factor solution can consist of one or more of the following options in any ratio deemed appropriate: osteocyte matrix growth factor extract, bone marrow growth factor extract, periosteal growth factor extract, and / or endosteal growth factor extract.

[0185] Reapply the growth factor extraction solution to the DBM fibers according to the following steps:

[0186] 1. Place 1 cc of DBM fibers in a lyophilization tray.

[0187] 2. Add the growth factor extract solution to the DBM fibers in the tray using a 1:3 ratio (cc of fibers to mL of extract).

[0188] 3. Place the lyophilization tray containing the DBM fibers and the growth factor extract solution in an -80°C freezer until the sample is completely frozen.

[0189] 4. Place the frozen sample in a roll print package for lyophilization.

[0190] 5. Pre-freeze the lyophilization rack to -40°C and then add the sample.

[0191] 6. When cooled, add the sample tray to the lyophilizer and use the cycle (lyophilization cycle parameters) mentioned in the following table.

[0192] 7. Remove the sample from the lyophilizer and place it in a Tyvek pouch for storage (Table 1).

[0193] Table 1. Lyophilization cycle parameters

[0194] Step Shelf Temperature (°C) Ramp (min) Hold (min) Pressure (mTorr) 1 -40 0 60 350 2 35 80 0 350 3 35 0 720 350 4 35 0 720 350 5 0 0 0 350 6 0 0 0 350

[0195] Sterilization. Submit packaging samples for sterilization (γ - radiation) according to the established company procedures for typical DBM fiber products.

[0196] Example 2. Growth factor extracts prepared from bone marrow or bone matrix

[0197] Prepare growth factor extracts from fresh tissue (e.g., bone marrow or bone matrix) obtained from a donor within 60 hours after receipt, frozen tissue (stored at - 80°C for more than one week), O / N frozen tissue (stored at - 80°C overnight), or O / N RT tissue (stored at room temperature (~20°C) overnight) using the method described in Example 1. The tissue is bone marrow or bone matrix.

[0198] Figure 2 It shows that when the source tissue is from a frozen donor source, the total protein concentration in growth factor extracts from bone marrow - derived and bone - matrix - derived sources both decrease. Quantify the total protein concentration using Pierce BCA protein assay and according to the manufacturer's instructions.

[0199] Figure 3 It shows that when the source tissue is from a frozen donor source, the concentration of BMP - 2 in growth factor extracts from bone marrow - derived and bone - matrix - derived sources both decrease. Quantify the BMP - 2 concentration using a BMP - 2 ELISA kit and according to the manufacturer's instructions.

[0200] Figure 4 It shows that the total protein concentration in growth factor extracts prepared from O / N frozen bone marrow or O / N RP bone marrow decreases compared to that from growth factor extracts prepared from fresh bone marrow. Quantify the total protein using Pierce BCA protein assay and according to the manufacturer's instructions.

[0201] Figure 5 It shows the expression of BMP2, BMP7, and PDGF growth factors in growth factor extracts prepared from O / N frozen bone marrow or O / N RP bone marrow compared to those from growth factor extracts prepared from fresh bone marrow. Quantify the growth factor concentration using an ELISA kit specific for the growth factor and according to the manufacturer's instructions.

[0202] Figure 6 It shows the concentration of BMP - 2 in growth factor extracts prepared from fresh bone marrow or fresh bone matrix. Measure the BMP - 2 concentration in the extract using a commercially available enzyme - linked immunosorbent assay (ELISA) kit and according to the manufacturer's instructions.

[0203] Figure 7Shows the concentrations of BMP-4, IGF, TGF-β, BMP-7, VEGF, aFGF, SDF-1, PDGF, and bFGF in growth factor extracts prepared from fresh bone marrow or fresh bone matrix. The growth factor concentrations in the extracts were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit and according to the manufacturer's instructions. These selected growth factors represent proteins involved in various stages related to bone healing and repair.

[0204] Example 3. Growth Factor Extract Prepared from Predemineralized Cortical Bone

[0205] Growth factor extracts can be prepared from intermediate processing steps of allograft tissue, such as from the acid wash solution of cortical bone intended for demineralization. In this example, the cortical bone was demineralized in HCl, and when the bone proceeded to subsequent steps of the process, the acid was not discarded. In this example, the HCl can be collected and the growth factor extract can be recovered from the acid. This is an intermediate process of bone matrix demineralization. Instead of discarding the HCl used to demineralize bone tissue, it was neutralized with NaOH, clarified by centrifugation, and the growth factors of the present application were retained, which were stripped together with calcium-containing minerals. Figure 8 Shows the total protein concentration yields of a proof-of-concept process using acetic acid when preparing growth factor extracts from bone matrix, periosteum, endosteum, or bone marrow.

