CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to United States Provisional Application No. 63/416,756, filed October 17, 2022, and Provisional Application No. 63/444,120, filed February 8, 2023, and the contents of each of which are incorporated herein by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The invention relates generally to bone grafts (e.g., allografts), including bone grafts impregnated with growth factors from living tissues.

BACKGROUND OF THE INVENTION

Patients with underlying diseases, for example, osteopenia, diabetes, smoker, obesity, suffer slower bone regeneration with poor graft healing outcomes. Bone grafts with added growth factors can promote bone healing after implantation. There remains a need for bone grafts, for example, impregnated with growth factors, to improve bone regeneration after implantation. It remains a challenge to develop technologies to release growth factors from tissues while keeping their biological activities, including bone formation and angiogenesis.

SUMMARY OF THE INVENTION

The present invention relates to bone grafts, for example, allografts, optionally impregnated with growth factors released from tissues having native living cells. The invention is based on the inventors' surprising discoveries that cortical, cancellous, or cortical cancellous bones from a donor may be machined, minimally processed and/or assembled as structural bone grafts containing viable bone cells for use in, for example, dental, foot and ankle and spine surgeries in the donor, and that more growth factors in greater amounts can be recovered from bone marrow, bone matrix, periosteum and/or endosteum having native living cells as compared with those from frozen tissues without native living cells.

An allograft is provided. The allograft comprises a bone from a donor and viable bone cells. The viable bone cells are native to the bone and on the surface of the bone. The allograft excludes demineralized bone matrix (DBM) and has a dimension greater than 3x3x3 mm ³ . The bone cells may be selected from the group consisting of osteocytes, osteoblast, bone lining cells, progenitor cells and combinations thereof. The bone may be selected from the group consisting of cancellous bones, cortical cancellous bones, cortical bones and combinations thereof. The allograft may have the viable bone cells at a concentration of at least 20,000 cells per cm ³ said allograft. The allograft may have a native lipid content lower than 10 mg said lipid per cm ³ said allograft. The bone may comprise bone marrow and at least 80% of native hematopoieitic cells may have been removed from the bone marrow. The allograft may have a pullout force for a surgical hardware in the bone of at least 1 N. The allograft may be assembled with a bone part, a metal implant, or a polymer implant. The allograft may be load-sharing.

A container comprising the allograft of the present invention and a liquid is provided. The liquid coats the allograft at a volume ratio of the liquid to the bone less than 1. The liquid may comprise the composition of the present invention.

A method of preparing a composition is provided. The preparation method comprises: (a) obtaining bone marrow from a first donor, wherein the bone marrow comprises first native living cells and has not been frozen; (b) incubating the bone marrow in a first aqueous solution to release first growth factors from the bone marrow into the first aqueous solution, whereby a first mixture is generated; (c) collecting a first liquid fraction from the first mixture, wherein the first liquid fraction comprises the first growth factors; (d) obtaining one or more additional tissues from a second donor, wherein the one or more additional tissues are selected from the group consisting of bone matrix, periosteum, endosteum and a combination thereof, and the one or more additional tissues comprise second native living cells; (e) incubating the one or more additional tissues in a second aqueous solution to release second growth factors from the one or more additional tissues into the second aqueous solution, whereby a second mixture is generated; (f) collecting a second liquid fraction from the second mixture, wherein the second liquid fraction comprises the second growth factors; and (g) combining the first liquid fraction and the second liquid fraction, whereby a composition is prepared, wherein the composition comprises the first growth factors and the second growth factors. The preparation method may further comprise comprising adjusting the pH of the composition to 6.5-7.5. The first donor and the second donor may be the same. The preparation method may further comprise measuring a protein concentration of the composition. The preparation method may further comprise increasing the protein concentration of the composition. The preparation method may further comprise removing a salt from the composition.

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

According to the preparation method, the first living cells may be selected from the group consisting of bone cells, stem cells, multipotent mesenchymal stromal cells, and combinations thereof. The second living cells may be selected from the group consisting of bone cells, stem cells, multipotent mesenchymal stromal cells, and combinations thereof. The bone cells may be selected from the group consisting of osteoblasts, osteocytes, osteoclasts, osteo-progenitor cells, bone lining cells and a combination thereof.

According to the preparation method, the growth factors released from the bone marrow may be selected from the group consisting of insulin-like growth factor (IGF), transformed growth factor (TGF), bone morphogenetic proteins (BMPs), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), stromal cell-derived factor-1 (SDF-1) and combinations thereof. The growth factors released from the one or more additional tissues may be selected from the group consisting of insulin-like growth factor (IGF), transformed growth factor (TGF), bone morphogenetic proteins (BMPs), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), stromal cell-derived factor-1 (SDF-1) and combinations thereof.

According to the preparation method, the bone matrix may comprise cellularized cortical chips, cortico-cancellous chips, cellularized cortical bone fibers, cellularized cancellous blocks, cellularized cortical blocks, or cortico-cancellous blocks. The one or more additional tissues may comprise the bone matrix prepared from a bone selected from the group consisting of cancellous bones, cortical cancellous bones, cortical bones and combinations thereof.

The preparation method may further comprise storing the composition.

The preparation method may further comprise freeze-drying or lyophilizing the composition.

The preparation method may further comprise sterilizing the composition. The preparation method may further comprise packaging the composition. A composition prepared according to the preparation method is provided. The composition may comprise the growth factors at a concentration of 0.001-42,000 ng/g based on the total weight of the composition.

A method for improving osteoinductivity of an implant is provided. The improvement method comprises incubating an implant with an effective amount of the composition of the present invention to generate an impregnated implant. The implant comprises a bone graft, a metal material, a synthetic material, or a combination thereof. The impregnated implant has greater osteoinductivity than the implant before being incubated. The implant may be incubated with the composition in the presence of an agent. The incubating may comprise agitating and/or ultrasonicating the implant and the composition. The improvement method may further comprise storing the impregnated implant in a container.

According to the improvement method, the implant may comprise the bone graft. The improvement method may further comprise procuring a bone from a donor and cryopreserving the procured bone to make the bone graft. The improvement method may further comprise freeze-drying the impregnated bone graft. The improvement method may further comprise demineralizing the impregnated bone graft before the freeze-drying. The improvement method may further comprise sterilizing the impregnated bone graft. The bone graft may be demineralized. The impregnated bone graft may comprise demineralized bone matrix (DBM) fibers, the first growth factors from the bone marrow, and the second growth factors from the one or more additional tissues, which one or more additional tissues may comprise bone cells on the surface of the one or more additional tissues. The bone graft may not be demineralized. The bone graft may be selected from the group consisting of cortical pin, block or plank, cancellous block or strip, cortical cancellous block or strip, cortical bone ring, and a combination thereof. The bone graft may be the allograft of the present invention. The impregnated bone graft may have a pullout force for a surgical hardware in the bone of at least 1 N. The impregnated bone graft may be load-sharing. The impregnated bone graft may be assembled with one or more additional bone grafts.

According to the improvement method, the implant may comprise the metal material.

According to the improvement method, the implant may comprise the synthetic material.

A product comprising the impregnated implant prepared according to the preparation method of the present invention is provided.

A container comprising the product of the present invention in a liquid is provided. The liquid may comprise the composition of the present invention. The liquid may be a cryopreservation, lyopreservation or radioprotectant solution. The impregnated implant may be an impregnated bone graft and the container may be a cannula for injection in a minimum invasive surgery (MIS). The cannula may have a particulate density of less than 1.2 g/cm ³ . The impregnated bone graft may have a maximum extrusion force of 160-200 N. The cannula may be loaded with the impregnated bone graft at a density of at least 0.2 g/cm ³ .

A treatment method is provided. The treatment method comprises treating cells with an effective amount of the composition of the present invention to reduce release of a pro-inflammatory cytokine by the cells. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating processing steps according to one embodiment of a growth factor preparation method.

FIG. 2 shows a comparison of protein concentration in growth factor extracts prepared from fresh or frozen bone marrow or bone matrix.

FIG. 3 shows BMP-2 Content in growth factor extracts prepared from fresh or frozen bone marrow or bone matrix.

FIG. 4 shows protein concentration in growth factor extracts prepared from fresh bone marrow, O/N Freeze bone marrow, or O/N RT bone marrow.

