Autologous bone graft substitute

Abstract

The present invention relates to an autologous bone graft substitute composition (ABGS) for inducing new bone formation, promoting bone growth, and treating bone defects, a method for preparing the autologous bone graft substitute composition, and a kit for preparing an implantable autologous bone graft substitute composition. In a particular embodiment, the present invention relates to an injectable / extrudable / implantable autologous bone graft substitute composition for treating bone defects, inducing new bone formation, and promoting bone growth for fracture healing and spinal fusion, and for repairing bone defects in bone reconstruction procedures in orthopedic surgery and oral and maxillofacial dental surgery.

Description

[Technical Field]

[0001] The present invention relates to compositions, methods for preparing compositions, and kits for preparing implant compositions. The compositions can mimic "biological autografts" used to treat bone defects, induce new bone formation, and promote bone growth. In a specific embodiment, the present invention relates to an injectable / extrudable / implantable autologous bone graft substitute composition (hereinafter referred to as ABGS) for treating bone defects, inducing new bone formation, and promoting bone growth for fracture healing and spinal fusion, and for repairing bone defects in bone reconstruction procedures in orthopedic surgery and oral and maxillofacial dental surgery. [Background technology]

[0002] Bone grafting is employed in a variety of surgical procedures designed to promote new bone growth to repair bone defects, enhance bone growth at specific sites, promote and reconstruct the fusion of adjacent bones, increase the stability of skeletal structures, and relieve back and leg pain experienced by affected individuals. Often, surgical procedures use bone grafts in combination with any of a variety of instrumentation, such as pedicle screws or rods, to stabilize the implanted bone graft and provide added stability as new bone grows to correct the defect.

[0003] Spinal fusion is a surgical procedure primarily used to alleviate back and leg pain in individuals with injured or inflamed spinal segments (vertebrae-discs-vertebrae). During spinal fusion, two or more adjacent spinal segments are fused together, limiting intersegmental motion and compression. This terminates or substantially reduces pain and further degeneration due to motion or compression of the injured adjacent segments. Therefore, the desired outcome of spinal fusion is to replace the original arrangement of two vertebrae separated by an articular fibrocartilaginous disc with a stable, continuous, load-bearing bony segment, thereby alleviating pain and mitigating further segmental degeneration (e.g., anterior interbody fusion, ALIF).

[0004] In addition to relieving pain and further stabilizing the spine to avoid compression, two transverse processes on both sides of a given segment (level) are fused by forming new functional bone in the heterotopic site using autograft, devitalized allograft segments, or allogeneic demineralized bone matrix enriched with patient bone marrow and containing devitalized allograft particles or patient bone marrow (e.g., posterolateral fusion, PLF). Surgeons use metal instruments such as cages, screws, and rods to assist in the fusion of both anterior (vertebral bodies) and posterior (vertebral processes) lumbar segments, since immobilization of motion (arthrodesis) minimizes compression of the spinal nerves passing between the two lumbar vertebrae. To increase fusion rates, instrumentation is supplemented with bone grafts to stimulate bone formation in either the intervertebral space (anterior interbody fusion, ALIF), the heterotopic site between the transverse processes (posterolateral fusion, PLF), or both (posterior interbody fusion, PLIF). See, e.g., Mobbs, RJ et al. Lumbar interbody fusion: techniques, indications and comparison of interbody fusion options including PLIF, TLIF, MI-TLIF, OLIF / ATP, LLIF and ALIF. J Spine Surg 1:2-18 (2015). A variety of disorders may be treated with spinal fusion, including, but not limited to, degenerative disc disease (DDD), spondylolisthesis, spinal stenosis, scoliosis, infection, fractures, spinal muscular atrophy, and various tumors.

[0005] Spinal fusion requires materials to help promote new bone growth to fuse adjacent vertebrae. For decades, standard spinal fusion procedures required an additional incision to harvest autologous bone fragments (autografts) from the patient's iliac crest in the hip. The harvested bone fragments are inserted into the affected intervertebral space and stabilized with appropriate instrumentation, such as pedicle screws or rods. This spinal fusion procedure using autografts from the patient is also known as intracorporeal bone grafting (ICBG). The advantage of using ICBG is that the harvested bone fragments contain native bone marrow cells and an extracellular matrix that is immunologically compatible with the patient's body and contains functional bone marrow elements such as red blood cells, white blood cells, and megakaryocytes. However, utilizing ICBG requires the patient to undergo an additional incision. Making an additional incision to harvest bone for use as an autograft lengthens the surgical procedure, resulting in the patient being under anesthesia for longer and experiencing postoperative pain at another site (see, e.g., Kim et al., Spine J. 9:886-92; 2009). This has also been a concern in the field because introducing another incision into the patient increases the risk of infection. Furthermore, the amount of IGBG that can be harvested is limited.

[0006] To reduce these additional risks to patients, many alternative implantable compositions have been developed and tested to replace the need for autografts during surgery. These compositions may include allografts (e.g., cadaveric bone from bone banks), demineralized bone matrix (DBM), various ceramics (calcium-based compounds), synthetic polymers, bone morphogenetic proteins (BMPs), and their analogs in combination with animal-derived collagen scaffolds, as well as various combinations thereof. For more information, see Gupta S, MV, Gupta MC (2017) Biology of spine fusion and application of osteobiologics in spine surgery in Vukicevic S, Sampath KT (eds.) Bone Morphogenetic Proteins: Systems Biology Regulators. Springer International Publishing. However, attempts to develop alternatives to autografts for spinal fusion surgery face unique challenges in gaining regulatory approval.

[0007] The INFUSE® Bone Graft / LT-Cage Tapered Lumbar Fusion Device (Medtronic Sofamor Danek, Memphis, Tennessee, USA) was approved in 2002 as an alternative to ICBG for DDD procedures (vertebra-disc-vertebra, ALIF) at the L2-S1 levels using an anterior approach in skeletally mature patients. The INFUSE® Bone Graft component is composed of human bone morphogenetic protein-2 (rhBMP-2) applied to an absorbable bovine Achilles tendon-derived collagen sponge carrier, which is used to fill the lordotic threaded (LT) cage component. Each device contains a sheet of collagen sheet soaked with 12 milligrams of rhBMP-2 (total amount), 6 mg of which is then inserted into the disc space, filling each of the LT cage components. For more information, see INFUSE Bone Graft Product Information, Lumbar. (2002) Medtronic Sofamor Danek USA, Inc. www.accessdata.fda.gov / cdrh_docs / pdf / P000058c.pdf. Accessed February 2010. The approved surgical procedure involves implanting the device on each side of the affected vertebral level to promote fusion stabilization on each side of the segment. The device's ability to facilitate spinal fusion in various off-label posterior approaches has also been the subject of considerable research. See, for example, Cahill et al., JAMA 302:58-66 (2009) and Carragee, et al., Spine J 11:471-491 (2011). However, off-label use has raised unwelcome safety concerns, leading to congressional hearings and FDA scrutiny.

[0008] The AMPLIFY® bone graft substitute (Medtronic) was initially designed and tested as an ICBG replacement for spinal fusion using the posterolateral approach (PLF). This ICBG replacement delivered 40 mg of rhBMP-2 in a porous synthetic slab. It consisted of a bovine Achilles tendon-derived collagen sponge filled with ceramic granules of hydroxyapatite (HA) and tricalcium phosphate (TCP) composite. Although the AMPLIFY® bone graft substitute provided moderate spinal fusion in DDD procedures, the U.S. Food and Drug Administration (FDA) denied approval of the product due to concerns about the continued low back and leg pain experienced by a significant number of patients, the possible cancer risk associated with the relatively large doses of rhBMP-2 administered, and the continued occurrence of undesired ectopic bone formation at sites distant from the implant due to the high doses of BMP-2 used in the device. See, e.g., P050036 Medtronic's AMPLIFY™ rhBMP-2 Matrix Orthopedic and Rehabilitation Devices Advisory Panel (2010) Food and Drug Administration Executive Summary.

[0009] The OP-1® Implant (Olympus / Stryker) is an implantable device for bone regeneration. The device contains recombinant human BMP-7 (rhBMP-7) dispersed within type I collagen derived from bovine bone. The OP-1® Implant has received FDA approval only for use under a Humanitarian Device Exemption (HDE) as a substitute for autograft in difficult long bone nonunion defects when the use of autograft is impractical due to the inability to obtain statistically significant differences in new bone formation over autograft and alternative treatments have failed. See, for example, Stryker Biotech OP-1 Implant® product information (2009) www.stryker.com / stellent / groups / public / documents / web_prod / 126737.pdf, accessed February 2010.

[0010] OP-1 Putty® is a composition combining rhBMP-7, bovine bone collagen, and a putty additive (carboxymethylcellulose). The OP-1 Putty® device was evaluated in a posterolateral fusion (PLF) clinical study, but no statistically significant difference was observed compared to autograft. Therefore, OP-1 Putty® is approved for use as an HDE in spinal fusion. See, for example, Stryker Biotech OP-1 Putty® product information (2009) www.stryker.com / stellent / groups / public / documents / web_prod / 127024.pdf, accessed February 2010.

[0011] In addition to the use of autografts in spinal fusion, there are a variety of other orthopedic and dental indications that are treated with autografts. These indications include various defects and fractures, such as fractures of the diaphysis, distal radius fractures, tibial nonunions, high tibial osteotomies, osteoporotic fractures (vertebral compression fractures, atypical femoral fractures), defects due to bone cysts, and defects due to bone tumors, as well as various oral, periodontal, and maxillofacial defects and abnormalities. Furthermore, autografts are also used to treat pseudarthrosis and pseudofractures associated with rare musculoskeletal disorders, such as hypophosphatasia (mutation of the tissue-nonspecific alkaline phosphatase gene), neurofibromatosis type I (mutation of the neurofibromin gene), and osteogenesis imperfecta (mutation of the type I collagen gene). Depending on the severity of bone loss in such conditions, the amount of autograft that can be safely obtained from the patient may be insufficient to provide the desired treatment, or may compromise bone quality (in at-risk smokers, diabetics, steroid users, and osteoporotic patients). In such cases, there is a great need for biological bone graft substitutes that can induce safe, robust bone formation localized to the defect site. For comprehensive reading, see Vukicevic S, Sampath TK, editors. Bone Morphogenetic Proteins: From Laboratory to Clinical Practice. Basel: Birkhauser Verlag, 2002; Vukicevic S, Sampath, KT, editor. Bone Morphogenetic Proteins: Systems Biology Regulators. Springer International Publishing; 2017. Additionally, WO2009129631A1 discloses a biocompatible implant for bone repair, comprising a flexible membrane adapted to fit around a bone defect, a platelet-rich plasma gel composition contained within a void space created by the membrane, and a platelet-rich plasma gel composition contained within the void space created by the membrane. However, WO 2008011192A2 discloses an induced whole blood-derived clot for use in treating bone defects, prepared by a process comprising: (a) combining (1) whole blood, (2) bone morphogenetic protein, (3) exogenously provided calcium ions, and (4) optionally, an exogenously provided fibrin-thrombin mixture; and (b) incubating the components combined in step (a) until a mechanically stable clot is formed, wherein the exogenously provided calcium ions are present at a concentration effective to provide a homogeneous, cohesive, syringeable, injectable, and flexible clot gel. WO 9119510A1 discloses an osteoinductive pharmaceutical formulation comprising an antifibrinolytic agent such as epsilon amino acid caproic acid (EACA) or other lysine analogue or a serine protease inhibitor and a cartilage and / or bone inductive protein. The efficacy of the formulation is based on the use of EACA in the treatment of cartilage and / or bone defects compared to samples lacking EACA. To date, patients have been limited to only a few potential options for undergoing autograft procedures for spinal fusion and other orthopedic surgeries. Clearly, there is a need for improved compositions that can promote the level and quality of new bone growth necessary for successful repair of bone defects and spinal fusion procedures without the need to subject patients to the additional risks, pain, and limitations associated with autograft procedures. [Prior art documents]

Non-licensed literature

[0012] [Non-licensed document 1] Mobbs,RJet al.Lumbar interbody fusion:techniques,indications and comparison of interbody fusion options including PLIF,TLIF,MI-TLIF,OLIF / ATP,LLIF and ALIF.J Spine Surg.2015 1:2-18 [Non-licensed document 2] Kim et al.,Spine J.9:886-92;2009 [Non-licensed document 3] Gupta S,MV,Gupta MC(2017) Biology of spine fusion and application of osteobiologics in spine surgery In:Vukicevic S, Sampath. KT.(ed) Bone Morphogenetic Proteins:Systems Biology Regulators.Springer International Publishing

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0013] The present invention solves the problems presented in the background art by providing an autologous bone graft substitute composition (ABGS) that functions as a mimic of ICBG for use in promoting new bone growth at the site of an individual in need of treatment. In certain aspects, the present invention relates to an injectable / extrudable / implantable autologous bone graft substitute composition (ABGS) for use in treating bone defects, inducing new bone formation and promoting bone growth for fracture healing, spinal fusion surgery, and repairing bone defects in bone reconstruction procedures in orthopedic surgery and oral and maxillofacial dental surgery. [Means for solving the problem]

[0014] In one embodiment, the present invention provides an autologous bone graft substitute composition (ABGS) that is an autograft substitute for promoting new bone growth, 1) Autologous blood (AB), 2) Bone morphogenetic proteins (BMPs) that cause bone formation and 3) Pharmacologically acceptable calcium salts and Ca in ionic solutions or nanoparticles ++ a blood coagulation agent selected from a microsphere complex; and 4) Compression-resistant base material (CRM) included; The autologous blood forms a clot gel containing bone morphogenetic proteins that carry out bone formation, calcium salts, and a compression-resistant matrix (hereinafter referred to as CRM).