[0206] Example 4. Bone Grafts Coated with Growth Factor Extracts

[0207] Inactivated and mineralized cancellous cubes of bone grafts coated with bone marrow growth factor extracts were prepared using the method described in Example 1. The bone grafts were incubated with bone marrow growth factor extracts for residence times of t = 0 hours (Method 1), t = 16 hours (Method 2), or t = 1 hour (Method 3), and then lyophilized. The coated bone grafts were placed in saline in an incubator, and the saline was sampled at 24-hour intervals for quantitative protein to show that release occurred. The positive control was a cube loaded with albumin (t = 0), and the negative control was an unloaded cube. Figure 9 Shows the total bone marrow protein release profile of the coated bone grafts.

[0208] Example 5. In Vitro Osteoinductivity

[0209] The in vitro osteoinductivity of growth factor extracts, which can be prepared from different tissues or grafts impregnated with growth factor extracts, was evaluated in in vitro alkaline phosphatase (ALP) assays and in vitro mineralization assays.

[0210] In vitro ALP assay. C2C12 cells were cultured in DMEM supplemented with 1% FBS for 3 days under standard cell culture conditions. After 3 days of exposure to the test samples, the alkaline phosphatase activity in the C2C12 cell lysates was measured and normalized to the total protein. Briefly, the cells were lysed on ice for 30 minutes in lysis buffer containing 0.125% Triton X-100. Then the cell lysates were combined with 3 mM p-nitrophenyl phosphate in a 96-well plate and incubated at 37 °C and 5% CO2 for 60 minutes. After 60 minutes, the reaction was terminated with 1N NaOH and the absorbance was read at 405 nm using a spectrophotometer. Alkaline phosphatase (ALK) activity is an early indicator of osteoblast formation, indicating the early stage of bone formation.

[0211] Figure 10 An increase in ALK activity was shown after the introduction of a liquid growth factor extract from bone marrow-derived (500 μg / cc), a liquid growth factor extract prepared from inactivated mineralized squares (500 μg / cc) (negative control), or a BMP-2 solution (200 ng / cc) (positive control).

[0212] Figure 11 An increase in ALK activity was shown after the introduction of liquid growth factor extracts prepared from representative bone marrow (columns 1 to 3), periosteum (column 4), or bone matrix (columns 5 to 8), each of the above liquid growth factor extracts having a total protein concentration of 500 μg / cc; a liquid growth factor extract prepared from inactivated mineralized squares (500 μg / cc) (negative control) or a BMP-2 solution (200 ng / cc) (positive control).

[0213] Figure 12 An increase in ALK activity was shown after the introduction of liquid growth factor extracts prepared from representative bone marrow components, the representative bone marrow components being bone marrow supernatant (liquid fraction, column 4), bone marrow pellet (cell fraction, column 5); undiluted bone marrow (bone marrow, column 9), periosteum (column 8) or bone matrix (column 10), each of the above liquid growth factor extracts having a total protein concentration of 500 μg / cc; a liquid growth factor extract prepared from inactivated mineralized squares (500 μg / cc) (negative control, column 2) or a BMP-2 solution (200 ng / cc) (positive control, column 3); extracts of high (column 6) and low (column 7) mass amounts of demineralized bone matrix (DBM) (each having a total protein concentration of 500 μg / cc) as a reference. Column 1 constituted the untreated cell population.

[0214] Figure 13Shows an increase in ALK activity after introduction of demineralized cancellous sponges impregnated with bone marrow growth factor extract, 500 μg of the sponge per cc of the extract. Columns 2, 3, and 4 describe three different bone marrow donors. Column 1 is the uncoated sponge. And columns 5 and 6 are liquid growth factor extracts (500 μg / cc) (negative control) or BMP-2 solution (200 ng / cc) (positive control) prepared from inactivated mineralized blocks, respectively.

[0215] In vitro mineralization assay. For this study, osteoblasts were exposed to discs loaded with bone marrow growth factor extracts from two separate donors (donor 1 and donor 2) using transwell inserts over a 21-day period in the presence of a medium containing ascorbic acid and β-glycerophosphate. Untreated controls represent the presence of mineralized bone discs without growth factor extract, and cell controls represent the absence of any loaded or unloaded mineralized bone discs. While ALK activity can represent an early indicator of in vitro bone formation through cell differentiation, the mineralization assay shows actual late-stage mineral formation in the culture.