FIG. 5 shows expression of BMP2, BMP 7 and PDGF growth factor in growth factor extracts prepared from growth factor extracts prepared from fresh bone marrow, O/N Freeze bone marrow, or O/N RT bone marrow.

FIG. 6 shows BMP-2 concentration in growth factor extracts prepared from fresh bone marrow or fresh bone matrix.

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

FIG. 8 shows protein concentration in growth factor extract prepared from predemineralized cortical bone.

FIG. 9 shows total protein release over time from coated devitalized cube bone grafts in culture.

FIG. 10 shows in vitro alkaline phosphatase (ALP) activity of a bone marrow growth factor extract (500ug/cc), a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control), or a BMP-2 solution (200ng/cc) (positive control) in an in vitro ALP assay.

FIG. 11 shows an increase in alkaline phosphatase activity after introduction of a liquid growth factor extract from representative bone marrow (bars 1-3), periosteum (bar 4), or bone matrix (bars 5-8); each at 500ug/cc; a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control), or a BMP-2 solution (200ng/cc) (positive control) in an in vitro ALP assay.

FIG. 12 shows ALK activity of a liquid growth factor extract prepared from Bone Marrow Supernatant (bar 4), Bone Marrow Pellet (bar 5), Undiluted Bone Marrow (bar 9), Periosteum (bar 8), or Bone Matrix (bar 10), each having a total protein concentration of 500 ug/cc; a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control, bar 2), or a BMP-2 solution (200ng/cc) (positive control, bar 3); extracts of demineralized bone matrix (DBM) in a high (bar 6) and low (bar 7) mass quantity (each having a total protein concentration of 500ug/cc) served as references. Bar 1 constituted an untreated cell population.

FIG. 13 shows ALK activity of demineralized cancellous sponges impregnated with a bone marrow growth factor extract from three different bone marrow donors (bars 2, 3, and 4), an uncoated sponge (bar 1), a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control, bar 5)), or a BMP-2 solution (200ng/cc) (positive control, bar 6).

FIG. 14 shows images osteoblast cultures after exposure to bone marrow growth factor extract- loaded mineralized bone discs after 21 days in an in vitro mineralization assay.

FIG. 15 shows calcium contents in osteoblasts exposed to uncoated (left bar) or 500ug/cc bone marrow growth factor extract coated mineralized bone discs (right bar) for 21 days.

FIG. 16 shows total protein concentration of mineralized cancellous cubes coated with a growth factor extract over time in response to different preservatives.

FIG. 17 shows BMP-2 concentration of mineralized cancellous cubes coated with a growth factor extract over time in response to different preservatives. While no lyoprotectant was the worst performer, sucrose performed as well or better than trehalose over all time points observed.

FIG. 18 shows viable cells in structural cellular grafts as visualized in situ using calcein staining (left panel), and outgrown cells under light microscopy after 2 weeks (middle panel) or 4 week (right panels).

FIG. 19 shows the cell concentration in the structural cellular prototype grafts.

FIG. 20 shows average expression of CD45 and CD166 markers in 30x30x15 mm ³ prototype grafts from three donors before and after processing.

FIG. 21 shows corrected proliferation of PMBC in response to bone cells lymphocytes (bar 1), Concanavalin A (bar 2), bone cells from structural cellular grafts (bar 4) or bone cells from a ViviGen® bone graft (bar 5).

FIG. 22 shows cells from outgrowth cultured were fixed and stained with Alizarin Red after 2 weeks (left panel), 3 weeks (middle panel), or 4 weeks (right panel).

FIG. 23 shows average BMP-2 or BMP-7 concentration from cells isolate from structural cellular prototype grafts in mineralization donor cells or control donor cells at day 3, 7, 14 or 21.

FIG. 24 shows structural cellular grafts for posterolateral fusion (PLF) procedure.

FIG. 25 shows viability map of structural cellular prototype grafts. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an allograft comprising a bone from a donor and living cells native to the bone, a substrate (e.g., bone graft) impregnated with growth factors released from tissues having living cells, as well as methods for recovering growth factors from tissues having living cells, and methods for impregnating the subject with the growth factors. The inventors have surprisingly discovered that cortical, cancellous, or cortical cancellous bones from a donor may be machined, minimally processed and/or assembled as structural bone grafts containing viable bone cells for use in, for example, dental, foot and ankle and spine surgeries in the donor. Such structural bone grafts may be allografts in any shape (e.g., blocks or particulates) as needed for a surgical site. The inventors have also surprisingly discovered that more active growth factors in greater amounts are recovered from tissues (e.g., bone marrow, bone matrix, periosteum and/or endosteum) having native living cells than those recovered from frozen tissues without native living cells. The inventors have further surprisingly discovered that implants impregnated with growth factors released from bone marrow, bone matrix, periosteum and/or endosteum comprising native living cells showed improved osteoinductivity.

The term "bone graft" used herein refers to a bone to be placed inside or on the surface the body of a subject. The bone has been obtained from a donor, and may have been processed. Where the subject is not the donor, the bone graft is also referred to herein as an allograft.

The term "implant" used herein refers to a device to be placed inside or on the surface of a subject. A metal implant is an implant made of a metal material. A synthetic implant is an implant made of a synthetic material, which excludes a metal material.

The term "subject" as used herein refers to an animal, for example, a mammal, which may be a human individual or non-human individual such as bovine, porcine, canine (e.g., dog), equine, ovine, or non-human primate (e.g., ape and gorilla).

The term "donor" as used herein refers to a subject from whom a tissue or cells are obtained.

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

The term "native" as used herein refers to the origin of cells. Cells native to a source, for example, a tissue or donor, are cells that are naturally occurring in the source. For example, cells native to a tissue are naturally occurring inside of or on the surface of the tissue. Cells native to a source may be separated from the source and then optionally processed. For example, cells native to a tissue may be separated from the tissue using any method, for example, a solution containing a protease such as trypsin and collagenase, without significantly affecting the biological activities of the cells.

The term "tissue" as used herein refers to a group of cells that possess a similar structure and perform a specific function. A tissue may be obtained from a donor. Examples of tissues include bone marrow, bone matrix, periosteum, and endosteum. A tissue from a donor may be processed, for example, to remove cells or cellular debris or to change a physical or biological property of the tissue.

The term "fresh tissue" as used herein refers to a tissue that has not been frozen after being obtained from a donor who has not been frozen or refrigerated. A fresh tissue has native living cells.

The term "frozen tissue" as used herein refers to a tissue that has been frozen after being obtained from a donor or has been obtained from a donor who has been frozen or refrigerated without the proper conditions to maintain cell viability. Native living cells in the tissue before being frozen are no longer viable after the tissue is frozen.

The term "bone marrow" as used herein refers to a tissue that is a spongy substance in the center of a bone. The bone marrow comprises bone progenitor cells, bone marrow stem cells, hematopoietic cells (HPCs) and red blood cells.

The term "bone matrix" as used herein refers to a tissue that is an intercellular substance of a bone. The bone matrix comprises bone cells on the surface or within the structure of the bone matrix. The bone cells in the bone matrix is also referred to as osteocytes. The bone matrix may be a mineralized and/or demineralized bone particulate and cellularized cortico-cancellous chips, or mineralized and/or demineralized bone fibers and cellularized cortico-cancellous chips.

The term "periosteum" as used herein refers to a tissue forming a fibrous sheath that covers a bone. The periosteum may comprise bone cells on the surface of the periosteum.

The term "endosteum" as used herein refers to a tissue forming a membrane that lines the center of a bone having bone marrow. The endosteum may comprise bone cells on the surface of the endosteum. The terms "living tissue" and "viable tissue" are used herein interchangeably and refer to a tissue having native living cells. For example, a bone graft having native living cells is referred to as a living or viable bone graft while a bone graft not having native living cells is referred to as a non-living or non-viable bone graft.

The term "cryopreservation" or "cryopreserved" as used herein refers to cooling a tissue or cells to preserve a biological activity of the tissue or cells. The cryopreserved tissue (e.g., bone graft) or cells may be coated with, or submerged, partially or completely, in a cryopreservation solution. The cryopreservation solution may comprise 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.