[0015] The bone morphogenetic protein that effects bone formation is selected from BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-6, BMP-9, BMP-12 and BMP-13, analogs thereof, heterodimers thereof and combinations thereof. Highly preferably, the bone morphogenetic protein that effects bone formation is BMP-6 or BMP-7, preferably BMP-6.

[0016] Furthermore, the autologous blood (hereinafter referred to as AB) may contain autologous or allogeneic platelet-rich plasma (hereinafter referred to as PRP), or the autologous blood (AB) may be substituted with platelet-rich plasma (PRP).

[0017] The compression resistant matrix (CRM) is selected from the group consisting of bone graft, calcium phosphate-calcium carbonate composite, bioabsorbable polymer or copolymer, calcium sulfate, bioabsorbable hydrogel, and combinations thereof.

[0018] In another embodiment of the present invention, an autologous bone graft substitute composition for treating bone defects, inducing new bone formation and promoting bone growth comprises: 1) autologous blood, 2) bone morphogenetic proteins that perform bone formation, selected from BMP-6, BMP-2, BMP-7, BMP-4, BMP-2, BMP-5, BMP-8, BMP-9, BMP-12 and BMP-13, analogs thereof, heterodimers thereof and combinations thereof; 3) Pharmacologically acceptable calcium salts and Ca in ionic solutions or nanoparticles ++ a blood coagulation agent selected from a microsphere complex; Here, the autologous blood forms a clot gel containing bone morphogenetic proteins and calcium salts that drive bone formation.

[0019] Suitably, the osteogenic protein that effects bone formation is BMP-6 or BMP-7, preferably it is BMP-6.

[0020] Additionally, the autologous blood (AB) may contain autologous or allogeneic platelet-rich plasma (PRP), or the autologous blood (AB) may be substituted with autologous platelet-rich plasma (PRP).

[0021] In another embodiment of the present invention, the autologous bone graft substitute composition of the implant for inducing new bone formation and promoting bone growth comprises: 1) autologous blood, 2) bone morphogenetic proteins that perform bone formation, selected from BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-8, BMP-9, BMP-12 and BMP-13, analogs thereof, heterodimers thereof and combinations thereof; 3) includes a compression resistant base material (CRM); Here, the autologous blood forms a clot gel containing bone morphogenetic proteins and CRMs that drive bone formation.

[0022] Preferably, the bone morphogenetic protein that causes bone formation is BMP-6. Instead of the bone morphogenetic protein that causes bone formation, a bone morphogenetic protein that causes bone formation selected from BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-8, BMP-9, BMP-12, BMP-13, analogs thereof, heterodimers thereof, and combinations thereof may be used.

[0023] Preferably, the compression resistant matrix (CRM) is selected from the group consisting of bone graft and / or calcium phosphate-calcium carbonate composite material.

[0024] In another embodiment, the present invention provides a kit for preparing an autologous bone graft substitute composition implant for inducing new bone formation.

[0025] In another embodiment, the autologous bone graft substitute compositions described herein may further comprise an exogenously provided population of a patient's bone marrow aspirate containing osteocyte progenitor cells.

[0026] In another embodiment, the autologous bone graft replacement compositions described herein may further comprise an exogenously provided population of ex vivo expanded mesenchymal stem cells (osteocyte progenitor cells) from the patient's bone marrow.

[0027] In another embodiment, the autologous bone graft replacement compositions described herein may further comprise an exogenously provided population of mesenchymal stem cells (osteocyte progenitor cells) expanded ex vivo from the patient's adipose tissue.

[0028] In another embodiment, the autologous bone graft replacement compositions described herein may further comprise an exogenously provided population of mesenchymal stem cells (osteocyte progenitor cells) expanded ex vivo from the patient's periosteal layer (periosteum).

[0029] In another embodiment, the autologous bone graft substitute compositions described herein may further comprise exogenously provided, finely divided tissue fragments prepared from the patient's adjacent local bone, suitable skeletal muscle and / or fascia.

[0030] In another embodiment, the autologous bone graft replacement compositions described herein may further comprise an exogenously provided population of ex vivo expanded mesenchymal stem cells (osteocyte progenitor cells) derived from allogeneic bone marrow or adipose tissue or periosteum from an immunocompatible individual of the patient.

[0031] In another embodiment, the autologous bone graft replacement compositions described herein may further comprise an exogenously provided population of mesenchymal stem cells (osteocyte progenitor cells) derived from allogeneic umbilical cord and expanded ex vivo.

[0032] In another embodiment, the autologous bone graft substitute compositions described herein may further comprise allogeneic or exogenously provided patient platelet-rich plasma (PRP), a source for fibrin microstructure and growth factors (e.g., PDGF, IGF, VEGF, and TGF-beta).

[0033] In another embodiment, the autologous bone graft substitute composition described herein as a compression-resistant matrix (CRM) is selected from the group consisting of bone grafts (e.g., prepared from cortical or cancellous bone or both), calcium phosphate-calcium carbonate composites (hereinafter abbreviated as ceramics), thermosensitive bioabsorbable polymers or copolymers (e.g., lactide or glycolide polymers and copolymers and combinations thereof), calcium sulfate, bioabsorbable hydrogels, and combinations thereof. The calcium phosphate-calcium carbonate composite is selected from the group consisting of hydroxyapatite (HA), tricalcium phosphate (TCP), and TCP / HA composites.

[0034] In yet another embodiment, the autologous bone graft substitute compositions described herein may further comprise allogeneic demineralized bone matrix.

[0035] In another embodiment of the present invention, the autologous bone graft substitute composition described herein may further comprise a bone graft substitute, which may further comprise a reverse-phase thermosensitive bioabsorbable polymer. The reverse-phase thermosensitive bioabsorbable polymer may be mixed with autologous blood (AB). This assumes the rheology of a composition comprising AB, BMP, and CRM. As a result, it can be injected in a liquid state at room temperature and forms a biocompatible gel at the delivery / implantation site as the temperature rises to body temperature (37°C). Thus, the composition provides a non-invasive, injectable composition containing BMP, CRM, and a reverse-phase thermosensitive bioabsorbable polymer in autologous blood. This formulation can be administered non-invasively, for example, by injection, thus overcoming limitations associated with currently commercially available individual bone graft substitutes. Injectable ABGS effectively induces new bone formation in a standard rat model of ectopic bone formation. This injectable composition allows new bone formation at relatively low BMP concentrations due to tight binding to plasma proteins that are retained by the reverse phase thermosensitive bioabsorbable polymer at body temperature for a sustained period of time.

[0036] In another embodiment, the present invention provides a method for preparing an autologous bone graft substitute composition for treating bone defects, inducing new bone formation, and promoting bone growth, the method comprising: (1) a) Autologous blood (AB), or autologous blood and platelet-rich plasma (PRP), or platelet-rich plasma (PRP); b) bone morphogenetic proteins that induce bone formation selected from BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-9, BMP-12 and BMP-13, analogs thereof, heterodimers thereof and combinations thereof; c) Pharmacologically acceptable calcium salts and Ca in ionic solutions or nanoparticles ++ a blood coagulation agent selected from a microsphere complex; and d) combining a compression resistant base material (CRM); (2) incubating the ingredients of step (1) for a time sufficient to form a coagulum gel.

[0037] In another embodiment, further ingredients are added prior to step (2).

[0038] In another embodiment, the present invention provides a method for preparing an autologous bone graft substitute composition for treating bone defects, inducing new bone formation, and promoting bone growth, the method comprising: (1) a) autologous blood; b) a bone-forming osteogenic protein selected from BMP-6, BMP-2, BMP-7, BMP-2, BMP-9, BMP-12 and BMP-13, heterodimers thereof and combinations thereof; c) Pharmacologically acceptable calcium salts and Ca in ionic solutions or nanoparticles ++ a blood coagulation agent selected from a microsphere complex; d) compression resistant base material (CRM); (d) optionally, an exogenously provided population of ex vivo expanded autologous or allogeneic mesenchymal cells (osteocyte progenitor cells) derived from bone marrow, adipose tissue, periosteal layer and / or umbilical cord; and / or (e) optionally binding demineralized bone matrix; (2) incubating the ingredients of step (1) for a time sufficient to form a coagulum gel.

[0039] In another embodiment, the present invention provides a method for preparing an autologous bone graft substitute composition for treating bone defects, inducing new bone formation, and promoting bone growth, the method comprising: (1) a) autologous blood; b) osteogenesis, which produces bone; c) a blood coagulant selected from pharmacologically acceptable calcium salts and Ca++ microsphere complexes in ionic solution or in nanoparticles; d) compression resistant base material (CRM); (d) reverse phase thermosensitive bioabsorbable polymers; (e) optionally, an exogenously provided population of ex vivo expanded autologous or allogeneic mesenchymal cells (osteocyte progenitor cells) derived from bone marrow, adipose tissue, periosteal layer or umbilical cord; and / or (f) optionally binding demineralized bone matrix; (2) incubating the components of step (1) for a time sufficient to form a coagulum gel having a rheology that allows it to be injected in a liquid phase at room temperature and forms a biocompatible gel at the delivery / implantation site as the temperature rises to body temperature (37°C).

[0040] The ABGS described herein may be implanted or injected (eg, using a syringe) at the site of a bone defect or at a site requiring new bone growth (eg, spinal fusion, skeletal reinforcement).

[0041] Preferably, the osteogenic BMP useful in the ABGS compositions described herein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-12, BMP-13, analogs thereof, heterodimers thereof, and combinations thereof. More preferably, the osteogenic BMP is BMP-6.

[0042] In another embodiment, the osteogenic BMP is present in the ABGS described herein in an amount ranging from 0.002 mg to 1 mg of bone-forming protein BMP (preferably BMP-6) per milliliter (ml) of autologous blood.

[0043] In another embodiment, the ABGS described herein comprises a compression-resistant matrix (CRM) selected from the group consisting of a bone graft, a calcium phosphate-calcium carbonate composite, a bioabsorbable polymer or copolymer, and combinations thereof. Preferably, the calcium phosphate-calcium carbonate composition is selected from the group consisting of hydroxyapatite (HA), tricalcium phosphate (TCP), and combinations thereof.

[0044] In another embodiment, the present invention provides A butterfly needle set for autologous blood collection, a freeze-drying container holder including a needle protected by a rubber sleeve; a sterile tube connecting the butterfly needle and the freeze-drying container holder; a tube clamp to release the vacuum or stop the blood flow; a freeze-drying container with a rubber stopper containing freeze-dried bone-forming bone morphogenetic protein (BMP) mixed with CRM; The kit for preparing an ABGS implant for inducing new bone formation is provided, wherein the CRM is selected from the group consisting of bone graft, hydroxyapatite, tricalcium phosphate, and combinations thereof, and the osteogenic protein that induces bone formation is selected from BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-8, BMP-9, BMP-12, BMP-13, analogs thereof, heterodimers thereof, and combinations thereof. Preferably, the osteogenic protein that induces bone formation is BMP-6.

[0045] In another embodiment, the present invention provides a method for preparing an ABGS implant for inducing new bone formation, the method comprising: (1) a. Autologous blood, b. A bone morphogenetic protein that induces bone formation selected from BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-8, BMP-9, BMP-12, BMP-13, analogs thereof, heterodimers thereof, and combinations thereof; c. combining a compression resistant matrix (CRM) selected from the group consisting of bone graft, hydroxyapatite, tricalcium phosphate, and combinations thereof; (2) mixing an osteogenic protein in an aqueous solution with a CRM in a lyophilization vial, the amount of the osteogenic protein in the aqueous solution added to the CRM being optimized to completely wet the CRM; (3) freeze-drying the bone morphogenetic protein and CRM that cause bone formation; (4) adding autologous blood; (5) incubating the components of step (1) for a time sufficient to form a biomechanically stable clot around the CRM.

[0046] Preferably, the bone morphogenetic protein that effects bone formation is BMP-6.

[0047] The autologous bone graft substitute composition described herein can also be used to treat any of various bone defects that require new bone formation or growth.The bone defects that can be treated by using the ABGS described herein include, but are not limited to, diaphyseal fractures, distal radius fractures, tibial fusion, osteoporotic fractures, bone cyst defects (e.g., when new bone needs to be generated to fill the void previously occupied by a cyst), bone tumor defects (e.g., when new bone needs to be generated to replace bone lost due to cancer or bone removed by surgery), pseudoarthrosis and pseudofractures associated with congenital skeletal diseases (e.g., hypophosphatasia, type I neurofibromatosis, osteogenesis imperfecta), osteoporotic fractures (e.g., vertebral compression fractures, atypical diaphyseal fractures), oral bone defects (e.g., various dental defects), periodontal defects, and maxillofacial defects or abnormalities (including bone augmentation of the jaw, face, and skull).