[0216] Figure 14 Shows an image of an osteoblast culture after 21 days of exposure to mineralized bone discs loaded with bone marrow growth factor extract (500 μg / cc) in an in vitro mineralization assay. The black nodules are von Kossa-stained calcium-containing mineralized deposits, indicating new bone formation in the culture.

[0217] Figure 15 Shows the calcium content in osteoblasts exposed to uncoated (left column) or mineralized bone discs coated with 500 μg / cc bone marrow growth factor extract (right column) for 21 days. Osteoblasts exposed to discs loaded with bone marrow extract expressed a higher amount of calcium-containing mineralized deposits, as confirmed by the images on the previous page (donor 1 triptych and donor 2 triptych), indicated by the presence of positive von Kossa staining (black nodules).

[0218] Example 6. Shelf-life of growth factor extract

[0219] Growth factor extracts prepared from the bone marrow of 3 separate donors were used to coat mineralized blocks (500 μg / cc), with sucrose, trehalose, or neither added as a cryoprotectant, followed by lyophilization and storage. After 1, 4, or 12 weeks, the growth factor extract was stripped by an acid step, and the total protein content was quantified by Pierce assay and the BMP-2 content was quantified by ELISA. Control columns represent the amount of protein loaded into each block.

[0220] Figure 16Shows total protein concentration over time in response to different cryoprotectants. While the two cryoprotectants showed similar performance at the first two time points, the untreated group had the least effect over the entire time course, and sucrose showed the best performance at the last time point evaluated. These results indicate that growth factor extract is well-preserved in the absence or presence of cryoprotectant when mineralized cancellous cubes coated with growth factor extract are used for up to 12 weeks.

[0221] Figure 17 Shows BMP-2 concentration over time in response to different cryoprotectants. While the performance without cryoprotectant was the worst, sucrose was observed to perform as well as or better than trehalose at all time points.

[0222] Example 7. Structural cell graft

[0223] Prepare a 10×10×10 mm 3 structural cell prototype graft. After obtaining donated allograft bone, the tissue is rinsed, attached soft tissue is removed, and unwanted bone tissue is cut off and then cut into the appropriate size and dimensions of the designed implant. The resulting graft can also be precisely machined into a specific shape using computer numerical control instruments (CNC). The graft is then rinsed to remove marrow and cleaned and disinfected with an antibiotic solution. The cleaned and disinfected graft is incubated with an appropriate cryopreservation solution and then cryopreserved to maintain the live cells in the graft. After incubation with the cryopreservation solution, the graft is dried or simply centrifuged to remove the excess cryopreservation solution and then inserted into a package and transferred to a -80 °C freezer or an LN2 freezer.

[0224] Figure 18 Shows live cells in a 10×10×10 mm structural cell prototype graft observed in situ using calcein staining (bright spots in the left figure), and outgrown cells observable under an optical microscope after 2 weeks (middle figure) or 4 weeks (right figure). The live cells are collected after growing out from the prototype graft and then used for all subsequent studies presented below.

[0225] Determine the cell concentration in the structural cell prototype graft (10×10×10 mm). The prototype graft contains live cells measured in situ by two different methods (Alamar Blue and LDH assay). The entire prototype graft is cultured in tissue culture on a medium containing Alamar Blue for 16 hours. A plate containing a known cell concentration is cultured along with it to provide a standard curve. The fluorescence readings of the unknown prototype graft are plotted against the known cell points to obtain the cells / cc of the tissue. For the LDH samples, the cells are divided into two groups: baseline and maximum release. The baseline group measures the LDH released by dead cells, and the maximum release sample is an artificially lysed sample that allows live cells to release LDH. This second sample measures the total LDH released by both dead and live cells. The difference between the two samples is the LDH content from live cells. Based on the LDH content, the number of live bone cells is determined. Figure 19 The cell concentrations in the structural cell prototype grafts prepared using Methods 1 to 4, which are similar with smaller differences, are shown.

[0226] An immune cell depletion study is conducted. The cells contained within the prototype graft (10×10×10 mm) before and after treatment are weighed, thoroughly washed with PBS, placed in 0.5 ml of 0.25% collagenase / 0.2 g of tissue, and digested for 4 hours with gentle stirring to obtain a single cell suspension. The cells are filtered through a 70 μm filter, centrifuged at 400 xg for 5 minutes, and washed once with PBS. Then the cells are treated with two different fluorophore-conjugated antibodies against CD45 (leukocytes) and CD166 (bone marrow hematopoietic cells) on ice for 30 minutes. Flow cytometry is used to identify the percentage of cells positive for each cell surface marker before and after treatment. The potentially immunoreactive cells are reduced by at least 85% or more. Figure 20 Shows the average expression of CD45 and CD166 markers in 30×30×15 mm 3 prototype grafts from three donors before and after treatment.