The term "lyopreservation" or "ly op reserved" as used herein refers to lyophilizing a frozen tissue or frozen cells, i.e., removing moisture from the frozen tissue or cells under vacuum. The lyopreserved tissue (e.g., bone graft) or cells may be coated with or submerged in a lyopreservation solution. The lyopreservation solution may comprise trehalose, HAS, BSA, mannitol, sucrose, glycerol, dextran, sodium alginate, EGCG, glucose, lactose, maltose, NFC, HES, PVP, and/or PEG.

The term "growth factor" as used herein refers to a substance that is naturally occurring in a tissue or cells, and is biologically active, for example, capable of stimulating cell proliferation, wound healing, and cellular differentiation. Examples of the growth factors include of insulin-like growth factor (IGF), transformed growth factor (TGF) (e.g., TGF ), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs) (e.g., aFGF and PFGF), stromal cell- derived factor-1 (SDF-1). The growth factors may be released from the tissue or cells, in which the growth factors are naturally occurring, and the released growth factors may remain biologically active. The released growth factors are also referred to as recovered growth factors. The released growth factors may be referred to with reference to their sources. For example, growth factors released from bone marrow are referred to as bone marrow growth factors (BMGFs); growth factors released from bone matrix are referred to as bone matrix growth factors (BGFs); growth factors released from periosteum are referred to as periosteum growth factors (BPGF); growth factors released from endosteum are referred to as endosteum growth factors (BEGF); and growth factors released from bone cells are referred to as bone cell growth factors (BCGF). A composition comprising growth factors released from a tissue or cells is also referred to as a growth factor extract or a growth factor extraction. The term "cannula" used herein refers to a thin tube used in a surgery for delivering a substance to a treatment site in a subject. The cannula has an exterior surface and an interior surface. The exterior surface defines the external diameter of the cannula. The interior surface forms a bore extending along the axis of the cannula and defines an inner diameter. The bore may be a cylinder.

The term "pullout force" as used herein refers to the force required to pull out an item from an object, for example, a bone graft or an impregnated bone graft.

The term "load-sharing" as used herein refers to distributing a force exerted on an object to one or more other objects. There may be one or more levels of force distribution among the objects.

The present invention provides a growth factor preparation method. According to the growth factor preparation method, a composition comprising growth factors from one or more tissues is prepared. The growth factors are native to the one or more tissues. Each of the one or more tissues is from a donor and has native living cells. The growth factor preparation method comprises incubating the one or more tissues in one or more aqueous solutions to release growth factors from the one or more tissues into the one or more aqueous solutions to generate one or more mixtures; and collecting one or more liquid fractions from the one or more mixtures. The collected one or more liquid fractions comprise growth factors released from the one or more tissues.

Where the one or more tissues consist of one tissue, the collected one liquid fraction according to the growth factor preparation method is a composition comprising the growth factors released from the one tissue.

Where the one or more tissues comprise two or more tissues, the growth factor preparation method may comprise incubating the two or more tissues in an aqueous solution to generate a mixture, and collecting a liquid fraction from the mixture. The liquid fraction is a composition comprising the growth factors released from the two or more tissues.

Where the one or more tissues comprise two or more tissues, the growth factor preparation method may comprise incubating the two or more tissues in two or more aqueous solutions to generate 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 generate a mixture; and collecting two or more liquid fractions each from one of the two or more mixtures. The growth factor preparation method may further comprise combining the two or more liquid fractions into a composition. The composition comprises growth factors released from the two or more tissues. According to the growth factor preparation method, the one or more tissues may be selected from the group consisting of bone marrow, bone matrix, periosteum, endosteum, and a combination thereof. The native living cells may be bone cells. Where the tissue is bone marrow, the native living bone cells may be in the bone marrow. Where the tissue is bone matrix, periosteum or endosteum, the native living bone cells may be on the surface of or within the bone matrix, periosteum or endosteum. Wherein the one or more tissues comprise bone marrow, the growth factor preparation method may further comprise removing red blood cells from the bone marrow by, for example, treating the bone marrow with ammonium chloride.

According to the growth factor preparation method, the one or more tissues may be obtained from one or more donors. Each donor may be an animal, for example, a human individual. Where the one or more tissues comprise two or more tissues, the two or more tissues may be from the same donor or two or more different donors. The one or more donors may not have been refrigerated or frozen before the one or more tissues are obtained from the one or more donors. At least one of the one or more donors may not have been refrigerated or frozen before a tissue is obtained from the at least one of the one or more donors.

According to the growth factor preparation method, the one or more tissues may not have been frozen, cryopreserved or lyopreserved. At least one of the one or more tissues may not have been frozen, cryopreserved and/or lyopreserved.

The growth factor preparation method may comprise obtaining bone marrow from a donor, wherein the bone marrow comprises native living cells; incubating the bone marrow in an aqueous solution to release growth factors from the bone marrow into the aqueous solution, whereby a mixture is generated; and collecting a liquid fraction from the mixture, wherein the liquid fraction comprises the growth factors released from the bone marrow. The growth factor preparation method may further comprise removing red blood cells from the bone marrow by, for example, treating the bone marrow with ammonium chloride. The bone marrow may not have been frozen, cryopreserved or lyopreserved. The donor may not have been refrigerated or frozen before the bone marrow is obtained from the donor. The living cells may be bone cells in the bone marrow.

The growth factor preparation method may comprise obtaining bone matrix from a donor, wherein the bone matrix comprises native living cells; incubating the bone matrix in an aqueous solution to release growth factors from the bone matrix into the aqueous solution, whereby a mixture is generated; and collecting a liquid fraction from the mixture, wherein the liquid fraction comprises the growth factors released from the bone matrix. The bone matrix may not have been frozen, cryopreserved or lyopreserved. The donor may not have been refrigerated or frozen before the bone matrix is obtained from the donor. The living cells may be bone cells, for example, on the surface of or within the bone matrix.

The growth factor preparation method may comprise obtaining periosteum from a donor, wherein the periosteum comprises native living cells; incubating the periosteum in an aqueous solution to release growth factors from the periosteum into the aqueous solution, whereby a mixture is generated; and collecting a liquid fraction from the mixture, wherein the liquid fraction comprises the growth factors released from the periosteum. The periosteum may not have been frozen, cryopreserved or lyopreserved. The donor may not have been refrigerated or frozen before the periosteum is obtained from the donor. The living cells may be bone cells, for example, on the surface of the periosteum.

The growth factor preparation method may comprise obtaining endosteum from a donor, wherein the endosteum comprises native living cells; incubating the endosteum in an aqueous solution to release growth factors from the endosteum into the aqueous solution, whereby a mixture is generated; and collecting a liquid fraction from the mixture, wherein the liquid fraction comprises the growth factors released from the endosteum. The endosteum may not have been frozen, cryopreserved or lyopreserved. The donor may not have been refrigerated or frozen before the endosteum is obtained from the donor. The living cells may be bone cells, for example, on the surface of the endosteum.

The growth factor preparation method may comprise obtaining one or more first tissues from a first donor, wherein the one or more first tissues comprise first native living cells; incubating the one or more first tissues in a first aqueous solution to release first growth factors from the one or more first tissues into the first aqueous solution, whereby a first mixture is generated; collecting a first liquid fraction from the first mixture, wherein the first liquid fraction comprises the first growth factors 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 comprise second native living cells; incubating the one or more second tissues in a second aqueous solution to release second growth factors from the one or more second tissues into the second aqueous solution, whereby a second mixture is generated; collecting a second liquid fraction from the second mixture, wherein the second liquid fraction comprises the second growth factors released from the one or more second tissues; and combining the first liquid fraction and the second liquid fraction to generate a composition, which composition comprises the first growth factors and the second growth factors, i.e. , growth factors from the one or more first tissues and the growth factors from the one or more second tissues. The one or more first tissues may be selected from the group consisting of bone marrow, bone matrix, periosteum, endosteum, and a combination thereof. The one or more second tissues may be selected from the group consisting of bone marrow, bone matrix, periosteum, endosteum, and a combination 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 a combination thereof. The one or more first tissues may not have been frozen, cryopreserved or lyopreserved. The one or more second tissues may not have been frozen, cryopreserved or lyopreserved. The first donor may not have been refrigerated or frozen before the one or more first tissues are obtained from the first donor. The second donor may not have been refrigerated or frozen before the one or more second tissues are obtained from the second donor. The first donor and second donor may be the same or different. The first native living cells may be bone cells. The second native living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