[0048] The "autologous bone graft substitute" (ABGS) described herein may also be used to promote new bone growth in a variety of orthopedic and dental indications, including, but not limited to, spinal fusion, high tibial osteotomy, and maxillofacial augmentation. In ABGS, BMPs are combined with autologous blood clots, which are then reinforced with a compression-resistant substrate to induce the formation of new bone tissue. BMP-6 is the preferred BMP because it does not tightly bind to the BMP antagonist noggin (Song et al., J Biol Chem.; 285(16):12169-80(2010)), which is abundant in bone. It also binds to most signaling BMP-6 type I and type II receptors (different from BMP2 and BMP7). Autologous blood clots (ABC) are the preferred substrate for BMP delivery because many plasma proteins tightly bind BMP6, resulting in a sustained and linear release of BMP6 over a 7-10 day period. ABC also provides a permissive environment for bone formation in the presence of CRM without eliciting an inflammatory or immune response, in stark contrast to the inflammation seen when BMPs are implanted onto animal-derived collagen scaffolds. Furthermore, autologous blood contains osteocyte precursor cells (mesenchymal stem cells), which readily respond to BMP-6 during clot formation and initiate new bone formation at the implant site.

[0049] The autologous bone graft substitute composition (ABGS) described above may also be used to increase bone density in the femoral head and vertebral bodies of patients with severe osteopenia, such as those suffering from postmenopausal, senile, or steroid-induced osteopenia. These patients with severe osteopenia may suffer hip and vertebral fractures when they fall. In these patients, small amounts of ABGS may be injected into the cancellous bone at multiple sites in small volumes to promote bone growth and increase bone density in the femoral head and vertebral bodies. [Brief explanation of the drawings]

[0050] [Figure 1]Micrographs of ABGS prepared by methods #2 or #3 and CRM derived from human blood from a volunteer are shown. Human allografts from a tissue bone bank are used as CRM. Overall view (left), X-ray (center), and micro-CT scan (right). [Figure 2A] (Allograft / autologous blood clot containing allogeneic bone from donor) ALLO plus ABC implants and ABC+ALLO+rhBMP6 implants at 7 and 35 days are shown. [Figure 2B] (Allograft / autologous blood clot containing allogeneic bone from donor) ALLO plus ABC implants and ABC+ALLO+rhBMP6 implants at 7 and 35 days are shown. [Figure 2C] MicroCT analysis of ABC / ALLO and ABGS (ABC / ALLO / rhBMP6) with implants at 7 and 35 days is shown. [Figure 2D] MicroCT analysis of ABC / ALLO and ABGS (ABC / ALLO / rhBMP6) with implants at 7 and 35 days is shown. [Figure 2E] 1 is a graph showing allograft volume and bone volume quantified using micro-CT analysis for ABC plus ALLO and ABC+ALLO+rhBMP6 at 7 and 35 days after subcutaneous implantation in rats. [Figure 2F] 1 is a graph showing allograft volume and bone volume quantified using micro-CT analysis for ABC plus ALLO and ABC+ALLO+rhBMP6 at 7 and 35 days after subcutaneous implantation in rats. [Figure 3A] 1 shows a photomicrograph of the histology from an autologous blood clot containing allograft (ALLO) implanted in a subcutaneous site in a rat. [Figure 3B] 1 shows photomicrographs of histology when ALLO particles were formulated into ABCs implanted in subcutaneous sites in rats. [Figure 3C]ABC+ALLO+rhBMP6, a photomicrograph of the histology of implants containing ALLO particles formulated and implanted in subcutaneous rat sites. [Figure 3D] Graph showing the mean number of multinucleated FBGCs morphometrically counted from three representative histological sections from ALLO, ALLO+ABC, and ALLO+ABC+rhBMP6 implants. [Figure 3E] Photomicrographs of tissue structures characterized by immunohistochemistry for acid phosphatase staining are shown. [Figure 3F] Photomicrographs of tissue structures characterized by immunohistochemistry for acid phosphatase staining are shown. [Figure 4A] 1 is a graph showing the amount of rhBMP6 released from ABGS without allograft and with allografts of two different particle sizes. [Figure 4B] 1 is a graph showing the cumulative release of rhBMP6 from ABGS without allograft and with allografts of two different particle sizes. [Figure 4C] 1 is a graph showing the cumulative release of rhBMP6 from ABGS without allograft and with allografts of two different particle sizes. [Figure 5] Figure 1 shows reproducibly induced new bone formation in rabbit ulnae, represented at 6, 9, 13, 16, 19, and 23 weeks, and reconstructed critical size defects measured radiographically in a dose-dependent manner. [Figure 6A] Radiographs and individual micro-CT rabbit ulna analyses are shown, demonstrating dose-dependent responses. [Figure 6B] X-ray scoring from the X-ray in Figure 6A is shown. [Figure 6C] Radiographs and individual micro-CT rabbit ulna analyses are shown, demonstrating dose-dependent responses. [Figure 6D] Morphometric analysis of the medullary canal and bone volume of the defect is shown. [Figure 7] 1 shows histological sections of rabbit ulnar defects at different rhBMP6 doses. [Figure 8A] 1 shows radiographs of rabbit ulnas treated with rhBMP7 in collagen and rhBMP6 in autologous blood clot (ABC), depicted at 6 weeks and 2 weeks. [Figure 8B] Graph showing the X-ray scores of rabbit ulnas treated with rhBMP7 in collagen and rhBMP6 in autologous blood clot (ABC) and rabbit ulnas treated with collagen only, represented at 6 weeks and 2 weeks. [Figure 8C] Graph showing bone volume (BV) of rabbit ulna treated with rhBMP7 in collagen and rabbit ulna treated with rhBMP6 collagen only in autologous blood clot (ABC) is shown, represented at 6 weeks and 2 weeks. [Figure 8D] 1 shows a graph depicting the activation of BMP6 and BMP7 by Noggin. [Figure 9] Figure 1 shows radiographs, micro-CT images, and macroscopic morphology of ABCS with and without ALLO at various doses of rhBMP6 per ml of ABC compared with ABC alone and ABC plus ALLO groups for spinal fusion in posterolateral fusion (PLF) in rabbits. [Figure 10A] Graphs showing quantitative measurements of bone volume, trabecular number and trabecular interconnectivity as measured by micro-CT analysis are shown. [Figure 10B] Graphs showing quantitative measurements of bone volume, trabecular number and trabecular interconnectivity as measured by micro-CT analysis are shown. [Figure 10C] Graphs showing quantitative measurements of bone volume, trabecular number and trabecular interconnectivity as measured by micro-CT analysis are shown. [Figure 10D] Graphs showing quantitative measurements of bone volume, trabecular number and trabecular interconnectivity as measured by micro-CT analysis are shown. [Figure 11A] Figure 1 shows the histology of the ABGS implant, showing new bone formation with typical remodeling and osseointegration at the interface between the newly formed bone and the proper transverse process. [Figure 11B]1 shows the histology of rabbit serum treated with ABC / rhBMP6 implants at day 21 after ABGS implantation compared to day 0. [Figure 12A] 1 shows radiographs of spinal fusions in sheep treated with 62.5 μg / ml rhBMP6 and 187.5 μg / ml rhBMP6 with allograft bone and allograft bone and instrumentation. [Figure 12B] The same sheep specimen is shown after μCT scanning. [Figure 12C] μCT quantitative analysis of bone volume (C), trabecular number (D), trabecular space (E), and connectivity density (F) of spinal fusion. [Figure 12D] μCT quantitative analysis of bone volume (C), trabecular number (D), trabecular space (E), and connectivity density (F) of spinal fusion. [Figure 12E] μCT quantitative analysis of bone volume (C), trabecular number (D), trabecular space (E), and connectivity density (F) of spinal fusion. [Figure 12F] μCT quantitative analysis of bone volume (C), trabecular number (D), trabecular space (E), and connectivity density (F) of spinal fusion. [Figure 13A] A macroscopic ovine spinal fusion specimen is shown, in which the newly formed bone is completely integrated with the lumbar transverse processes (white arrows). [Figure 13B] Figures 13C-13E show rectangular regions on either side of the lumbar vertebrae (white boxes), transverse processes (black boxes), and newly formed bone between the transverse processes (gray boxes) within which morphometric parameters were measured. A more accurate transverse section, fully integrating the transverse processes with the newly formed bone, is shown in the right panel of Figure 13B (white arrows). [Figure 13C] μCT quantitative analysis of bone volume (C), trabecular morphology (D), and bone thickness (E) are shown. [Figure 13D] μCT quantitative analysis of bone volume (C), trabecular morphology (D), and bone thickness (E) are shown. [Figure 13E] μCT quantitative analysis of bone volume (C), trabecular morphology (D), and bone thickness (E) are shown. [Figure 14]1 shows X-ray and μCT scans of an anterior interbody fusion (ALIF) model in sheep 11 weeks after surgery. [Figure 15] Blood clot volume for BMP6 binding is shown. [Figure 16] The release of BMP from BMP-incorporated ceramics, as measured by ELISA assay, and the effect of adding ABC on the release time at 3-day intervals are shown. [Figure 17] FIG. 1 shows a schematic perspective view of a kit for preparing ABGS for inducing new bone formation. [Figure 18] FIG. 1 shows a schematic side view of a kit for preparing ABGS to induce new bone formation. [Figure 19] A freeze-drying container containing the freeze-dried contents (BMP+CRM) is shown. [Figure 20] 1 shows a freeze-dried container containing freeze-dried rhBMP-6 and a mechanically stable clot formed around a compression-resistant substrate. [Figure 21] Tricalcium phosphate (TCP) implanted in ABC with rhBMP6 at a subcutaneous site in rats and analyzed at days 1, 3, 7, and 35. At day 35, new bone was formed between the TCP particles (last row). [Figure 22] High magnification of implants from FIG. 21 at 35 days is shown showing newly formed bone surrounding the TCP particles (creeping displacement) (white arrows) showing signs of TCP displacement with respect to the bone. [Figure 23] Platelet-rich plasma (PRP) gels prepared from 2 ml of rat blood and containing no, 5, or 20 μg of BMP6 were implanted under rat skin. Newly formed bone in the BMP6-enriched PRP samples is marked with a white circle. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention provides an autologous bone graft substitute composition (ABGS) for use in treating bone defects, inducing new bone formation, and promoting new bone growth at desired sites. The ABGS of the present invention may be advantageously used in place of autologous grafts in surgeries to generate or repair bone at specific sites in individuals in need of such treatment. According to one embodiment of the present invention, the ABGS is formed by combining (mixing) a series of components including a sample of autologous blood, an osteogenic bone morphogenetic protein (preferably BMP-6), and a compression-resistant matrix (CRM). The components are incubated for a time sufficient to form a clot gel. According to another embodiment of the present invention, the ABGS may be formulated by first precipitating or lyophilizing the osteogenic bone morphogenetic protein (preferably BMP-6) onto a compression-resistant matrix of a specific geometric shape (e.g., particle, cylinder, or slab). Autologous blood is added and incubated for a time sufficient to form a biomechanically stable clot around the lyophilized combination of osteogenic bone morphogenetic protein and CRM.

[0052] According to a further embodiment of the present invention, an ABGS is formed by combining (mixing) a series of components including a sample of autologous blood, a bone morphogenetic protein (preferably BMP-6) that effects bone formation, and a blood clotting agent.

[0053] In accordance with the present invention, the ABGS described herein can be easily implanted, injected, or otherwise applied to the site where new bone growth is needed or desired.

[0054] Autologous bone graft substitutes (ABGS) essentially mimic "living autografts" in the following way: 1) It uses autologous blood, which does not trigger an inflammatory cytokine storm and foreign body reaction. This is because the compression-resistant substrate surface (either as particles, cylinders, or slabs) masks the foreign body and blocks foreign body recognition by T cells (e.g., high-mineral content ceramics at ectopic sites). 2) This provides circulating osteoprogenitor cells trapped in the blood clot that readily respond to BMPs. 3) It does not induce an immune response with the production of antibodies, unlike conventional animal-derived collagen, which may act as an adjuvant for the immune response to recombinant BMP. 4) This allows BMPs to bind tightly to plasma proteins within the fibrin microstructure, while remaining locally available or being slowly released at the implantation site over several days to activate BMP receptors on recruited mesenchymal stem cells for an osteogenic response. 5) The compression-resistant matrix particles in the implant are biocompatible and also provide good handling characteristics. Furthermore, the mineral content (hydroxyapatite) undergoes creeping replacement as it is replaced by newly formed bone. 6) BMP-6 is a preferred BMP because it does not bind tightly to noggin, a natural BMP antagonist that is abundant in bone (Song et al., J Biol Chem.; 285(16):12169-80(2010)). This allows for lower doses compared to BMP-2 or BMP-7, reducing safety concerns and avoiding excessive doses in the clinic.

[0055] An ABGS according to another embodiment of the present invention may be formulated by combining (mixing) a series of components including a sample of autologous blood, a bone forming bone morphogenetic protein (BMP), a compression resistant matrix (CRM), and a sample of a reverse phase thermosensitive bioabsorbable polymer, and incubating the components for a time sufficient to form a clot gel having a rheology such that the composition can be injected in a liquid state at room temperature and forms a biocompatible gel at the delivery / implantation site as the temperature rises to body temperature (37°C).