[0227] Evaluate the reactivity of PBMCs with the structural cell prototype graft. Lymphocytes and osteocytes isolated from the structural cell donor are separately plated in 96-well plates to form a stimulating layer of cells, and treated with mitomycin C to inhibit proliferation from the stimulating layer. The cells are then co-cultured with PBMCs that form a reactive layer. PBMCs treated with concanavalin A or lymphocytes are used as positive controls. The cells are placed at 37 °C and 5% CO2 for 2 days. Cell proliferation is evaluated by BrdU ELISA according to the manufacturer's protocol. PBMCs in contact with osteocytes isolated from the structural cell donor do not proliferate compared to PBMCs alone. Figure 21Shows the corrected PMBC proliferation in response to osteocyte lymphocytes (column 1), concanavalin A (column 2), osteocytes from structural cell grafts (column 4), or osteocytes from bone grafts (column 5). For this example, the term bone graft means a bone void filler having a combination of DBM and mineralized bone containing living cells. The bone graft is prepared as follows Bone graft: The cleaned bone mass (cancellous bone) containing living osteocytes is ground into particles, then mixed with demineralized bone fibers or particles from the same donor, incubated with a cryopreservation solution, and then cryopreserved in an LN2 refrigerator.

[0228] Evaluate the osteoblast population. Cells released from the prototype graft are cultured in basal or mineralized medium (containing ascorbic acid and β-glycerophosphate) under standard cell culture incubation conditions for up to 4 weeks. Figure 22 Shows cells from outgrowth cultures that have been fixed and stained with alizarin red after 2 weeks (left panel), 3 weeks (middle panel), or 4 weeks (right panel). Positive staining with alizarin red (dark red areas) indicates positive cultures with calcium-containing mineralized deposits. Control cells (inset) show no staining.

[0229] Evaluate growth factor (BMP) release. Cells are released from the prototype graft, collected, and cultured in basal or mineralized medium (containing ascorbic acid and β-glycerophosphate) for up to 21 days. After 3, 7, 14, or 21 days, BMP-2 and BMP-7 ELISA kits are used for the medium samples. Figure 23 Shows the average BMP-2 or BMP-7 concentration of cells isolated from structural cell prototype grafts in mineralized donor cells or control donor cells at day 3, 7, 14, or 21. At each time point tested, cells in mineralized medium showed higher BMP-2 and BMP-7 concentrations than those in basal medium.

[0230] Example 8. Structural cell grafts in PLF applications

[0231] Structural cell grafts can have different sizes and shapes. Cylindrical and / or bar-shaped (30×15×5 mm 3 ) structural cell grafts containing living cells are placed over two spinal transverse processes for posterolateral fusion (PLF) surgery, as Figure 24 shown. The trabecular structure of the structural cell graft provides direct structural stability and eliminates the concern of graft migration, which is commonly observed in putty-like grafts due to compression of the surrounding tissue after implantation. This method promotes healing for optimal fusion results.

[0232] Example 9. Activity map of structural cell grafts

[0233] The structural cell prototype graft (30×30×15 mm) contains viable cells determined in situ by Alamar Blue assay. As Figure 25 shown, the graft was sectioned and the viable cells of each sub-section were measured. The entire prototype was cultured in tissue culture plates seeded with media containing Alamar Blue for 16 hours. Then, the bulk prototype was cut as per Figure 25 each figure in for measuring cells / cc. Plates containing known cell concentrations were cultured along with the unknown prototype grafts to provide a standard curve. The fluorescence readings of the unknown prototype grafts were plotted against the known cell points to obtain the cells / cc of the tissue. These findings showed that cell viability and cell count were consistent and uniform throughout the structural cell graft.

[0234] All documents, books, manuals, papers, patents, published patent applications, guidelines, abstracts, and / or other references cited herein are hereby incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art in light of the specification and practice of the invention disclosed herein. The specification and examples are to be considered exemplary, with the actual scope and spirit of the invention being indicated by the appended claims.