The growth factor preparation method may comprise obtaining bone marrow from a first donor, wherein the bone comprises first native living cells; incubating the bone marrow in a first aqueous solution to release first growth factors from the bone marrow into the first aqueous solution, whereby a first mixture is generated; collecting a first liquid fraction from the first mixture, wherein the first liquid fraction comprises the first growth factors released from the bone marrow; obtaining one or more additional tissues from a second donor, wherein the one or more additional tissues comprise second native living cells, and are selected from the group consisting of bone matrix, periosteum, endosteum, and a combination thereof; incubating the one or more additional tissues in a second aqueous solution to release second growth factors from the one or more additional tissues into the second aqueous solution, whereby a second mixture is generated; collecting a second liquid fraction from the second mixture, wherein the second liquid fraction comprises the second growth factors released from the one or more additional tissues; and combining the first liquid fraction and the second liquid fraction to generate a composition, which composition comprises the first growth factors and the second growth factors, i.e., growth factors from the bone marrow and the one or more additional tissues. The growth factor preparation method may further comprise removing red blood cells from the bone marrow by, for example, treating the bone marrow with ammonium chloride. The bone marrow may not have been frozen, cryopreserved or lyopreserved. The one or more additional tissues may not have been frozen, cryopreserved or lyopreserved. The first donor may not have been refrigerated or frozen before the bone marrow is obtained from the first donor. The second donor may not have been refrigerated or frozen before the one or more additional tissues are obtained from the second donor. The first donor and second donor may be the same or different. The first native living cells may be bone cells. The second native living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

The growth factor preparation method may comprise obtaining bone matrix from a first donor, wherein the bone comprises first native living cells; incubating the bone matrix in a first aqueous solution to release first growth factors from the bone matrix into the first aqueous solution, whereby a first mixture is generated; collecting a first liquid fraction from the first mixture, wherein the first liquid fraction comprises the first growth factors released from the bone matrix; obtaining one or more additional tissues from a second donor, wherein the one or more additional tissues comprise second native living cells, and are selected from the group consisting of bone marrow, periosteum, endosteum, and a combination thereof; incubating the one or more additional tissues in a second aqueous solution to release second growth factors from the one or more additional tissues into the second aqueous solution, whereby a second mixture is generated; collecting a second liquid fraction from the second mixture, wherein the second liquid fraction comprises the second growth factors released from the one or more additional tissues; and combining the first liquid fraction and the second liquid fraction to generate a composition, which composition comprises the first growth factors and the second growth factors, i.e., growth factors from the bone matrix and the one or more additional tissues. The bone matrix may not have been frozen, cryopreserved or lyopreserved. The one or more additional tissues may not have been frozen, cryopreserved or lyopreserved. The first donor may not have been refrigerated or frozen before the bone matrix is obtained from the first donor. The second donor may not have been refrigerated or frozen before the one or more additional tissue is obtained from the second donor. The first donor and second donor may be the same or different. The first native living cells may be bone cells. The second native living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

The growth factor preparation method may comprise obtaining periosteum from a first donor, wherein the bone comprises first native living cells; incubating the periosteum in a first aqueous solution to release first growth factors from the periosteum into the first aqueous solution, whereby a first mixture is generated; collecting a first liquid fraction from the first mixture, wherein the first liquid fraction comprises the first growth factors released from the periosteum; obtaining one or more additional tissues from a second donor, wherein the one or more additional tissues comprise second native living cells, and are selected from the group consisting of bone marrow, bone matrix, endosteum, and a combination thereof; incubating the one or more additional tissues in a second aqueous solution to release second growth factors from the one or more additional tissues into the second aqueous solution, whereby a second mixture is generated; collecting a second liquid fraction from the second mixture, wherein the second liquid fraction comprises the second growth factors released from the one or more additional tissues; and combining the first liquid fraction and the second liquid fraction to generate a composition, which composition comprises the first growth factors and the second growth factors, i.e. , growth factors from the periosteum and the one or more additional tissues. The periosteum may not have been frozen, cryopreserved or lyopreserved. The one or more additional tissues may not have been frozen, cryopreserved or lyopreserved. The first donor may not have been refrigerated or frozen before the periosteum is obtained from the first donor. The second donor may not have been refrigerated or frozen before the one or more additional tissues are obtained from the second donor. The first donor and second donor may be the same or different. The first native living cells may be bone cells. The second native living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

The growth factor preparation method may comprise obtaining endosteum from a first donor, wherein the bone comprises first native living cells; incubating the endosteum in a first aqueous solution to release first growth factors from the endosteum into the first aqueous solution, whereby a first mixture is generated; collecting a first liquid fraction from the first mixture, wherein the first liquid fraction comprises the first growth factors released from the endosteum; obtaining one or more additional tissues from a second donor, wherein the one or more additional tissues comprise second native living cells, and are selected from the group consisting of bone marrow, bone matrix, periosteum, and a combination thereof; incubating the one or more additional tissues in a second aqueous solution to release second growth factors from the one or more additional tissues into the second aqueous solution, whereby a second mixture is generated; collecting a second liquid fraction from the second mixture, wherein the second liquid fraction comprises the second growth factors released from the one or more additional tissues; and combining the first liquid fraction and the second liquid fraction to generate a composition, which composition comprises the first growth factors and the second growth factors, i.e., growth factors from the endosteum and the one or more additional tissues. The endosteum may not have been frozen, cryopreserved or lyopreserved. The one or more additional tissues may not have been frozen, cryopreserved or lyopreserved. The first donor may not have been refrigerated or frozen before the endosteum is obtained from the first donor. The second donor may not have been refrigerated or frozen before the one or more additional tissues are obtained from the second donor. The first donor and second donor may be the same or different. The first native living cells may be bone cells. The second native living cells may be bone cells. The first aqueous solution and the second aqueous solution may be the same or different.

The growth factor preparation method may further comprise adjusting the pH of the composition to a neutral pH, for example, about 6.5-7.5, 6.5-7.0, 7.0-7.5, 6.8-7.2, 6.8-7, 7-7.2, 6.9-7.1, 6.9-7.0 or 7.0-7.1.

The growth factor preparation method may further comprise measuring a protein concentration of the composition, the first liquid fraction and/or the second liquid fraction, and/or increasing the protein concentration of the composition, the first liquid fraction and/or the second liquid fraction.

The growth factor preparation method may further comprise removing one or more salts from the composition, the first liquid fraction and/or the second liquid fraction.

According to the growth factor preparation method, the aqueous solution may have a pH of 0-6.9, 0-6.5, 0-6, 0-5, 0-4, 0-3, 0-2, 0-1, 1.0-6.9, 1.0-6.5, 1-6, 1-5, 1-4, 1-3, 1-2, 2.0-6.9, 2.0-6.5, 2-6, 2-5, 2-4, 2-3, 3.0-6.9, 3.0-6.5, 3-6, 3-5, 3-4, 4.0-6.9, 4.0-6.5, 4.0-6.4, 4-5, 5.0-6.9, 5.0-6.5, 5-6, 6.0-6.9 or 6.0-6.5. The aqueous solution may comprise an acid selected from the group consisting of acetic acid, hydrochloric acid, citric acid, sulfuric acid, and formic acid. The aqueous solution may be the first and/or second aqueous solution.

According to the growth factor preparation method, the incubation step may comprise disrupting the living cells. The disrupting may comprise agitating (e.g., freeze-thaw cycle(s), stirring, rocking, spinning, shaking), cryomilling, centrifuging, and/or ultrasonicating the tissue. The living cells may be the first or second living cells.

According to the growth factor preparation method, the living cells may be selected from the group consisting of bone cells, stem cells, multipotent mesenchymal stromal cells, and combinations thereof. The bone cells may be selected from the group consisting of osteoblasts, osteocytes, osteoclasts, osteo-progenitor cells, bone lining cells and a combination thereof. The living cells may be the first and/or second living cells.