[0056] To more clearly understand the present invention, the following terms are defined. The terms "bone morphogenetic protein," "BMP," "osteogenic BMP," and "morphogen" are synonymous and refer to any member of a specific subclass (i.e., the BMP family) of the transforming growth factor-□ (TGF-□) superfamily of proteins (see, e.g., Massague J (1998) TGF-β signal transduction. Annu Rev Biochem 67:753-791; Sampath TK, Rueger DC (1994) Structure, function, and orthopedic application of osteogenic protein-1 (OP-1). Complications in Orthopedics 9:101-107 (1994); U.S. Pat. Nos. 4,968,590, 5,011,691, 5,674,844, and 6,333,312). All such BMPs have a signal peptide, a prodomain, and a carboxy-terminal (mature) domain. The carboxyl-terminal domain is the mature form of the BMP monomer and contains a highly conserved region characterized by seven cysteines, termed the "7-cysteine domain," which is characteristic of BMP family proteins that form a cysteine knot (see Griffith et al., Proc. Natl. Acad. Sci. USA, 93:878-883 (1996)).

[0057] BMPs were originally isolated from mammalian bone using protein purification methods (see, e.g., Sampath, et al., Proc. Natl. Acad. Sci. USA 84:7109-7113 (1987); Wang et al., Proc. Natl. Acad. Sci. USA 85:9484-9488 (1988); Sampath, et al., J. Biol. Chem. 265:13198-13205 (1990); U.S. Patent No. 5,496,552). However, BMP has also been detected or isolated in other mammalian tissues and organs, including kidney, liver, lung, brain, muscle, tooth and intestinal tract.BMP can be produced by using standard in vitro recombinant DNA technology to express in prokaryotic or eukaryotic cell culture (see, for example, Wang et al., Proc. Natl. Acad. Sci. USA, 87:2220-2224 (1990); Wozney et al., Science, 242:1528-1534 (1988)).Similarly, some BMPs are commercially available for local use (for example, BMP-7 is produced and sold by Stryker (Kalamazoo, Michigan, US) for the treatment of long bone nonunion fractures). BMP-2 is also manufactured and sold for acute fractures of long bones by Wyeth (Madison, New Jersey, US) and for spinal fusion with InFUSE® bone graft, which uses a processed bovine type I collagen sponge carrier in combination with an implantable lordotic threaded cage (LT / CAGE® Lumbar Tapered Fusion Device by Medtronic Sofamor Danek USA, Inc. (Memphis, Tennessee, US)).

[0058] BMPs usually exist as dimers of identical monomeric polypeptides (homodimers), held together by hydrophobic interactions and at least one interchain (intermonomer) disulfide bond. The BMPs useful in the compositions and methods described herein have osteogenic activity, i.e., the ability to stimulate bone formation. Osteogenic (or "osteoinductive") activity can be detected using any of a variety of standard assays. Such osteogenic assays include the ectopic bone formation assay, in which a carrier matrix containing collagen and BMP is implanted into an ectopic site of a rodent, and then bone formation is monitored (Sampath TK and Reddi AH Proc.Natl.Acad.Sci.USA, 78:7599-7603(1981)). In a variation of such an assay, the matrix can be implanted into an ectopic site, and BMP can be administered to the site, for example, by intravenous injection into a rodent. Another method for assaying BMP osteogenic activity is to incubate cultured mesenchymal progenitor cells with BMP and then monitor the cells for differentiation into chondrocytes and / or osteoblasts (see, e.g., Asahina et al., Exp. Cell. Res., 222:38-47 (1996)). BMPs that have osteogenic activity and are therefore useful in the compositions and methods described herein include, but are not limited to, BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, BMP-12, BMP-13, and heterodimers thereof, if present, purified from natural sources, recombinantly produced in eukaryotic (e.g., mammalian, yeast, insect, fish) or prokaryotic (bacterial) cells, or produced in whole or in part by in vitro protein synthesis methods. BMPs with osteogenic activity may also possess one or more other beneficial pharmacological activities, such as the ability to rebuild or regenerate damaged soft tissues or organs, e.g., ischemic kidneys (Vukicevic et al., J. Clin. Invest., 102:202-214 (1998)).

[0059] The term "pharmaceutically acceptable" refers to a material that is biologically, chemically, or otherwise compatible with the chemistry and metabolism of the living body and that does not adversely affect the desired, effective activity of an osteogenic BMP or other components in a composition that may be administered to an individual to promote bone growth in accordance with the present invention. One or more elements or steps so designated also describe corresponding, more limited compositions or methods that "consist essentially of" (or "which consists essentially of") the similarly designated elements or steps. This means that the composition or method includes the designated essential elements or steps, and may also include additional elements or steps that do not significantly adversely affect one or more of the basic and novel characteristics of the composition or method. It is also understood that any composition or method described herein, such as "comprising" or "consisting essentially of" one or more elements or steps, also describes the corresponding, more limited, and close-ended composition or method "consisting of" (or "which consists of") the stated elements or steps, to the exclusion of other unstated elements or steps. In any composition or method disclosed herein, known or disclosed equivalents of any stated essential element or step may be substituted for that element or step. Unless otherwise specified, other terms have the same meaning as understood and used by those skilled in the art, including those in the fields of orthopedics, medicine, immunology, biochemistry, molecular biology, and tissue regeneration.

[0060] The BMPs present in the bone graft substitutes described herein promote new bone growth from progenitor cells present in or migrating to the defect site when the bone graft substitute is implanted. Any bone-forming bone morphogenetic protein (BMP), including analogs thereof, heterodimers of two BMPs, and combinations (mixtures) of two or more BMPs, may be used in the compositions and methods described herein. Preferred osteogenic BMPs useful in the bone graft substitutes described herein include, but are not limited to, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-9, BMP-12, BMP-13, heterodimers thereof, and combinations thereof. Even more preferred for use in the bone graft substitutes described herein are osteogenic BMPs selected from BMP-2, BMP-4, BMP-6, BMP-7, analogs thereof, heterodimers thereof, and combinations thereof. Most preferably, the BMP used in the bone graft substitutes described herein is BMP-6.

[0061] The compression-resistant matrix (CRM) present in the bone graft substitutes described herein provides a biocompatible scaffold that not only provides structural support but is gradually replaced by new bone growth stimulated by the osteogenic BMP component of the implanted autologous bone graft substitute. CRMs useful in the autologous bone graft substitutes described herein include any of the compression-resistant matrixes currently used in devices approved for use in spinal fusion. A characteristic of the CRMs in currently approved spinal fusion devices is their ability to withstand the local forces exerted on the implanted bone graft substitute by the local spinal musculature and vertebrae. CRMs useful in the autologous bone graft substitutes described herein include allografts (bone grafts prepared from bone from individuals other than the individual requiring treatment), bioabsorbable polymers or copolymers (e.g., polylactide, polyglycolide, etc.), calcium phosphate-calcium carbonate composites such as hydroxyapatite (HA), tricalcium phosphate (TCP), and combinations thereof. CRMs useful in the bone graft substitutes described herein may also include allograft and one or more calcium phosphate-calcium carbonate composites and / or bioabsorbable polymers or copolymers, or combinations thereof. CRM granules for use in bone graft substitutes according to the present invention may have a granule size of 74 μm to 8 mm, obtained using a sieve for such granule sizes.

[0062] The CRM granule size for use in the autologous bone graft substitute described herein ranges from 74 μm to 8 mm. Preferred CRM shapes or forms include cylinders, slabs, sheets, or meshes of specific dimensions depending on the bone defect. The CRM shape or form may be any one selected from cylinders, slabs, sheets, meshes, or other shapes depending on the bone defect.

[0063] The geometry of the compression-resistant substrate is dictated by the medical application. The CRM particle and pore size in a composition containing ABC with BMP6 should preferably match the size of the bone defect. For example, in dental applications to fill bone defects after tooth extraction, the CRM particle size should be in the range of 72–420 μm to cover approximately 30% of the total ABGS volume. For alveolar ridge augmentation in dentistry, which allows for the insertion of more dental implant ceramics, a CRM particle size in the range of 0.5–4 mm may be preferred. For long bone defects, such as tibial nonunions, defects 1–3 cm in length can be filled with CRMs having particle sizes of 3–8 mm. For long bone defects greater than 3 cm in length, up to 10 cm in length, cylindrical or slab-shaped CRMs can be used in combination with ABC and BMP6. The pore size of the CRM particles must be large enough to allow vascular ingrowth, which will further promote new bone formation at either eutopic (interepiphyseal) or ectopic (away from the skeleton) sites.

[0064] Similarly, for bone defects in long bones, such as tibia nonunions, defects 1–3 cm in length can be filled with CRMs consisting of synthetic calcium phosphate-calcium carbonate (ceramics) or allografts with particle sizes of 3–8 mm. For bone defects in long bones larger than 3 cm in length, up to 10 cm in length, ceramics in the form of cylinders or slabs can be used in combination with ABC and BMP6. Preferably, the cylinders or slabs are made of 20% hydrohydrapite (HA) and 80% tricalcium phosphate (TCP). These are freeze-dried with BMP (preferably BMP6) in a sterile freeze-drying container connected to a blood collection unit that allows a suitable amount of blood to cover the cylinders or slabs. They are then inserted between large bone defects or between the transverse processes of the spine (preferably between the transverse processes of the lumbar vertebrae) to induce ectopic bone formation and support the fusion of two adjacent lumbar vertebrae. A similar principle can be used to fuse the thoracic vertebrae. The pore size of the ceramic particles must be large enough to allow vascular ingrowth, which will further promote new bone formation at orthotopic (interepiphyseal) or ectopic (away from the skeleton) sites. The ceramic cylinder has a central cored stem intended to connect the individual ceramic particles without overlapping along the core element. This provides a more biomechanically stable structure for spinal fusion of two or more vertebrae in patients with partial bone defects and lumbar back pain due to degenerative disc disease.

[0065] The inverse thermosensitive polymer is poloxamer, a nonionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). See generally U.S. Pat. No. 3,740,421. Poloxamers include products such as Synperonics (Croda Inc., Edison, NJ), specifically poloxamer 407, Pluronic (BASF Corporation, Florham Park, NJ), and Kolliphor (BASF Corporation, Tarrytown, NY), LeGoo® vaso-occlusive gel, composed of polyethoxylated castor oil and 20% (weight percent in saline) purified poloxamer 407. Poloxamer 407 / Pluronic F-127 copolymer (ethylene oxide and propylene oxide block copolymer) used in this study was purchased from BASF (Mount Olive, NJ). The polymer was solubilized in phosphate-buffered saline (PBS) to a final polymer concentration of 20-40% w / v. At this concentration, the polymer exhibits thermoreversible properties, remaining liquid at room temperature and gel at body temperature. For example, 20 g of Pluronic F-127 was added to 100 ml of chilled PBS to prepare a 20% gel, which was stirred overnight at 4°C for proper solubilization. The solution was then filtered through a 0.22 μm filter for sterilization. Poloxamers are a family of biocompatible, water-soluble polymers with inverse thermosensitivity (i.e., their viscosity increases with increasing temperature). In particular, the poloxamers used are non-toxic, biocompatible, and water-soluble, and their viscosity decreases with increasing temperature within the range of use. At room temperature, the composition is injectable but viscous. Upon heating to body temperature, it forms a plug or slab of temperature-induced phase-change polymer without effective denaturation (no hardening) of the chemical composition. Below room temperature, the composition's viscosity is suitable for injection with a syringe. For example, ABGS refers to a syringe delivered by a 5-15 cc syringe with a 20G-1.5 needle size. ABGS can be a flexible putty. The composition contains 50-80% liquid by weight.The mean particle size of the CRM ranges from 70-425 μm, or 1-5 mm as measured by particle sieve. The composition components are dissolved / suspended in autologous blood. The ABGS may include a contrast agent.

[0066] Autologous bone graft substitute compositions (autologous blood clot / BMP-6 / CRM or BMP6 / CRM / autologous blood clot or BMP6 / CRM / autologous platelet-rich plasma or allogeneic blood / BMP-6 / CRM / reverse-phase thermosensitive polymer), with or without autologous bone marrow aspirate, or with or without MSCs expanded from autologous bone marrow, autologous fat, autologous periosteum, or allogeneic umbilical cord as described herein, provide a permissive microenvironment and induce robust new bone formation by overcoming the undesirable responses that can occur with the use of grafted bone or highly mineralized synthetic ceramic (calcium phosphate, calcium carbonate, or calcium sulfate) animal-derived collagen or collagen-CRM composite scaffolds approved for use with BMP in humans. Notably, implantation of the ABGS described herein does not provoke the rapid and powerful inflammatory and immune responses ("inflammatory storm") and foreign body reaction (formation of multinucleated giant cells) that can occur at the site of implantation of currently approved bone graft substitutes, especially in ectopic sites. The "inflammatory storm" involves abundant infiltrating, inflammatory, cytokine-producing cells (e.g., monocytes, polymorphonuclear leukocytes, macrophages, myofibroblasts, and fibrocytes), immune responses, or foreign body reactions (formation of multinucleated giant cells). To overcome the inflammatory storm induced by CRM / CRM-collagen composites and the immune and fibrogenic responses caused by animal-derived collagen, current devices use high concentrations of BMPs, which can cause undesirable safety issues, such as ectopic bone formation away from the implantation site. During the "inflammatory storm," the population of such inflammatory / immunological (non-progenitor) cells significantly outnumbers the population of mesenchymal stem cells (osteocyte progenitors), which respond to osteogenic BMP contact to form new bone. The primary trigger of this "inflammatory storm" of non-progenitor cells is thought to be the presence of CRM components currently used as scaffolds in BMP bone graft substitutes.