Claims

1. A method for preparing a composition, comprising: (a) obtaining bone marrow from a first donor, wherein the bone marrow contains first natural living cells and is not frozen; (b) incubating the bone marrow in a first aqueous solution to release a first growth factor from the bone marrow into the first aqueous solution, thereby producing a first mixture; (c) collecting a first liquid portion from the first mixture, wherein the first liquid portion contains the first growth factor; (d) obtaining one or more other tissues from a second donor, wherein the one or more other tissues are selected from bone matrix, periosteum, endosteum, and combinations thereof, and the one or more other tissues contain second natural living cells; (e) incubating the one or more other tissues in a second aqueous solution to release a second growth factor from the one or more other tissues into the second aqueous solution, thereby producing a second mixture; (f) collecting a second liquid portion from the second mixture, wherein the second liquid portion contains the second growth factor; and (g) combining the first liquid portion and the second liquid portion, thereby preparing a composition, wherein the composition contains the first growth factor and the second growth factor.

2. The method according to claim 1, further comprising adjusting the pH of the composition to 6.5 to 7.

5.

3. The method according to claim 1 or 2, wherein the first donor and the second donor are the same.

4. The method according to any one of claims 1 to 3, further comprising measuring the protein concentration of the composition.

5. The method according to claim 4, further comprising increasing the protein concentration of the composition.

6. The method according to any one of claims 1 to 5, further comprising removing salts from the composition.

7. The method according to any one of claims 1 to 6, wherein step (a) includes removing red blood cells from the bone marrow.

8. The method according to any one of claims 1 to 7, wherein the first aqueous solution has a pH of 0 to 6.

9.

9. The method according to any one of claims 1 to 8, wherein the second aqueous solution has a pH of 0 to 6.

9.

10. The method according to any one of claims 1 to 9, wherein step (b) includes disrupting the first living cells.

11. The method according to any one of claims 1 to 9, wherein step (d) includes disrupting the second living cells.

12. The method according to any one of claims 1 to 11, wherein the first living cells are selected from osteocytes, stem cells, multipotent mesenchymal stromal cells, and combinations thereof.

13. The method according to any one of claims 1 to 12, wherein the second living cells are selected from osteocytes, stem cells, multipotent mesenchymal stromal cells, and combinations thereof.

14. The method according to claim 12 or 13, wherein the osteocytes are selected from osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, bone lining cells, and combinations thereof.

15. The method according to any one of claims 1 to 14, wherein the growth factors released from the bone marrow are selected from insulin-like growth factor (IGF), transforming growth factor (TGF), bone morphogenetic protein (BMP), angiogenic factor, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), stromal cell-derived factor-1 (SDF-1), and combinations of the foregoing.

16. The method according to any one of claims 1 to 15, wherein the growth factors released from the one or more other tissues are selected from insulin-like growth factor (IGF), transforming growth factor (TGF), bone morphogenetic protein (BMP), angiogenic factor, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), stromal cell-derived factor-1 (SDF-1), and combinations of the foregoing.

17. The method according to claim 1, wherein the bone matrix comprises cellularized cortical fragments, cortico-cancellous fragments, cellularized cortical bone fibers, cellularized cancellous blocks, cellularized cortical blocks, or cortico-cancellous blocks.

18. The method according to any one of claims 1 to 17, wherein the one or more other tissues comprise bone matrix prepared from bone selected from cancellous bone, cortico-cancellous bone, cortical bone, and combinations of the foregoing.

19. The method according to any one of claims 1 to 18, further comprising storing the composition.

20. The method according to any one of claims 1 to 19, further comprising lyophilizing or freeze-drying the composition.

21. The method according to any one of claims 1 to 20, further comprising sterilizing the composition.

22. The method according to any one of claims 1 to 21, further comprising packaging the composition.

23. A composition prepared by the method according to any one of claims 1 to 22.

24. The composition according to claim 23, wherein the composition comprises the growth factor at a concentration of 0.001 ng / g to 42,000 ng / g based on the total weight of the composition.

25. A method for improving the osteoinductivity of an implant, comprising incubating the implant with an effective amount of the composition according to claim 23 or 24 to produce an impregnated implant, wherein the implant comprises a bone graft, a metallic material, a synthetic material, or combinations of the foregoing, and wherein the impregnated implant has greater osteoinductivity than the implant before incubation.

26. The method according to claim 25, wherein the implant is incubated with the composition in the presence of a reagent.

27. The method according to claim 25 or 26, wherein the incubation comprises agitating and / or sonication of the implant and the composition.

28. The method according to any one of claims 25 to 27, further comprising storing the impregnated implant in a container.