According to the growth factor preparation method, the growth factors released from one or more tissues may be selected from the group consisting of insulin-like growth factor (IGF), transformed growth factor (TGF) (e.g., TGFP), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet- derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs) (e.g., aFGF and PFGF), stromal cell-derived factor-1 (SDF-1) and combinations thereof.

According to the growth factor preparation method, the growth factors released from bone marrow may be selected from the group consisting of insulin-like growth factor (IGF), transformed growth factor (TGF) (e.g., TGFP), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs) (e.g., aFGF and PFGF), stromal cell-derived factor-1 (SDF-1) and combinations thereof.

According to the growth factor preparation method, the growth factors released from bone matrix may be selected from the group consisting of insulin-like growth factor (IGF), transformed growth factor (TGF) (e.g., TGFP), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs) (e.g., aFGF and PFGF), stromal cell-derived factor-1 (SDF-1) and combinations thereof. In one embodiment, a composition or extraction comprising growth factors released from bone matrix may further comprise calcium ions (Ca ⁺⁺ ) from the bone matrix, and the released growth factors and Ca ⁺⁺ may be impregnated onto a bone graft, a metal implant or a polymer implant.

According to the growth factor preparation method, the growth factors released from periosteum may be selected from the group consisting of insulin-like growth factor (IGF), transformed growth factor (TGF) (e.g., TGF ), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs) (e.g., aFGF and PFGF), stromal cell-derived factor-1 (SDF-1) and combinations thereof.

According to the growth factor preparation method, the growth factors released from endosteum may be selected from the group consisting of insulin-like growth factor (IGF), transformed growth factor (TGF) (e.g., TGFP), bone morphogenetic proteins (BMPs) (e.g., BMP-4 and BMP-7), angiogenic factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs) (e.g., aFGF and PFGF), stromal cell-derived factor-1 (SDF-1) and combinations thereof.

According to the growth factor preparation method, the bone matrix may comprise cellularized cortical chips, cortico-cancellous chips, cellularized cortical bone fibers, cellularized cancellous blocks, cellularized cortical blocks, or cortico-cancellous blocks. For example, the bone matrix may comprise (i) mineralized and/or demineralized bone particulates and cellularized cortico-cancellous chips, or (ii) mineralized and/or demineralized bone fibers and cellularized cortico-cancellous chips. The bone matrix may be prepared from a bone selected from the group consisting of cancellous bones, cortical cancellous bones, cortical bones and combinations thereof.

The growth factor preparation method may further comprise storing the composition; freeze-drying or lyophilizing the composition; sterilizing the composition; and/or packaging the composition. The composition may be sterilized by gamma irradiation, electron beam (e-beam), x-ray or ethylene oxide (EtO).

In one embodiment, the growth factor preparation method comprises processing steps shown in FIG. 1 : Obtaining a tissue, for example, bone marrow, from a donor; subjecting the tissue to an ammonium chloride treatment, 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 generate a sample; centrifuging the sample and neutralizing the pH of the sample; desalting the pH neutralized sample; concentrating the desalted sample using stirred cell concentrator; and measuring protein content in the concentrated sample. As a result, a growth factor extract is recovered from a donor tissue.

For each growth factor preparation method of the present invention, a composition prepared according to the growth factor preparation method is provided. The growth factors in the composition may comprise BMGFs, BGFs, BPGF, BEGF, and/or BCGF. For example, the composition may comprise BCGFs, and bone cells 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 dry composition may be in the form of powder. The dry composition may be rehydrated.

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

The composition may be packaged, freeze dried, and/or sterilized until being used in a surgery, for example, applied to a surgical site to improve bone formation. For example, the composition may be applied to a metal or polymer implant surface prior to a surgical procedure.

The present invention also provides a method for improving osteoinductivity of an implant. The improvement method comprises incubating the implant with an effective amount of the composition of the present invention to generate an impregnated implant. The implant comprises a bone graft, a metal material, a synthetic material, or a combination thereof. The impregnated implant has greater osteoinductivity than the implant before being incubated.

According to the improvement method, the implant may be incubated with the composition in the presence of an agent. The agent may be a solvent, a preservative or a combination thereof. The incubating may comprise agitating (e.g., stirring, rocking, spinning, shaking), and/or ultrasonicating the implant and the composition. The improvement method may further comprise storing the impregnated implant in a container.

Where the implant comprises the bone graft, the improvement method may further comprise procuring a bone from a donor and cryopreserving the procured bone to make the bone graft. The improvement method may further comprise freeze-drying the impregnated bone graft. The improvement method may further comprise demineralizing the impregnated bone graft before the freeze-drying. The improvement method may further comprise sterilizing the impregnated bone graft. The bone graft may be the allograft of the present invention.

The bone graft may be demineralized. The bone graft may comprise demineralized bone matrix (DBM) particulates, DBM fibers, DBM blocks from, for example, a cortical bone plank, DBM sponges from, for example, cancellous bone blocks, and/or DBM putties, optionally with carriers.

The impregnated bone graft may comprise impregnated demineralized bone matrix (DBM) particulates, DBM fibers, DBM blocks from, for example, a cortical bone plank, DBM sponges from, for example, cancellous bone blocks, and/or DBM putties, optionally with carriers. The impregnated DBM putties may be flowable and delivered into a deep defects through an injection needle. The carriers may be derived from a bone. For example, the impregnated bone graft may comprise impregnated DBM fibers.

The impregnated bone graft may comprise the growth factors from one or more tissues selected from the group consisting of bone marrow, bone matrix, periosteum, endosteum, and a combination thereof. For example, the impregnated bone graft may comprise growth factors from bone marrow (BMGFs) and growth factors from one or more additional tissues selected from the group consisting of bone matrix, periosteum, endosteum, and a combination thereof. The bone marrow may comprise native living bone cells in the bone marrow. The one or more additional tissues may comprise native living bone cells on the surface of the one or more additional tissues selected from the group consisting of bone matrix, periosteum, endosteum, and a combination thereof. The one or more additional tissues may comprise periosteum.

The bone graft may not be demineralized. The bone graft may comprise cortical, cancellous, or cortical cancellous particulates. The bone graft may be selected from the group consisting of cortical pin, block or plank, cancellous block or strip, cortical cancellous block or strip, cortical bone ring, and a combination thereof.

The impregnated bone graft may have a pullout force for a surgical hardware in the impregnated bone graft of at least about 0.1, 0.5, 1, 5, 10, 20 or 50 N. The impregnated bone graft may be load-sharing. The implanted bone graft may share force with one or more load-bearing constructs such as plate(s) and/or screw(s). There may be different levels of feree distribution between the implanted bone graft and the load-bearing construct(s). The impregnated bone graft may have machinability, for example, being capable of further being machined, cut, drilled, or shaped. The impregnated bone graft may be assembled with one or more additional bone grafts.

According to the improvement method, the implant may comprise the metal material. Such a metal implant may be a joint implant in, for example, a knee, shoulder, elbow, ankle, or finger; a spinal cage, which may be expandable and/or 3D printed; or a metal plate, screw, or dental implant.

According to the improvement method, the implant may comprise the synthetic material. The synthetic material may be a synthetic bone void filler, for example, tricalcium phosphate (TCP) or bioglass. The synthetic implant may be a polymer implant, for example, a PEEK implant.

For each improvement method of the present invention, a product comprising the impregnated implant prepared according to the method is provided.

For each product of the present invention, a container is provided. The container comprises the product in a liquid. The liquid may comprise the composition of the present invention. The liquid may be a cryopreservation, lyopreservation or radioprotectant solution. The impregnated implant may be an impregnated bone graft and the container may be a cannula for injection in a minimum invasive surgery (MIS). The cannula may have a particulate density of less than about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 g/cm ³ . The impregnated bone graft may have a maximum extrusion force of about 10-500, 10-400, 10-300, 10-200, 100-500, 100-400, 100-300, 100-200, 150-500, 150-400, 150-300, 150-250, 150-200, or 160-200 N, for example, about 180 N. The cannula may be loaded with the impregnated bone graft at a density of at least about 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5 or 1.0 g/cm ³ .

The present invention further provides a treatment method. The treatment method comprises treating cells with an effective amount of the composition of the present invention to reduce release of a pro-inflammatory cytokine by the cells.