[0067] According to this view, multinucleated foreign body giant cells at ectopic sites are large enough to obstruct or otherwise inhibit the ability of osteocyte progenitor cells to be recruited by the CRM of many currently approved bone graft substitutes. They are either absent or present in significantly lower numbers to respond to osteogenic BMPs and generate sufficient new bone growth required to treat bone defects. In contrast, the autologous bone graft substitutes (ABGS) described herein, which contain similar or equivalent CRM components found in currently approved bone graft substitutes, do not induce a cellular inflammatory storm when implanted or otherwise applied to a site requiring new bone growth. Instead, the CRM of the ABGS described herein initially provides a mineral-like structure (within a clot gel). The CRM is absorbed and replaced in an orderly manner with the amount and type of new bone growth required to achieve the desired treatment, such as repair of a bone defect or fusion of adjacent bone segments. This unexpected biological finding indicates that the autologous blood components of the ABGS described herein are capable of responding to prevent or inhibit the onset of an "inflammatory storm" by inhibiting the CRM components and the foreign body response by inhibiting the formation of multinucleated giant cells that may occur if a clot is not present.

[0068] The creation of a robust "inflammatory storm" may be due to the fact that currently approved non-autologous blood clot bone graft substitutes, such as those approved for spinal fusion, use relatively large amounts of osteogenic BMPs to promote new bone growth (e.g., 12-40 mg or more per level for spinal indications). The fact that the use of such relatively large amounts of BMP can result in bone formation at sites distal to the local implant site has raised regulatory concerns about potentially harmful side effects. In contrast, the ABGS described herein, which comprises an autologous blood clot gel, advantageously uses significantly lower amounts of BMP to promote new bone growth than levels found in currently approved bone graft substitute devices. Typically, the amount of BMP present in the autologous bone graft substitute compositions described herein is 5-20 times less than the amount of BMP used in currently approved bone graft substitute devices. As a non-limiting example, the ABGS of the present invention may contain 0.002 mg to 1 mg of BMP per ml of autologous blood. Preferably, the BMP is BMP-6, a naturally occurring BMP that is abundant in bone and across most type I and type II BMP receptors. Antagonist It does not bind to Noggin.

[0069] Optionally, the ABGS described herein may further comprise allogeneic demineralized bone matrix (or "demineralized bone matrix"), which is a gel composition derived from allogeneic bone and contains residual factors that enhance new bone growth. Due to this gel state, the ABGS described herein may be administered to a site requiring new bone growth by implantation (placement of a bone graft substitute in or at the site) or injection (e.g., using a syringe). Unless otherwise specified, the terms "implanted" and "implantation" are also understood to encompass application of a bone graft substitute described herein to a defect site by injection.

[0070] Optionally, ABGS can be enriched with mesenchymal stem cells (osteocyte progenitor cells) obtained or expanded from autologous or allogeneic bone marrow or adipose tissue. In some cases, ABGS can be enriched with tissue fragments from local bone, muscle, or fascia. This level of cell / tissue-enhanced ABGS minimizes BMP-responsive cells at a given site (e.g., the distal tibia), which is advantageous in rare genetic disorders (hypophosphatasia, neurofibromatosis type I, and osteogenesis imperfecta). Furthermore, ABGS containing a reverse-phase thermosensitive bioresorbable polymer provides biocompatibility and handling properties that allow injection at room temperature, resulting in a solid gel-like structure at body temperature. The presence of autologous blood provides protection against the inflammatory storm induced by the CRM and biocompatibility added to the ABGS, providing an "autograft" tissue equivalent.

[0071] In some settings, allogeneic or autologous platelet-rich plasma (PRP) may be used to prepare ABGS instead of autologous blood (AB). Due to its various properties, the autologous bone graft substitute composition described herein may be advantageously used in place of "autograft" in one or more of a variety of procedures that utilize autografts harvested from individuals in need of treatment. These procedures include, but are not limited to, spinal fusion, skeletal fracture repair, high tibial osteotomy, dental restoration, periodontal repair, pseudarthrosis, pseudofractures associated with rare skeletal diseases, and maxillofacial augmentation. In particular, the ABGS described herein is intended for use in treating any of a variety of bone defects. Such bone defects include, but are not limited to, diaphyseal fractures, distal radius fractures, tibia nonunion fractures, osteoporotic fractures (such as vertebral compression fractures and atypical diaphyseal fractures), bone cysts (where bone must be generated to fill the void), bone tumors (where new bone must replace bone lost due to cancer or removed by surgery), oral bone defects, periodontal defects, and various maxillofacial abnormalities.The use of the autologous bone graft substitutes described herein advantageously extends the use of such procedures beyond the limit of the amount of autograft that can be safely harvested from individuals who require such treatment.Only pharmaceutically acceptable ingredients are used to prepare the implantable composition of the present invention.

[0072] The term "allograft" is a term of art and refers to bone from a cadaver that has been sterilely prepared for implantation in a patient. Allograft may be obtained commercially from tissue bone banks.

[0073] The terms "disease" and "disorder" are synonymous and refer to any pathological condition, regardless of cause or etiology. A "defect" in bone or other tissue refers to an area of abnormal tissue growth or lack of tissue growth. A "disease" or "disorder" may be characterized by one or more "defects" in one or more tissues. As used herein, the terms "treatment" and "treating" refer to any therapeutic regimen that alleviates one or more symptoms or manifestations of a disease or disorder, inhibits, suppresses, or reverses (causes regression of) a disease or disorder, or prevents the onset of a disease or disorder. The term "treatment" includes preventing (blocking) one or more symptoms or manifestations of a disease, and includes ameliorating or inhibiting the severity of a symptom or manifestation, including pain, that may otherwise characterize a disease in the absence of treatment.

[0074] A "therapeutically effective amount" is an amount of compound (e.g., osteogenic BMP protein) that promotes bone growth at a desired location, and is the amount desired to achieve a desired endpoint, such as, but not limited to, stable spinal fusion of adjacent vertebrae, filling a bone defect with new bone, filling a void at the distal end of a bone defect, or correcting or regenerating oral or maxillofacial trauma or abnormality. Such endpoints can be measured following new bone growth using standard methods, such as x-ray or visual inspection, by the attending surgeon or other highly skilled practitioner.

[0075] Compositions or methods described herein as "comprising" one or more stated elements or steps are open-ended. The stated elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid multiplicity, any composition or method described herein as "comprising" (or "which comprises") is also understood to be intended to be illustrative and not restrictive. Ceramic Emission Testing Explained

[0076] For comparison, clots formed in the presence of rhBMP6 without calcium phosphate-calcium carbonate composites (hereafter referred to as ceramics) were used. It was revealed that ABGS containing rhBMP6 / ABC initially released a large amount of ABGS, which steadily decreased to a very low level after 10 days.

[0077] In contrast, ABGS containing rhBMP6 / ABC / ceramics still showed significant release after 10 days, thus demonstrating the long-lasting effect of the ceramics.

[0078] Tricalcium phosphate (TCP)-hydroxyapatite (HA) particle size has some effect, but all sizes bind to BMP.

[0079] Using pre-loaded BMP ceramics without ABC, no early release was observed until at least 6 days had elapsed, with release increasing by 10 days and possibly longer.

[0080] However, when pre-loaded ceramics were mixed with ABC, the release interval became shorter after a certain period of release. This resulted in a combination of the release curves of pre-loaded ceramics only and ABC without ceramics. The initial release was presumably due to the addition of ABC containing plasmin protease, which is involved in the clotting reaction. This resulted in a partial modification of BMP in a way that eliminated its ability to bind to ceramics.

[0081] A reliable modification of BMP6 that allows release is the removal of the arginine-rich N-terminal loop, which also contains the heparin-binding site. Initial release was not observed without the addition of ABC. Therefore, release upon ABC addition was due to proteins and proteases present in the clot interacting with the BMP, triggering release. However, not all BMP was immediately converted in this way, which corresponds to BMP remaining in the ceramics. Ultimately, this would be released even if residual proteases were allowed to act for a longer period.

[0082] This is claimed as an improvement in that the release is slower and lasts longer.

[0083] Furthermore, the binding to CRM is very strong (release occurs with 100 mM phosphate, but not with saline / medium alone). It is concluded that the ceramics bind BMP very tightly via ionic interactions, with little release occurring during the first few days. This is advantageous for dense localization of new bone formation.

[0084] This tight bond then prevents excessive release and spread of bone formation.

[0085] Figure 16 shows the BMP6 release results obtained from ceramics using particles of various sizes. The ceramics were manufactured by CaP, Biomaterials LLC. Ceramics of various sizes were placed in Eppendorf tubes, 140 mg per tube. Next, 200 μl of a solution containing 50 μg of BMP6 drug substance in 20 mM glycine buffer (pH 6) was added to the dry ceramics, which was immediately absorbed. The interaction was allowed to continue for 15 minutes at room temperature.

[0086] The 200 μl of liquid was rapidly absorbed by the finer ceramic particles, while the larger particles significantly reduced the liquid adsorption capacity.

[0087] The tubes with the wet ceramics were placed in a -80°C freezer for 30 minutes, then transferred to a GEA SL-2 freeze dryer with a shelf temperature of -18°C and freeze-dried for 24 hours. By this time, the material was completely dry and the vacuum remained stable at a residual pressure of 20 microbars.

[0088] The ceramics preloaded with BMP6 were then evaluated in a 10-day BMP release study using a BMP6 Elisa assay by the RnD system. Blood was placed in tubes containing the preloaded lyophilized matrix, which allowed it to clot after gentle mixing with the ceramic particles. Serum was removed from the clots, and these samples were also evaluated for BMP release over a 10-day period.

[0089] To compare the release kinetics, a sample of "ABC+BMP6", ie, 50 μg of BMP and 200 μl of blood, was also coagulated to form a clot, and the serum was also removed from the clot.

[0090] To measure BMP6 release, the described clot samples and each ABC-free BMP6 matrix sample were first rinsed once with 1 ml of tissue culture medium, and 1 ml of fresh medium was added for 1 day. This 1 ml was collected for ELISA assays and replaced with fresh medium after 3 days. The medium was collected and replaced again on day 6 for ELISA assays, and then collected again on day 10.

[0091] The results of the study are shown in Figure 16, panels A–G, which demonstrate the release of rhBMP6 from ABGS during the first 10 days in vitro, as measured by ELISA assay. Panel A shows the release from ABC+BMP6 clots, while panels B, D, and E show the release from ceramics with various particle size ranges: 1–4 mm (B), 0.5–2.5 mm (D), and 0.5–1.5 mm (E). Panels C, F, and G show the corresponding release from ceramics containing clots, while comparing coarse, medium, and fine particles.

[0092] Seven different BMP carriers (140 mg each) containing 50 ng of rhBMP6 are compared for BMP6 release over 10 days. The Y-axis shows micrograms of BMP6 released into 1 ml of serum-free cell culture medium used to soak the carriers, with the medium replaced every few days (X-axis) and assayed by Elisa.

[0093] Panel A: 0.14 ml ABC + BMP6, no ceramics. Panels B, D, and F show ceramics containing BMP6 but without ABC. Panels C, E, and G show ceramics containing BMP6 with ABC added. Panel B: large particle size ceramics (1000-4000 μm); Panel C: same as B but with ABC added; Panel D: medium particle size ceramics (500-2500 μm); Panel E: same as D but with ABC added; Panel F: small particle size ceramics (500-1500 μm); and Panel G: same as F but with ABC added. The first data point (W) in each panel represents the BMP6 obtained from the first wash (1 ml) of the support.

[0094] The conclusion was that ceramics pre-loaded with BMP6 without ABC showed delayed release of BMP, whereas ceramics plus ABC showed a biphasic release that was initially similar to ABC + BMP6 but then sustained much longer than ABC + BMP6. Thus, this combination provided a more uniform and prolonged release.

[0095] Example 1 Preparation of Autologous Bone Graft Substitute Composition (ABGS) Autologous bone graft substitutes (ABGS) are: 1) recombinant human BMP6 (rhBMP6); 2) Autologous blood and 3) Calcium or strontium or magnesium salts (low mM) in aqueous solution or in the form of nanoparticles or microspheres or 1) recombinant human BMP6 (rhBMP6); 2) autologous blood, 3) calcium or strontium or magnesium salts (low mM) in aqueous solution or in the form of nanoparticles or microspheres; and 4) Composed of compression-resistant base material (CRM).

[0096] Preparation method Method #1: Comparative bone graft without compression-resistant matrix (CRM) components The comparative bone grafts are: 1) Recombinant human BMP6 (rhBMP6) 2) Autologous blood 3) Consisting of calcium or strontium or magnesium salts (low mM) in the form of aqueous solutions, nanoparticles, or microspheres.