29. The method according to any one of claims 25 to 28, wherein the implant comprises the bone graft.

30. The method according to claim 29, further comprising obtaining bone from a donor and cryopreserving the obtained bone to fabricate the bone graft.

31. The method according to claim 29 or 30, further comprising lyophilizing the impregnated bone graft.

32. The method according to claim 31, further comprising demineralizing the impregnated bone graft prior to the lyophilization.

33. The method according to any one of claims 29 to 32, further comprising sterilizing the impregnated bone graft.

34. The method according to claim 29, wherein the bone graft is demineralized.

35. The method according to claim 34, wherein the impregnated bone graft comprises demineralized bone matrix (DBM) fibers, a first growth factor from the bone marrow, and a second growth factor from the one or more other tissues, wherein the one or more other tissues comprise osteocytes on the surface of the one or more other tissues.

36. The method according to claim 29, wherein the bone graft is not demineralized.

37. The method according to claim 29, wherein the bone graft is selected from cortical pins, blocks or plates, cancellous blocks or strips, cortico-cancellous blocks or strips, cortical bone rings, and combinations of the foregoing.

38. The method according to any one of claims 29 to 37, wherein the impregnated bone graft has a pull-out force of at least 1 N against a surgical instrument in the bone.

39. The method according to any one of claims 29 to 38, wherein the impregnated bone graft is load-sharing.

40. The method according to any one of claims 29 to 39, wherein the impregnated bone graft is assembled with one or more other bone grafts.

41. The method according to any one of claims 25 to 28, wherein the implant comprises the metallic material.

42. The method according to any one of claims 25 to 28, wherein the implant comprises the synthetic material.

43. A product, comprising an impregnated implant prepared by the method according to any one of claims 25 to 42.

44. A container, comprising the product according to claim 43 in a liquid.

45. The container according to claim 44, wherein the liquid comprises the composition according to claim 23 or 24.

46. The container according to claim 44 or 45, wherein the liquid is a cryopreservation, lyophilization preservation or radiation protection agent solution.

47. The container according to any one of claims 44 to 46, wherein the impregnated implant is an impregnated bone graft, and the container is a cannula for injection in minimally invasive surgery (MIS).

48. The container according to claim 47, wherein the sleeve has a particulate density of less than 1.2 g / cm 3 .

49. The container according to claim 47 or 48, wherein the impregnated bone graft has a maximum extrusion force of 160 N to 200 N.

50. The container according to any one of claims 47 to 49, wherein the sleeve is loaded with the impregnated bone graft having a density of at least 0.2 g / cm 3 of the impregnated bone graft.

51. A method, comprising treating cells with an effective amount of the composition according to claim 23 or 24 to reduce the release of pro-inflammatory cytokines from the cells.

52. An allograft comprising bone and viable bone cells from a donor, wherein the viable bone cells are naturally present in and on the surface of the bone, and wherein the allograft does not include demineralized bone matrix (DBM), and has dimensions greater than 3×3×3 mm 3 in size.

53. The allograft according to claim 52, wherein the bone cells are selected from osteocytes, osteoblasts, bone lining cells, progenitor cells, and combinations thereof.

54. The allograft according to claim 52 or 53, wherein the bone is selected from cancellous bone, cortico-cancellous bone, cortical bone, and combinations thereof.

55. An allograft as claimed in any one of claims 52 to 54, wherein the allograft has said live bone cells at a concentration of at least 20,000 cells / cm 3 The allograft.

56. An allograft according to any one of claims 52 to 55, wherein the allograft has a natural lipid content of less than 10 mg of the lipid / cm 3 of the allograft.

57. The allograft according to any one of claims 52 to 56, wherein the bone comprises bone marrow, and at least 80% of the native hematopoietic cells have been removed from the bone marrow.

58. The allograft according to any one of claims 52 to 57, wherein the allograft has a pull-out force of at least 1 N against a surgical instrument in the bone.

59. The allograft according to any one of claims 52 to 58, wherein the allograft is assembled with a bone portion, a metal implant, or a polymer implant.

60. The allograft according to any one of claims 52 to 59, wherein the allograft is load-sharing.

61. A container comprising the allograft according to any one of claims 52 to 60 and a liquid, the liquid coating the allograft at a volume ratio of the liquid to the bone of less than 1.

62. The container according to claim 61, wherein the liquid comprises the composition according to claim 23 or 24.

63. The method according to any one of claims 25 and 29 to 40, wherein the bone graft is the allograft according to any one of claims 52 to 60.

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