The present invention further provides an allograft. The allograft comprises a bone from a donor and viable bone cells. The viable bone cells are native to the bone and on the surface of the bone. The allograft excludes a demineralized bone matrix (DBM). The allograft has a dimension greater than about Ixlxl, 2x2x2, 3x3x3, 4x4x4, 5x5x5, or 10x10x10 mm ³ . The bone cells may be selected from the group consisting of osteocytes, osteoblast, bone lining cells, progenitor cells and combinations thereof. The bone may be selected from the group consisting of cancellous bones, cortical cancellous bones, cortical bones and combinations thereof. The allograft may 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, 1 million, 2 million, or 3 million viable bone cells per cm ³ of the allograft. The allograft may have a native lipid content lower than about 1, 5, 10, 50 or 100 mg said lipid per cm ³ said allograft. The bone may comprise bone marrow, and at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of native hematopoieitic cells may have been removed from the bone marrow. The allograft may have a pullout force for a surgical hardware in the bone of at least about 0.1, 0.5, 1, 5, 10, 20 or 50 N. The allograft may be assembled with a bone part, a metal implant, or a polymer implant (e.g., a cage, plate, or screw). The bone part may comprise native viable bone cells. The allograft may be load-sharing. The allograft may share force with one or more load-bearing constructs such as plate(s) and/or screw(s). There may be different levels of force distribution between the allograft and the load-bearing construct(s). The allograft may have machinability, for example, being capable of further being machined, cut, drilled, or shaped.

For each allograft of the present invention, a container is provided. The container comprises the allograft in a liquid coating the allograft. The liquid comprises 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 may be a cryopreservation, lyopreservation or rad io protecta nt solution.

As used herein, the term "about" modifying, for example, the dimensions, volumes, quantity of an ingredient in a composition, concentrations, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term "about" also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term "about" the claims appended hereto include equivalents to these quantities. The term "about" further may refer to a range of values that are similar to the stated 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 percent or less of the stated reference value.

Example 1. Preparation of growth factor extractions from tissues

A procedure is provided for preparing a growth factor extraction and subsequent reapplication. This procedure applies to tissues, including Bone Matrix (e.g., Bone Cell Matrix), Bone Marrow, Periosteum and/or Endosteum, whereby growth factors are extracted from the tissues, potentially combined, and subsequently reapplied to a target implant, comprising, for example, bone graft such as DBM fibers.

Growth Factor Extraction from Bone Cell Matrix. Bone Cell Matrix may include demineralized bone particulate and cellularized cortico-cancellous chips or demineralized bone fibers and cellularized cortico-cancellous chips. These products are then freeze-dried prior to further facilitate growth factor extraction. Growth factors extracted from other bone matrix may be prepared using the same procedure. In one aspect, the following steps may be carried out in a clean room or a laminar flow hood to maintain aseptic conditions:

1. Create a 10% solution of acetic acid by diluting with deionized water.

2. Produce a volume equivalent to twice the bone cell matrix product being extracted.

3. Example: for 50cc of bone cell matrix product produce lOOcc of 10% acetic acid by mixing 90cc acetic acid with lOcc dHzO.

4. Remove bone cell matrix product from pouch, if applicable, and wash twice with HBSS for 30 seconds in a sterile basin.

5. Place bone cell matrix product in suitable sealed sterile container and add correct volume of 10% acetic acid solution.

6. Place on shaker at 4°C for 20-24 hours.

7. Transfer samples to 50 mL conical tubes.

8. Balance and centrifuge at 4,000xg at 4°C for 15 minutes.

9. Collect supernatants, and neutralize with 0.3x(recorded volume) 5N NaOH.

10. Verify pH.

11. Retrieve sufficient number of Zeba™ Spin Desalting Columns for total volume of extract. 12. For each column: Twist off column's bottom closure and loosen cap; Place column in 50 mL conical tube; Add 5 mL PBS to column; Centrifuge at l,000xg for 2 minutes, discard buffer, repeat twice more; and Place column in new 50 mL tube.

13. Apply sample fraction to column and centrifuge at l,000xg for 2 minutes.

14. Pool fraction.

Growth Factor Extraction from Bone Marrow. Bone Marrow is collected by rasping the long bones (e.g., femur, tibia, and/or humerus) marrow contents into a sterile basin and subsequently transferring into sterile 50 mL conical tubes to facilitate further extraction. In one aspect, the following steps may be carried out in a clean room or a laminar flow hood to maintain aseptic conditions:

1. Create a 10% solution of acetic acid by diluting with deionized water.

2. Produce a volume equivalent to twice the bone marrow volume being extracted.

3. Example: for 50cc of bone marrow produce lOOcc of 10% acetic acid by mixing 90cc acetic acid with lOcc dbW.

4. Combine bone marrow and correct volume of 10% acetic acid solution in a sterile sealed container.

5. Place on shaker at 4°C for 20-24 hours.

6. Transfer samples to 50 mL conical tubes.

7. Balance and centrifuge at 4,000xg at 4°C for 15 minutes.

8. Collect supernatants, record volume, and neutralize with 0.3x(recorded volume) 5N NaOH.

9. Verify pH.

10. Retrieve sufficient number of Zeba™ Spin Desalting Columns for total volume of extract.

11. For each column: Twist off column's bottom closure and loosen cap; Place column in 50 mL conical tube; Add 5 mL PBS to column; Centrifuge at l,000xg for 2 minutes, discard buffer, repeat twice more; and Place column in new 50 mL tube.

12. Apply sample fraction to column and centrifuge at l,000xg for 2 minutes.

13. Pool fraction.

Growth Factor Extraction from Periosteum. Periosteum is recovered by using an osteotome on long bones (e.g., femur, tibia, and/or humerus) to recover the thin tissue on the surface of the bone. The soft tissue fragments are collected over a sterile basin and transferred into sterile 50 mL conical tubes to facilitate further extraction. In one aspect, the following steps may be carried out in a clean room or a laminar flow hood to maintain aseptic conditions:

1. Create a 10% solution of acetic acid by diluting with deionized water. 2. Produce a volume equivalent to twice the periosteum being extracted.

3. Example: for 50cc of periosteum produce lOOcc of 10% acetic acid by mixing 90cc acetic acid with lOcc dHzO.

4. wash twice with HBSS for 30 seconds in a sterile basin.

5. Place periosteum in suitable sealed sterile container and add correct volume of 10% acetic acid solution.

6. Place on shaker at 4°C for 20-24 hours; record start time.

7. Transfer samples to 50 mL conical tubes.

8. Balance and centrifuge at 4,000xg at 4°C for 15 minutes.

9. Collect supernatants, record volume, and neutralize with 0.3x(recorded volume) 5N NaOH.

10. Verify pH.

11. Retrieve sufficient number of Zeba™ Spin Desalting Columns for total volume of extract.

12. For each column: Twist off column's bottom closure and loosen cap; Place column in 50 mL conical tube; Add 5 mL PBS to column; Centrifuge at l,000xg for 2 minutes, discard buffer, repeat twice more; and Place column in new 50 mL tube.

13. Apply sample fraction to column and centrifuge at l,000xg for 2 minutes.

14. Pool fraction.

Growth Factor Extraction from Endosteum. Endosteum is recovered by using an osteotome or reamer on long bones (e.g., femur, tibia, and/or humerus) having bone marrow to recover the thin tissue that lines the center of the bones. The soft tissue fragments are collected over a sterile basin and transferred into sterile 50 mL conical tubes to facilitate further extraction. In one aspect, the following steps may be carried out in a clean room or a laminar flow hood to maintain aseptic conditions:

1. Create a 10% solution of acetic acid by diluting with deionized water.

2. Produce a volume equivalent to twice the endosteum being extracted.

3. Example: for 50cc of endosteum produce lOOcc of 10% acetic acid by mixing 90cc acetic acid with lOcc dHzO.

4. wash twice with HBSS for 30 seconds in a sterile basin.