[0097] Autologous blood was collected from patients either peripherally or locally (local blood). For preclinical studies, autologous blood was collected from the marginal ear vein in rabbits or the jugular vein in sheep into tubes that did not contain any anticoagulants. The tubes were supplemented with specific volumes of autologous blood supplemented with 5–50 mM calcium, strontium, or magnesium salts (e.g., chloride, carbonate, bicarbonate, gluconate) per ml of blood, either in solution or in nanoparticle or microsphere solutions. Lyophilized rhBMP6 was dissolved in a small volume of water for injection (10–500 μL) and then mixed with autologous blood (0.2–10 ml). Immediately after mixing (within 1 min), rhBMP6 solidified at room temperature with defined structural and rheological properties as measured by stiffness, elasticity, and strain.

[0098] Method #2: Comparative bone grafts containing compression-resistant matrix (CRM) components Autologous bone graft substitutes (ABGS) are: 1) Recombinant human BMP6 (rhBMP6) 2) Autologous blood 3) Consisting of calcium or strontium or magnesium salts (low mM) in the form of aqueous solutions, nanoparticles, or microspheres. 4) Composed of compression-resistant base material (CRM).

[0099] Autologous blood was collected from the patient either peripherally or locally (local blood). For preclinical studies, autologous blood was collected from the marginal ear vein in rabbits or the jugular vein in sheep into tubes containing no anticoagulant and supplemented with a specific volume of 0.1 ml to 50 mM CaCl2 solution, depending on the indication. Lyophilized rhBMP6 was dissolved in a small volume of water for injection (10 to 200 μl) and mixed with the autologous blood. Immediately after mixing the rhBMP6 with the autologous blood, particulate CRM was added and the mixture was allowed to stand at room temperature to clot with defined structural and rheological properties as measured by stiffness, elasticity, and strain (ref).

[0100] Method #3: Autogenous bone graft substitute containing compression-resistant matrix (CRM) components CRMs (in the form of microparticles, cylinders, slabs, or meshes) were immersed in or thoroughly wetted with a small amount (10–1000 μL) of rhBMP6 dissolved in water for injection and then freeze-dried under vacuum. The freeze-dried CRM-rhBMP6 composites were supplemented with 0.1 ml of 50 mM CaCl2, and then sufficiently added with autologous blood without anticoagulants and allowed to stand at room temperature to solidify with defined structural and rheological properties as measured by stiffness, elasticity, and strain.

[0101] Method #4 Autogenous bone graft substitute containing compression-resistant matrix (CRM) components 1. A method for preparing ABGS for inducing new bone formation, the method comprising: (1) a. Autologous blood, b. recombinant human bone morphogenetic protein BMP-6 (rhBMP-6); c. combining a compression resistant matrix 12 (CRM) selected from the group consisting of bone graft, hydroxyapatite, tricalcium phosphate, and combinations thereof; (2) mixing rhBMP-6 in an aqueous solution with a compression-resistant matrix (CRM) 12 in a freeze-drying container 10, the amount of rhBMP-6 aqueous solution added to the CRM being optimized to completely wet the CRM; (3) freeze-drying the rhBMP-6 and compression-resistant matrix 12 (CRM); (4) adding autologous blood; (5) incubating the freeze-dried bone-forming bone morphogenetic protein and compression-resistant matrix 12 (CRM) and autologous blood for a time sufficient to form a biomechanically stable clot 13 around the freeze-dried rhBMP-6 and compression-resistant matrix 12 (CRM).

[0102] First, rhBMP6 in aqueous solution is mixed with a compression-resistant matrix (CRM) 12 in a freeze-drying container 10, which can be sealed under vacuum with a rubber stopper 9 and secured by crimping with an aluminum cap or an additional screw cap. The amount of rhBMP6 aqueous solution added to the selected compression-resistant matrix 12 (CRM) is optimized to ensure complete wetting of the CRM. Depending on the CRM's shape (porous vs. non-porous, particulate vs. cylindrical), the amount of rhBMP6 aqueous solution used for wetting only needs to be sufficient for or equal to the volume of the CRM. If the water content exceeds the wetting volume, some dried rhBMP6 may form along the surface of the freeze-drying container 10. Freeze-drying is continued until completely dry. During freeze-drying, the RhBMP6 / CRM freeze-drying container 10 is closed under vacuum (the rubber stopper is fully depressed) and stored refrigerated, preferably at -20°C or 4°C.

[0103] Next, autologous blood is collected from the patient's vein, as in a typical phlebotomy, into a vacutainer. The vacutainer contains lyophilized rhBMP6-CRM (shown in Figure 19). A long line of sterile tubing 5 with a clamp 3 likely facilitates blood processing into a freeze-drying container 10. The freeze-drying container 10 containing the freeze-dried contents (rhBMP6+CRM) 12 is placed into the freeze-drying container holder 2, ensuring safe puncture of the freeze-drying container's rubber stopper 9. This ensures sterile insertion of the needle 1 through the septum. The needle 1 may be protected by another rubber sleeve 4 to further ensure sterility. The freeze-drying container holder 2 is connected to the phlebotomy needle 7 by sterile tubing 5, initially secured with a small plastic hose clamp.

[0104] 17 to 20 show kits for preparing ABGS of implants for inducing new bone formation. For example, the kits for preparing ABGS of implants for inducing new bone formation include butterfly needle sets 6, 7, and 8 for self-blood collection; a freeze-drying container holder (2) containing a needle (1) protected by a rubber sleeve (9); a sterile tube 5 connecting the butterfly needle sets 6, 7, and 8 to the freeze-drying container holder 2; a tube clamp 3 for releasing the vacuum or stopping the blood flow; and a freeze-drying container 10 equipped with a rubber stopper 9 containing freeze-dried rhBMP6 mixed with a compression-resistant matrix 12 (CRM).

[0105] The compression resistant matrix 12 (CRM) is selected from the group consisting of bone graft, hydroxyapatite, tricalcium phosphate, and combinations thereof.

[0106] The freeze-drying container 10 may have any shape suitable for receiving the compression-resistant substrate 12 (CRM), which may have any shape selected from a cylinder, a slab, a sheet, a mesh, or any other shape depending on the bone defect.

[0107] Example 2: CRM evaluation method (subcutaneous implant in rats) The following CRMs are used in the preparation of ABGS: allograft TCP HA TCP / HA complex calcium sulfate Calcium phosphate-calcium carbonate composite material (ceramics) Bioabsorbable polymers Bioabsorbable Hydrogels

[0108] The following outlines of the given CRM used in the manufacture of ABGS: Particle size ranges from 74 μm to 8 mm The CRM may be in the form of particles or may have any one of the shapes selected from a cylinder, slab, sheet, mesh, or other shape depending on the bone defect.

[0109] CRMs with different physical properties were formulated with rhBMP6 using either method #2 or #3, as described in Example 1. The cellular responsiveness and osteoinductive activity of the autologous bone graft substitute composition ABGS were measured by implanting it subcutaneously or injecting it percutaneously into the abdominal fascia or skeletal muscle pouch of rodents. At 1, 3, 7, and 12-35 days after implantation / injection, the implants were harvested and assayed for cellular responsiveness and osteogenic activity by histology as described (Sampath, TK. and Reddi, AH. PNAS 1981).

[0110] ABGS were prepared from 0.25–0.5 ml of rat whole blood mixed with an appropriate amount of BMP (e.g., 2–200 μg of recombinant BMP-6 per ml of blood). Next, CRM (allograft, donor-derived allogeneic bone (ALLO), or tricalcium phosphate (TCP) or TCP and hydroxyapatite (HA) composite) was left to clot in a 1 ml syringe for 60 minutes. After serum removal, approximately 125–300 μl of ABC was implanted. Most of the rhBMP-6 (>95%) bound to the ABC. The osteogenic response of each rhBMP-6 dose was tested in 4–8 implants in 2–4 rats each. A small pocket was created under the abdominal skin in the axillary region to implant the ABGS prepared with autologous blood / rhBMP-6 / CRM. The implants were filled with ABGS (approximately 125-300 μl without serum) and sealed with one suture to the fascia and three sutures to the skin. To analyze ectopic bone formation, animals were scanned using a 1076 micro-CT device (SkyScan, Belgium) 28 days after implantation. Ectopic bone formation was observed in all groups of animals and quantified by micro-CT analysis. Quantification of ectopic bone showed a dose-dependent relationship. Cell response and bone formation were measured by histology at various time intervals after creeping displacement, more clearly at higher magnifications.

[0111] Subcutaneous implantation of autologous blood clot (ABC) alone recruits migrating mesenchymal stem cells (osteocyte progenitor cells) within 1–3 days and forms a tissue capsule that dissolves by 7–9 days. ABC containing rhBMP6 implants induce MSC differentiation into endochondral bone by day 7, while the newly formed bone then progresses to remodeling by days 21–35, filling with ossicles containing functional bone marrow elements. Histological observations of ALLO plus ABC and ABC + ALLO + rhBMP6 implants at days 7 and 35 are shown in Figures 2A and 2B. By day 7, signs of endochondral bone formation are already present in the rhBMP6-containing implants. At day 35, robust bone formation has occurred between the ABC / ALLO / rhBMP6 implants and attached to the ALLO particles. This bone formation was typical of bone remodeling by creeping replacement (allograft resorption replaced by newly formed bone). Micro-CT analysis of ABC / ALLO and ABGS (ABC / ALLO / rhBMP6) implants at days 7 and 35 visualized comprehensive micro-CT images. Specifically, newly formed and remodeled bone was observed with ALLO particles and without ALLO particles, as shown in Figure 2C and Figure 2D. ALLO and ABC implants alone did not induce bone formation but instead formed a capsule-like fibrous tissue around the implant, which subsequently resorbed by days 18–21. Allograft bone formation was observed with graded resolution in ABC / ALLO implants containing rhBMP6. Figure 2E and Figure 2F show allograft volume and bone volume quantified using micro-CT analysis of ABC plus ALLO and ABC + ALLO + rhBMP6 implants at days 7 and 35 after subcutaneous implantation in rats.

[0112] Example 3: Unexpected biological activity of autologous blood A rat subcutaneous implantation assay was used to evaluate the role of autologous blood in overcoming the inflammatory and foreign body responses induced by ABGS containing CRM (allograft and / or synthetic ceramics (tricalcium phosphate or hydroxyapatite, or a combination of these) used in generating ABGS using Methods 2 or 3). The osteogenic activity of rhBMP6 was tested at various doses. ABC / rhBMP6 / ALLO implants were prepared by adding 74-420 μm rat allograft particles at 0.1-0.5 g / ml to autologous blood.

[0113] To identify inflammation and foreign body rejection in implant-containing allografts, implants were harvested from days 1, 3, and 7. Paraffin-embedded sections were stained with H&E and / or toluidine blue staining and acid phosphatase detection by histochemistry. Groups tested included allograft alone, allograft mixed with ABC, or allograft containing 25 μg of rhBMP6 per ml of autologous blood. Implants were removed from animals on days 7 or 14, and foreign body giant cell analysis was performed.

[0114] The results demonstrated that allograft (ALLO) particles, when implanted subcutaneously in rats, induced inflammation and a foreign body response due to their high mineral content at the ectopic site by recruiting mononuclear phagocytes by days 1–3, followed by fusion to form multinucleated foreign body giant cells (FBGCs) by days 7–14 (Figure 3A). When ALLO particles were formulated with ABC, the number of inflammatory lesions and fused multinucleated FBGCs was significantly reduced (Figure 3B and Figure 3D). In ABGS implants containing ABC+ALLO+rhBMP6, few or no FBGCs adhered to the ALLO particles and formed endochondral bone (Figure 3C and Figure 3D). Figure 3D shows the average number of multinucleated FBGCs morphometrically counted from three representative histological sections of ALLO, ALLO+ABC, and ALLO+ABC+rhBMP6 implants. The multinucleated FBGC cells recruited by the ALLO implant were further characterized by immunohistochemistry for acid phosphatase staining, which was reduced by the addition of ABC (Figures 3E and 3F). Similar results were observed when ceramics (tricalcium phosphate or hydroxyapatite or a combination of these) were used as CRMs (see Figure 21).

[0115] Example 4: Pharmacokinetics and cumulative release of rhBMP6 from ABGS in vitro and retention of rhBMP6 in implants. ABGS containing human blood and allografts was formulated as described in either Method #2 or #3. Blood samples from healthy human volunteers were collected from the antecubital vein into anticoagulant-free tubes. At the time of collection, the blood was mixed with allografts (particle size 2-5 mm or 5-8 mm) and rhBMP6 at two concentrations (62.5 or 125 μg per ml of blood). After clotting was complete (60 min), the ABC+rhBMP6 and allografts were rinsed with 1 ml of basal medium. Each implant was placed in a Falcon tube containing 3 ml of Dulbecco's modified Eagle's medium. The tubes were incubated at 37°C for 10 days, with medium changes on days 1, 3, 6, 8, and 10. The amount of BMP6 released from the ABC+ allografts in the medium was measured by rhBMP6-specific ELISA (R&D Systems, DY507).

[0116] The amount of rhBMP6 released from ABGS without allograft and with two different particle sizes of allograft is shown in Figure 4A. ABGS were prepared using human blood from volunteers. The CRM was a human allograft from an in-clinic bone tissue bank reserved for patients. After reaching a stable state, rhBMP6 was readily released over the first 3–6 days. The ABGS containing the allograft showed a small amount of rhBMP6 release, but this was not statistically significant. The cumulative release measured on days 1, 3, 6, 8, and 10 and the total release calculated from the ABGS were approximately 3–9% of the total rhBMP6 dose (Figures 4B–4C). The addition of the allograft appeared to result in slightly greater cumulative release. As tested in vitro, rhBMP6 is primarily bound to plasma proteins in ABGS, both with and without the allograft. The pharmacokinetics of rhBMP6 may change at the implantation site as the protein is taken up by responsive cells and induces endochondral bone differentiation.

[0117] rhBMP6 binding and release characteristics for ABGS containing ceramics or ALLO: The added rhBMP6 bound tightly and specifically to the ceramics on the surface and inside the pores through ionic and hydrophobic interactions. 100 mM phosphate was required to elute the bound protein. The binding of rhBMP6 to the clot is protein-protein, based on hydrophobic interactions between plasma proteins and cell membranes on the red blood cell surface. Furthermore, because BMP6 is known to bind to heparin sulfate, this may involve high-affinity binding to heparin-like glycosoaminoglycans. Therefore, two (orthogonal) structures exist for rhBMP6 held in the ABGS-containing CERAMIC-rhBMP6-ABC.

[0118] The extremely high binding capacity of the clots was demonstrated by SDS-PAGE and immunoblotting using anti-BMP6 (note: high molecular weight background is seen at the top of all lanes due to antibody cross-reactivity). Figure 15 shows the clot volume of BMP6 binding. Lanes 2–13 show increasing amounts of BMP that can be added due to BMP binding in the clot and subsequently quantitatively released by solubilization with SDS sample buffer. Even with 800 μg per ml of blood, no significant amounts of BMP6 were found in the serum supernatant, and there was no sign of clot saturation. This is important because the absence of widespread release of BMP prevents excessive bone formation. Figure 16 shows BMP release from preloaded ceramics at 3-day intervals, as measured by ELISA assay.

[0119] Example 5: ABGS Target Product Profile: Autograft Mimics We demonstrate that autologous bone graft substitutes (ABGS) containing recombinant human BMP6 (rhBMP6) dispersed within autologous blood clots (ABC) using allograft (ALLO) particles can induce new bone formation. BMP6 was selected as the preferred morphogenetic protein because it does not strongly bind to noggin, a natural BMP antagonist abundant in bone. BMP6 also binds to most type I and type II BMP receptors and exhibits high specific alkaline phosphatase activity in osteoblast cell cultures. This allows for the use of lower doses compared to BMP2 or BMP7. Autologous blood clots (ABC) were selected as the carrier because they: 1) reduce inflammation; 2) provide circulating osteoprogenitor cells; 3) promote tight binding of rhBMP6 to plasma proteins within the fibrin microstructure, allowing for its slow release as an intact protein; 4) reduce immune responses and avoid the generation of antibodies against rhBMP6; and 5) ultimately provide a permissive environment for endochondral bone differentiation. The ALLO particles were uniformly loaded throughout the ABC, providing biocompatibility, good handling properties and compression resistance.

[0120] ABGS containing ABC and ALLO are manufactured with defined rheological properties. The addition of allograft particles shortens the time required to achieve coagulation and improves handling characteristics. We first observed that ABC possesses unexpected unique biological properties by overcoming the foreign body response induced by high Ca / P-containing minerals (ALLO) at ectopic sites. ABC significantly reduces the formation of multinucleated foreign body giant cells around allograft particles, enabling mesenchymal stem cell recruitment, which then undergoes endochondral bone differentiation in response to rhBMP6. The binding and release properties of rhBMP6, as well as the dose of rhBMP6 required to induce optimal bone formation, are comparable in ABGS with and without allograft. Newly formed bone in ABGS containing allograft is dense and undergoes typical bone remodeling, as examined by microCT analysis and histology of subcutaneous implants in rats. This mimics the autograft assimilation observed in orthotopic sites. ABGS (ABC / allograft / rhBMP6) induced endochondral bone, and ALLO particles were assimilated into newly formed bone by creeping displacement (Figure 22). ABGS containing synthetic ceramics (ABC / TCP / rhBMP6) is shown in Figure 22. ABGS induces bone in a dose-dependent manner, with an effective rhBMP6 dose of 100 μg of ABC per ml.

[0121] Bovine-derived collagen is used as a carrier to deliver BMPs and is available as InFuse® 31 , or Amplify (registered trademark) 38 Acid-soluble reconstituted type I collagen meshwork derived from bovine Achilles tendon, such as a slab-shaped composite containing synthetic ceramics, is used to deliver rhBMP2. Bovine diaphyseal bone-derived insoluble type I collagen combined with granular and / or additional CM-cellulose (injectable putty) is used as a composite material. 39 and OP1-Putty® 40Sterilization of these bovine collagens by chemical methods or gamma radiation for clinical use also results in undesirable denaturation of the collagen carrier. 40 Here, we present autologous blood clot as a natural carrier for delivering rhBMP6 (ABGS) containing allografts (ALLO) as a compression-resistant matrix to facilitate posterolateral fusion. We also demonstrate that delivery with animal-derived collagen, which acts as an adjuvant, minimizes adverse events such as: 1) the generation of antibodies against BMP; 2) neuroinflammation as a result of the high doses of BMP used and readily released from the implantation site; and 3) nerve compression due to functional impairment of biomechanical bone support.

[0122] Example 6: ABGS (AB vs. autologous platelet-rich plasma) ABGS was formulated using platelet-rich plasma (PRP) protein containing rhBMP6 derived from autologous blood (AB) and compared with ABGS formulated with autologous blood clot (ABC). Additionally, ABGS was formulated with ABC and PRP. ABGS-ABC (formulated with ABC), ABGS-PRP (formulated with PRP), and ABGS-ABC / PRP (formulated with both ABC and PRP) were tested in a rat subcutaneous implantation assay. Figure 23 shows that adding 5–20 μg of BMP-6 to autologous PRP (shown as open circles) induced new bone formation depending on the dose, whereas autologous PRP prepared from rats and implanted under the rat skin failed to form bone after 14 days.

[0123] Example 7: Preclinical studies conducted to predict clinical outcomes Rabbit Diaphyseal Segment Defect Model: The efficacy of ABGS in reducing critical-size bone defects was tested in a rabbit ulnar segmental defect model. ABC was collected from the rabbit marginal ear vein in a volume of 1.5 ml. rhBMP6 was added to the blood at 25 μg, 50 μg, or 100 μg doses along with 50 mM calcium chloride, and the tube was mixed by swirling. ABC + rhBMP6 was prepared in a syringe and allowed to solidify at room temperature for 60–90 minutes. The liquid portion (serum) was removed, leaving a homogeneous, viscous, injectable, flexible ABGS gel ready for use.

[0124] The study protocol was performed on 10-week-old (2.3-2.5 kg) male New Zealand laboratory rabbits (Bratella ruficollis). Animals were randomly divided into four groups (n = 5 each): A) control, defects filled with ABC alone, B) defects filled with ABC + rhBMP6 (25 μg / ml), C) defects filled with ABC + rhBMP6 (50 μg / ml), and D) defects filled with ABC + rhBMP6 (100 μg / ml). In a separate experiment (n = 5 per group), ABC + rhBMP6 (100 μg / ml) was compared with collagen (150 mg) + rhBMP7 (100 μg / ml) at 2 and 8 weeks after implantation.

[0125] A 17 mm (large defect) ulnar segment was removed and ABGS was implanted into the defect site. No internal or external fixation devices were used, and the radius was left intact for mechanical stability. Radiographs of the right forelimb were taken immediately after surgery and during the 23-week bone healing period. No adverse events were observed during the experiment. Healing outcomes were analyzed by radiography, micro-CT quantification, and histology.

[0126] ABC implanted alone did not result in new bone formation and failed to fill the defect (Figure 5). However, ABC containing rhBMP6 (ABGS) reproducibly induced new bone formation and reconstructed the defect as measured by radiography. New bone formation was induced in a dose-dependent manner, as shown at weeks 6, 9, 13, 16, 19, and 23 (Figure 5), and all rabbit ulnae are shown at week 23 (Figures 6A and 6C). Micro-CT analysis demonstrated a dose-dependent increase in bone mass, as measured by bone volume (BV) and marrow volume (MV) compared to the contralateral untreated bone (Figure 6D). Bone quality was further confirmed by histology, as shown in representative samples from each group (Figure 7). Administration of rhBMP6 at a dose of 100 μg per ml of ABC resulted in complete reconstruction, with the cortex fully established and the medullary canal remodeled. Histological evaluation confirmed that critical size defects in rabbit ulnae treated with 100 μg of rhBMP6 per ml of ABC were completely filled (Fig. 7 ).

[0127] In a similar model, we performed a side-by-side comparison of rhBMP7 / bovine bone collagen devices containing ABGS (rhBMP6 / ABC). Collagen alone did not induce bone formation, whereas rhBMP7 containing collagen induced new bone formation (Figure 8A). The commercially available rhBMP7 / bovine bone collagen device contains 3.5 mg of rhBMP7 per gram of collagen. To fill a rabbit ulnar defect, we used 300 mg of rhBMP7, corresponding to a total of 1.06 mg of rhBMP7 in the collagen carrier. As shown in Figure 8A, we compared ulnar defects filled with rhBMP7 / collagen induction with those filled with ABGS (100 μg of rhBMP6 in 1.5 ml of blood). At weeks 2 and 6, ABGS induced the formation of new uniform bone and remodeled the new cortex to the adjacent host bone, whereas rhBMP7 / collagen-induced bone formation was delayed. Micro-CT analysis confirmed that rhBMP6-containing ABGS induced approximately two times more bone volume at 8 weeks after surgery (Figures 8B-8C).

[0128] Results from this preclinical study allowed us to evaluate a first-in-human (FIH) randomized, placebo-controlled, and double-blind Phase I safety study in patients with distal radius fractures (DRF) (1) and a Phase I / II efficacy and safety study in patients with high tibial osteotomy (HTO) (2).

[0129] A study of posterolateral fusion (PLF) in rabbits: The bone-inductive activity of ABGS was evaluated in a posterolateral fusion (PLF) rabbit model. The study protocol was performed in male New Zealand white laboratory rabbits (Australia rufoca) aged 14 weeks and weighing 3-5 kg. Twenty-eight skeletally mature rabbits were implanted at the lumbar L5-L6 vertebrae. L6 Animals were divided into seven test groups (four animals each): ABC alone (which served as a control); ABC with 50 μg (0.05 mg / ml, 125 μg per device) of rhBMP6; ABC with 100 μg (0.1 mg / ml, 250 μg per device) of rhBMP6; ABC with 200 μg (0.2 mg / ml, 500 μg per device) of rhBMP6; ABC with 200 μg (0.2 mg / ml, 500 μg per device) of rhBMP6 and devitalized rabbit bone graft (0.3 g / ml); ABC with 400 μg (0.4 mg / ml, 1000 μg per device) of rhBMP6 and devitalized rabbit bone graft (0.3 g / ml); and ABC without rhBMP6 and devitalized rabbit bone graft (0.3 g / ml) (which served as a control). The volume of ABC for device preparation was 2.5 ml per implantation side.

[0130] Figure 9 shows radiographs, microCT images, and macroscopic photographs of ABGS with and without ALLO at various doses of rhBMP6 per ml of ABC compared with the ABC alone and ABC plus ALLO groups. The newly formed bone between the two transitional processes in the ABGS was dense, uniformly dispersed throughout the implant, and separated by surrounding soft tissue around the implant as examined at 14 weeks. At 50 μg per ml of ABC (125 μg / implant), the group containing rhBMP6 demonstrated new bone formation, but union was achieved in only two of four rabbits. This suggests that the amount of rhBMP6 may not have been sufficient. At 100 μg per ml of ABC (250 μg / implant), complete union was observed in all rabbits. At 200 μg of rhBMP6 per ml of ABC (500 μg / implant), all rabbits demonstrated bilateral union, but the amount was less than that observed in the 100 μg / ml ABC group. Because ALLO can induce inflammation and form multinucleated FBGCs, ABGS implants containing either double the amount of rhBMP6 at 200 μg per ml of ABC or quadruple the amount at 400 μg per ml of ABC (500 or 1000 μg / implant) were formulated. ABGS plus ALLO containing 200 μg per ml of ABC (500 μg / implant) induced complete union with increased bone volume compared with ABGS containing 100 μg per ml of ABC (250 μg / implant). Increasing the amount of rhBMP6 to 400 μg per ml of ABC (1000 μg / implant) did not further increase bone volume. The groups receiving ABC alone or ABC plus ALLO did not fuse at all, despite the fact that ABC contains endogenous circulating osteoprogenitor cells and growth factors within the ALLO particles and that ALLO can release endogenous BMPs upon resorption. The contact areas between the transverse processes and the newly formed bone were indistinguishable, and the bone fused into a single, continuous bone section. Quantitative measurements of bone volume, trabecular number, and trabecular interconnectivity, as measured by microCT analysis, are shown in Figures 10A-10D.ABGS at 100 μg of rhBMP6 per ml of ABC appears to be the optimal dose, and doubling the dose does not necessarily increase bone formation parameters. Compared to ABGS without ALLO at 100 μg per ml of ABC, ABGS plus ALLO implants containing either 200 μg or 400 μg per ml of ABC also result in new bone formation.