5. Place endosteum in suitable sealed sterile container and add correct volume of 10% acetic acid solution.

6. Place on shaker at 4°C for 20-24 hours; record start time.

7. Transfer samples to 50 mL conical tubes.

8. Balance and centrifuge at 4,000xg at 4°C for 15 minutes.

9. Collect supernatants, record volume, and neutralize with 0.3x(recorded volume) 5N NaOH. 10. Verify pH.

11. Retrieve sufficient number of Zeba™ Spin Desalting Columns for total volume of extract.

12. For each column: Twist off column's bottom closure and loosen cap;

Place column in 50 mL conical tube; Add 5 mL PBS to column; Centrifuge at l,000xg for 2 minutes, discard buffer, repeat twice more; and Place column in new 50 mL tube.

13. Apply sample fraction to column and centrifuge at l,000xg for 2 minutes.

14. Pool fraction.

Growth Factor Extract Solution. For reapplication of the extracted growth factors onto a recipient implant, for example, a bone graft such as DBM fibers, the final growth factor solution may consist of one or more of the following selections: Bone Cell Matrix Growth Factor Extract, Bone Marrow Growth Factor Extract, Periosteum Growth Factor Extract, and/or Endosteum Growth Factor Extract in any ratio deemed appropriate.

Reapplication of Growth Factor Extract Solution onto DBM Fibers following the steps below:

1. Place 1 cc of DBM Fibers into a freeze dry tray.

2. Add Growth Factor Extract Solution onto DBM Fibers in tray using a 1 :3 ratio (cc of fibers to mL of extract).

3. Place freeze dry tray with DBM Fibers and Growth Factor Extract Solution into a -80C freezer until samples are completely frozen.

4. Place frozen samples in roll print package for freeze drying.

5. Pre-freeze the freeze drier shelf to -40C before adding samples.

6. Once cooled, add sample tray into freeze drier and use the cycle referred in table below (Freeze Dry Cycle Parameters).

7. Remove samples from freeze dryer and place in Tyvek pouch for storage (Table 1).

Table 1. Freeze Dry Cycle Parameters Sterilization. Submit packaged samples for sterilization (gamma irradiation) according to established company procedures for typical DBM Fiber products.

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

Growth factor extracts were prepared from a fresh tissue (e.g., bone marrow or bone matrix) (obtained from a donor within 60 hours of receipt), a frozen tissue (stored for more than one week at -80°C), an O/N Freeze tissue (stored overnight at -80°C), or an O/N RT tissue (stored overnight at room temperature (~20C)) using the methods described in Example 1. The tissue was bone narrow or bone matrix.

FIG. 2 shows that the total protein concentration was reduced in both marrow sourced and bone matrix sourced growth factor extracts when the source tissue was derived from a frozen donor source. The total protein concentration was quantified using the Pierce BCA protein assay and following manufacturer's instructions.

FIG. 3 shows that the BMP-2 concentration was reduced in both marrow sourced and bone matrix sourced growth factor extracts when the source tissue was derived from a frozen donor source. The BMP-2 concentration was quantified using a BMP-2 ELISA kit and following manufacturer's instructions.

FIG. 4 shows that the total protein concentration was reduced in the growth factor extract prepared from O/N Freeze bone marrow or O/N RP bone marrow as compared with the growth factor extract prepared from fresh bone marrow. The total protein was quantified using the Pierce BCA protein assay and following manufacturer's instructions.

FIG. 5 shows that the expression of BMP2, BMP 7 and PDGF growth factor in the growth factor extract prepared from O/N Freeze bone marrow or O/N RP bone marrow as compared with the growth factor extract prepared from fresh bone marrow. The growth factor concentrations were quantified using an ELISA kit specific to that growth factor and following manufacturer's instructions.

FIG. 6 shows BMP-2 concentration in growth factor extracts prepared from fresh bone marrow or fresh bone matrix. BMP-2 concentration was measured in extracts using a commercially available enzyme-linked immunosorbent assay (ELISA) kit and following manufacturer's instructions.

FIG. 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. The growth factor concentration was measured in extracts using commercially available enzyme-linked immunosorbent assay (ELISA) kits and following manufacturer's instructions. These select growth factors represent proteins which participate in the various stages associated with bone healing and repair.

Example 3. Growth factor extract prepared from pre-demineralized cortical bone Growth factor extracts may be prepared from allograft tissue intermediate processing steps, such as the acid rinsate from cortical bone that is intended for demineralization. In this example, cortical bone was demineralized in HCI and that acid was not discarded as the bone proceeded to the next steps of the process. In the example, the HCI may be collected and a growth factor extract may be recovered from the acid. This is an intermediate process in the demineralization of bone matrix. Instead of discarding the HCI used to demineralize a bone tissue, it is instead neutralized with NaOH, cleared with centrifugation, and kept for the present growth factors that were stripped alongside calcium-containing minerals. FIG. 8 illustrates a total protein concentration yield from a proof-of-concept process utilizing acetic acid when a growth factor extract is prepared from bone matrix, periosteum, endosteum or bone marrow.

Example 4. Bone grafts coated with growth factor extracts

Devitalized and mineralized cancellous cubes of bone grafts coated with a bone marrow growth factor extract was prepared using the method described in Example 1. The bone grafts were incubated with the bone marrow growth factor extract for dwell times, t=0 hours (Method 1), t= 16 hours (Method 2), or t=l hour (Method 3), respectively) before lyophilization. The coated bone grafts were placed in saline in an incubator and the saline was sampled after 24-hour intervals for quantifying protein to show release was occurring. Positive controls were an albumin loaded cube (t=0) and the negative control was an unloaded cube. FIG. 9 shows the total bone marrow protein release profile of the coated bone grafts.

Example 5. In vitro osteoinductivity

In vitro osteoinductivity of growth factor extracts, which may be prepared from different tissues or grafts impregnated with growth factor extracts, was evaluated in an in vitro alkaline phosphatase (ALP) assay and an in vitro mineralization assay.

In vitro ALP assay. C2C12 cells were cultured in DMEM supplemented with 1% FBS under standard cell culture conditions for 3 days. Alkaline phosphatase activity was measured in C2C12 cell lysates after exposure to a test sample for 3 days and normalized to total protein. Briefly, cells were lysed in lysis buffer containing 0.125% Triton X-100 on ice for 30 minutes. Cell lysates were then combined with 3mM para- Nitrophenyl Phosphate in a 96 well plate and incubated at 37C and 5% CO2 for 60 minutes. After 60 minutes, the reaction was stopped with IN NaOH and the absorbance was read at 405 nm with a spectrophotometer. Alkaline phosphatase (ALK) activity is an early indicator of osteoblastogenesis signaling the early phase of bone formation.

FIG. 10 shows an increase in ALK activity after introduction of a liquid growth factor extract from a bone marrow source (500ug/cc), a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control), or a BMP-2 solution (200ng/cc) (positive control).

FIG. 11 shows an increase in ALK activity after introduction of a liquid growth factor extract prepared from representative bone marrow (bars 1-3), periosteum (bar 4), or bone matrix (bars 5-8); each having a total protein concentration of 500 ug/cc; a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control), or a BMP-2 solution (200ng/cc) (positive control).

FIG. 12 shows an increase in ALK activity after introduction of a liquid growth factor extract prepared from representative bone marrow components: Bone Marrow Supernatant (liquid component, bar 4), Bone Marrow Pellet (cell component, bar 5); Undiluted Bone Marrow (bone marrow, bar 9), Periosteum (bar 8), or Bone Matrix (bar 10); each having a total protein concentration of 500 ug/cc; a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control, bar 2), or a BMP-2 solution (200ng/cc) (positive control, bar 3); extracts of demineralized bone matrix (DBM) in a high (bar 6) and low (bar 7) mass quantity (each having a total protein concentration of 500ug/cc) served as references. Bar 1 constituted an untreated cell population.

FIG. 13 shows an increase in ALK activity after introduction of demineralized cancellous sponges impregnated with a bone marrow growth factor extract at 500 ug said sponges per cc said extract. Bars 2, 3, and 4 depict three different bone marrow donors. Bar 1 is an uncoated sponge. And bars 5 and 6 are a liquid growth factor extract prepared from a devitalized mineralized cube (500ug/cc) (negative control), or a BMP-2 solution (200ng/cc) (positive control), respectively.

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 a transwell insert over the course of 21 days in the presence of media containing ascorbic acid and p-glycerophosphate. Untreated Control represented the presence of mineralized bone discs without a growth factor extract and Cell Control represented the absence of any mineralized bone disc, loaded or unloaded. Whereas ALK activity may represent an early indicator of bone formation in vitro through cell differentiation, the mineralization assay showcases actual late stage mineral formation in culture.

FIG. 14 shows images osteoblast cultures after exposure to bone marrow growth factor extract- loaded (500ug/cc) mineralized bone discs after 21 days in an in vitro mineralization assay. Black nodules are von Kossa stained calcium-containing mineralized deposits indicative of new bone formation in culture. FIG. 15 shows calcium content in osteoblasts exposed to uncoated (left bar) or 500ug/cc bone marrow growth factor extract coated mineralized bone discs (right bar) for 21 days were quantified. Osteoblasts exposed to discs loaded with bone marrow extracts expressed higher amounts of calcium-containing mineral deposits as corroborated by the images from the previous page (Donor 1 triplets and Donor 2 triplets) indicated by the presence of positive von Kossa staining (black nodules).

Example 6. Shelf-life of growth factor extracts

Growth factor extracts prepared from bone marrow of 3 separate donors were used to coat mineralized cubes (500ug/cc) with the addition of either sucrose, trehalose, or neither serving as a lyoprotectant 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 a Pierce assay and the BMP-2 content was quantified by ELISA. The control bar represents the amount of protein loaded into each cube.

FIG. 16 shows total protein concentration over time in response to different preservatives. While both lyoprotectants performed similarly at the first two time points with no treatment being the least effective over the whole-time course, sucrose performed best at the last time point evaluated. These results demonstrate that the growth factor extract was well preserved when it was used to coat mineralized cancellous cubes in the absence or presence of a lyoprotectant for up to 12 weeks.

FIG. 17 shows BMP-2 concentration over time in response to different preservatives. While no lyoprotectant was the worst performer, sucrose performed as well or better than trehalose over all time points observed.

Example 7. Structural cellular grafts

Structural cellular prototype grafts of 10x10x10 mm ³ were prepared. After obtaining the donated allograft bones the tissues were rinsed, debrided of the attached soft tissues, and cut off of undesired bone tissues and then cut into proper sizes and dimensions of implants as designed. The resulting grafts may also be machine cut using computer numerical control instrument (CNC) into certain shapes with precision. Then the grafts were rinsed to remove bone marrow and with antibiotics solutions to clean and disinfect the grafts. The cleaned and disinfected grafts were incubated with proper cyropreservation solutions then cryopreserved to maintain viable cells in the grafts. Post incubation with cryopreservation solutions, the grafts were blotted or centrifuged briefly to remove extra cryopreservation solution prior to insertion into a package and transferring into a freezer at -80oC or a LN2 freezer.

FIG. 18 shows viable cells in lOxlOxlOmm structural cellular prototype grafts as visualized in situ using calcein staining (bright dots in left panel), and outgrown cells visible under light microscopy after 2 weeks (middle panel) or 4 week (right panels). The viable cells were collected after they outgrew from the prototype grafts, and then used for all subsequent studies presented hereafter.

The cell concentration in the structural cellular prototype grafts (lOxlOxlOmm) was determined. The prototype grafts contained viable cells as determined in situ by two different methods (Alamar Blue and LDH assays). The whole prototype grafts were cultured in tissue culture plated with media containing Alamar Blue for 16 hours. Plates containing known cell concentrations were cultured alongside in order to provide a standard curve. Fluorescence readings of unknown prototype grafts were plotted against the known cell points to obtain cells/cc of tissue. For the LDH samples, the cells were 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 which allows viable 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 the live cells. Based on the LDH content, the number of viable bone cells is determined. FIG. 19 shows the cell concentration in the structural cellular prototype grafts prepared using Processes 1-4, which were similar with minor differences.

Immune cell depletion study was carried out. The cells contained within the prototype grafts (lOxlOxlOmm) before and after processing were weighed, washed thoroughly with PBS, placed in 0.5 ml 0.25% collagenase per 0.2 g of tissue and digested for 4 h with gentle agitation to obtain single-cell suspensions. The cells were filtered through a 70 pm filter and centrifuged at 400 x g for 5 minutes and washed once with PBS. The cells were then treated with CD45 (white blood cells) and CD166 (bone marrow hematopoietic cells) antibodies conjugated to 2 different fluorophores on ice for 30 minutes. Flow cytometry was used to identify the percentage of cells positive for each cell surface marker before and after processing. Reduction in potentially immunoreactive cells was at least 85% or greater. FIG. 20 shows average expression of CD45 and CD166 markers in 30x30x15 mm ³ prototype grafts from three donors before and after processing.

The PBMC reactivity with the structural cellular prototype grafts was evaluated. Both lymphocytes and bone cells isolated from the structural cellular donors were plated separately in a 96-well plate to form a stimulating layer of cells, and treated with mitomycin C to inhibit proliferation from the stimulating layer. Cells were then cocultured with PBMCs, which formed a responding layer. PBMCs treated with Concanavalin A or lymphocytes were used as positive controls. The cells were left for 2 days at 37°C and 5% CO2. Cell proliferation was assessed via a BrdU ELISA following the manufacturers protocol. PBMCs in contact with bone cells isolated from the structural cellular donors did not proliferate when compared to PBMCs alone. FIG. 21 shows corrected proliferation of PMBC in response to bone cells lymphocytes (bar 1), Concanavalin A (bar 2), bone cells from structural cellular grafts (bar 4) or bone cells from a ViviGen® bone graft (bar 5). For this example, reference to the ViviGen® bone graft means a bone void filler with a combination of DBM and mineralized bone having viable cells. A ViviGen® bone graft was prepared by grinding cleaned bone blocks (cortical cancellous bone) containing viable bone cells into particulates, then mixed with demineralized bone fibers or particulates from the same donor, incubated with a cryopreservation solution, and then cryopreserved in a LN2 freezer.

Osteogenic Cell Population was evaluated. Cells released from the prototype grafts were cultured in either basal or mineralization media (containing ascorbic acid and p-glycerophosphate) under standard cell culture incubated conditions for up to 4 weeks. FIG. 22 shows cells from outgrowth cultured were fixed and stained with Alizarin Red after 2 weeks (left panel), 3 weeks (middle panel), or 4 weeks (right panel). Cultures positive for calcium-containing mineral deposits were denoted by positive staining for Alizarin Red (dark red regions). Control cells (insets) did not exhibit any staining.

Growth factor (BMP) release was evaluated. Cells were released from the prototype grafts, collected, and cultured in either basal or mineralization media (containing ascorbic acid and p-glycerophosphate) for up to 21 days. After 3, 7, 14, or 21 days, media samples were subject to a BMP-2 and BMP-7 ELISA kit. FIG. 23 shows average BMP-2 or BMP-7 concentration from cells isolate from structural cellular prototype grafts in mineralization donor cells or control donor cells at day 3, 7, 14 or 21. Cells in mineralization media exhibited higher concentrations of BMP-2 and BMP-7 over those in basal media at every time point tested.

Example 8. Structural cellular grafts in PLF application

Structural cellular grafts may have different sizes and shapes. Structural cellular grafts in cylinder and/or strip shapes (30x15x5 mm ³ ) containing viable cells overlaid between two spine transverse processes for posterolateral fusion (PLF) procedure as shown in FIG. 24. Trabecular structure of Structural Cellular grafts provides immediate structural stability and no graft migration concern which usually observed in putty like graft due to compression from surrounding tissue after implantation. This approach will promote healing to get optimal fusion outcome.

Example 9. Viability map of structural cellular grafts

Structural cellular prototype grafts (30x30x15mm) contained viable cells as determined in situ by an Alamar Blue assay. Grafts were sectioned as shown in FIG. 25 and each subsection was measured for viable cells. Whole prototypes were cultured in tissue culture plated with media containing Alamar Blue for 16 hours. Then the block prototypes were sectioned per diagram in FIG. 25 for cells per cc measurements. Plates containing known cell concentrations were cultured alongside in order to provide a standard curve. Fluorescence readings of unknown prototype grafts were plotted against the known cell points to obtain cells/cc of tissue. These study results showed cell viability and cell count are consistent and homogeneous throughout the structural cellular grafts.

All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and/or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.