[0131] The histology of the ABGS implants showed new bone formation, with typical remodeling and osseointegration at the interface between the newly formed bone and the proper transverse process (Figure 11A). In the ABGS containing ALLO implants, the newly formed bone rapidly remodeled, completely assimilated with the ALLO particles, and eventually replaced by creeping displacement, as shown by ABC / rhBMP6 / ALLO at 200 μg per ml of ABC (500 μg / implant) at low and high magnification (Figure 11A). The histology of ABC / ALLO implants showed a lack of new bone, except for the formation of fibrous tissue. Furthermore, it is noteworthy to note that anti-rhBMP6 antibodies were not detected in the serum of rabbits treated with ABC / rhBMP6 implants at 21 days after ABGS implantation compared with day 0 (Figure 11B). Figures 11C and 11D show the uptake of rhBMP6 at 1 mg and 2 mg doses at different serum dilutions on days 0 and 21.

[0132] A study of posterolateral fusion (PLF) in sheep The study protocol was carried out on 2-3 year old mixed breed ewes (Ovis spp.) with hygiene certificates and weighing 60-70 kg.

[0133] Two PLF experiments were performed in sheep. In the first experiment, eight ewes were treated with ABC alone (n = 2) and ABC containing 62.5 μg of rhBMP6 per ml (0.5 mg total) without instrumentation (n = 6). In the second experiment, 14 sheep were administered ABC containing 187.5 μg of rhBMP6 per ml (1.5 mg total). Animals were assigned to three groups: Group A, ABC alone (n = 2); Group B, ABC / rhBMP6 with instrumentation (n = 6); and Group C, ABC / rhBMP6 with instrumentation and devitalized ovine allograft (2 g / implant) (n = 6). Surgery was performed under general anesthesia and by the same surgical team for all animals. Autologous blood samples (16 mL) for implant preparation were collected from the animals' jugular vein, and two implants (8 mL each) were prepared bilaterally per animal. The animals were then implanted after surgical decortication of the lateral aspect of the transverse process until bleeding was evident. The sheep were randomly assigned to groups and received the appropriate implants placed on both sides of the transverse process and vertebral arch. Clinical and radiographic controls were performed by a veterinarian immediately after surgery, at 8 and 27 weeks after the study ended. No adverse events were observed in any of the experimental groups during the study.

[0134] We evaluated ALLO-free ABGS at doses of 62.5 μg of rhBMP6 per ml of ABC (0.5 mg / implant) or 187.5 μg of rhBMP6 per ml of ABC (1.5 mg / implant). Both groups induced new bone formation, as assessed by X-ray (Figure 12A) and micro-CT (Figure 12B), resulting in complete unions harvested 6 months after implantation. There was no apparent difference in the amount of bone formed between the two doses, suggesting that an effective dose of rhBMP6 in the range of approximately 100 μg per ml of ABC is an effective dose. Furthermore, PLF studies in both rabbits and sheep did not significantly differ in effective dose. Tests of ALLO-containing ABGS with 187.5 μg of rhBMP6 per ml of ABC (1.5 mg / implant), with or without instrumentation, also induced new bone formation and demonstrated significantly greater ABGS than ALLO-free ABGS (Figures 12C-12F). Sheep treated with ABGS and instrumentation had approximately 83% success in bilateral new bone formation (2 of 12 implants did not fully heal), whereas the addition of allogeneic bone graft to ABGS increased the union success rate to approximately 93% (1 of 12 implants did not fully heal) (see Figure 12).

[0135] Photomicrographs of the macroscopic morphology of spinal fusions from sheep PLF studies of the newly formed bone bilaterally and in the intertransverse process space are shown in Figure 13A. A similar microCT analysis from a representative sheep is shown in Figure 13B. Osseointegration between the newly formed bone at the ectopic site and the proper transverse process was indistinguishable, and the fusion was dense and robust. Quantitative measurements of bone volume, trabecular morphology, and bone thickness of the newly fused bone and the adjacent proper transverse process and lumbar vertebrae, as examined by microCT analysis, are shown in Figures 13C-13E. The newly formed bone has increased bone volume, trabecular number, and trabecular interconnectivity compared with the proper transverse process and adjacent lumbar vertebrae. Studies are ongoing to define the dosage and physical properties of ALLO particles and the required amount (mass per volume of ABC) required for successful, long-term lumbar fusion between two transverse processes at individual and multiple levels in sheep. The results of PLF studies in rabbits and sheep allowed us to evaluate randomized, double-blind, controlled stage II posterior interbody fusion (PLIF) versus autograft ( 3 ).

[0136] Anterior interbody fusion study in sheep Using rhBMP6 ABGS The efficacy of this compound was tested in sheep anterior lumbar fusion after implantation of DePuy Cervical CFRP I / F cages. Ten 3-4 year old female sheep (Merinolandschaf breed) weighing 50-60 kg were used in the study and divided into two experimental groups: control: cages filled with ABC without rhBMP6 (n=4) and experimental: cages filled with autologous blood clot containing 250 μg of rhBMP6 per ml (n=5).

[0137] Surgery was performed under general anesthesia by the same surgical team for all animals. Blood samples (2 ml) for implant preparation were collected from the jugular vein. The cages were constructed from carbon fiber-reinforced polymer and measured 15 mm x 12 mm x 5 mm in width, depth, and height. The intervertebral discs (L5-L6) were excised, the endplate cartilage was scraped off, and the cervical cage was implanted and filled with approximately 1 cc of blood clot, with another 1 cc distributed bilaterally around the cage perimeter. The sheep were clinically and radiologically monitored immediately after surgery, and at 7 and 11 weeks.

[0138] The time course of fusion maturation was measured by surgical examination, radiography, and μCT analysis. Anteroposterior and lateral plain radiographs were taken of the spine under controlled conditions. Fusion status was assessed on plain radiographs. Radiographs were independently measured by three blinded orthopedic surgeons. Computed tomography scans were performed to assess fusion in cross section and in three dimensions.

[0139] The experiment was terminated at 11 weeks, and all animals survived except for one sheep in the control group, which died after surgery due to a respiratory infection. No side effects were recorded regarding mobility, partial or complete paralysis, nerve irritation and / or pain, decreased food intake, or weight loss. No ectopic bone formation, edema, or other visible morphological changes were observed. In sheep receiving rhBMP6, newly formed bone was present inside and outside the cage (n=5), and the bone was fused to both vertebral bodies. Some bone formed in the cage of control animals, but fusion was not complete. The bone volume and bone thickness of the newly formed bone were significantly greater in sheep receiving rhBMP6 than in control animals. ABGS The results of the sheep anterior interbody fusion (ALIF) model after 11 weeks are shown in Figure 14.

[0140] Referring to Figure 14, a radiograph of a control sheep shows the space between two vertebrae (black arrow) has not fused to the newly formed bone. This is indicated on the μCT scan by the white arrow pointing to the bone inside the cage and the red arrow pointing to the space outside the cage (side and bottom views). In the radiograph of a sheep treated with ABGS and rhBMP6, newly formed bone is present between two adjacent vertebrae. This is confirmed on the μCT scan by the continuous bone (white arrows in the side and bottom views) and the open space outside the cage (black arrows in the side and bottom views). This sheep experiment using a cervical cage for ALIF (human) demonstrated that ABGS containing rhBMP6 (250 μg / ml) resulted in complete fusion of two adjacent lumbar vertebrae compared to incomplete fusion in control animals.

[0141] (References) Asahina, I., Sampath, TK, Nishimura, I. and Hauschka, PV (1993). Human Osteogenic Protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria. J. Cell. Biol . 123 :921-933. Cahill, KS, Chi, JH, Day, A., Claus, EB (2009) Prevalence, complications, and hospital charges associated with use of bone-morphogenetic proteins in spinal fusion procedures. JAMA 302:58-66 Carragee, E. J., Hurwitz, E. L., Weiner, B. K. (2011) A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J 11:471-491 Executive Summary for P050036 Medtronic’s AMPLIFY TM rhBMP-2 and analogs Matrix Orthopedic and Rehabilitation Devices Advisory Panel (2010) Food and Drug Administration. Gupta S, M. V., Gupta MC (2017) Biology of spine fusion and application of osteobiologics in spine surgery. In: Vukicevic S, Sampath. KT. (ed) Bone Morphogenetic Proteins: Systems Biology Regulators. Springer International Publishing Griffith, D.L., Oppermann, H., Rueger, D.C., Sampath, T.K., Tucker, R.F. and Carlson, W.D. (1994). Crystallization and preliminary crystallographic data of recombinant human Osteogenic Protein-1 (hOP-1). J. Mol. Biol . 244 : 657-8. Kim DH, Rhim R, Li L, Martha J, Swaim BH, Banco RJ, et al. Prospective study of iliac crest bone graft harvest site pain and morbidity. Spine J. 9:886-92, (2009) Massague J (1998) TGF-βsignal transduction. Annu Rev Biochem 67: 753-791 Medtronic Sofamor Danek USA, Inc. INFUSE Bone Graft product information: Oral / Facial. Memphis, TN; 2006. Available online at www.accessdata.fda.gov / cdrh_docs / pdf5 / P050 053c.pdf. Last accessed February 2010 Mobbs, R. J. et al. (2015) Lumbar interbody fusion: techniques, indications and comparison of interbody fusion options including PLIF, TLIF, MI-TLIF, OLIF / ATP, LLIF and ALIF. J Spine Surg 1:2-18 Sampath TK. The Systems Biology of Bone Morphogenetic Proteins. In: Vukicevic S, Sampath, KT, editor. Bone Morphogenetic Proteins: Systems Biology Regulators. Springer International Publishing; 2017. p. 15-38 Sampath TK, Reddi AH. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci U S A. 1981;78(12):7599-603. Sampath, T.K., Muthukumaran, N. and Reddi, A.H. 1987. Isolation of osteogenin, an extracellular matrix-associated, bone-inductive protein, by heparin affinity chromatography. Proc. Natl. Acad. Sci. USA 84 : 7109-7113. Sampath, T.K., Coughlin, J.E., Whetstone, R.M., Banach, D., Corbett, C., Ridge, R.J., Ozkaynak, E., Oppermann, H. and Rueger, D.C. 1990. Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the Transforming Growth Factor - βsuperfamily. J. Biol. Chem . 265 : 13198-13205. Sampath TK, Maliakal JC, Hauschka PV, Jones WK, Sasak H, Tucker RF, et al. (1992) Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem. 267(28):20352-62. Sampath TK, Rueger DC (1994) Structure, function and orthopedic application of osteogenic protein-1 (OP-1) Complications in Orthopedics 9:101-107 Song K, Krause C, Shi S, Patterson M, Suto R, Grgurevic L, et al. (2010) Identification of a key residue mediating bone morphogenetic protein (BMP)-6 resistance to noggin inhibition allows for engineered BMPs with superior agonist activity. J Biol Chem. 285(16):12169-80 Stryker Biotech: OP-1 Implant product information (2009). www.stryker.com / stellent / groups / public / documents / web_prod / 126737.pdf. Accessed February 2010 Stryker Biotech: OP-1 Putty product information (2009) www.stryker.com / stellent / groups / public / documents / web_prod / 127024.pdf. Accessed February 2010 Vukicevic S, Sampath TK, editors. Bone Morphogenetic Proteins: from Laboratory to Clinical Practice. Basel: Birkhauser Verlag, 2002. Vukicevic, S., Basic, V., Rogic, D., Basic, N., Shih, M-S., Shepard, A., Jin, D., Dattatreyamurty, B., Jones, W., Dorai, H., Ryan, S., Griffiths, D., Maliakal, J., Jelic, M., Pastorcic, M., Stavljenic, A. and Sampath, T.K. (1998). Osteogenic Protein-1 reduces severity of injury in ischemic acute renal failure. J. Clin. Invest . 102 : 202-214. Vukicevic S, Sampath, TK, editors, Bone Morphogenetic Proteins: Systems Biology Regulators. Springer International Publishing; 2017. Wang EA, Rosen V, D'Alessandro JS, Bauduy M, Cordes P, Harada T, et al. (1990) Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A. 87(6):2220-4. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. (1988) Novel regulators of bone formation: molecular clones and activities. Science. 242(4885):1528-34.

Claims

1. 1. An autologous bone graft substitute composition for inducing new bone formation, promoting bone growth, and treating bone defects, said composition comprising: (i) autologous blood; (ii) a bone-forming osteogenic protein selected from the group consisting of BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-8, BMP-9, BMP-12, BMP-13, analogs or heterodimers thereof, and combinations thereof, in the range of 0.002 to 1 mg per ml of autologous blood; (iii) a compression-resistant substrate, wherein the compression-resistant substrate is selected from the group consisting of bone autograft, bone graft, hydroxyapatite, tricalcium phosphate, and combinations thereof; the autologous blood contains the bone-forming osteogenic proteins within a fibrin microstructure and forms a clot gel reinforced with the compression-resistant matrix, the clot gel having structural and rheological properties that provide sustained release of the bone-forming osteogenic proteins over a period of 7 to 10 days; An autologous bone graft replacement composition that does not contain a lysine analog or a serine protease inhibitor.

2. The composition of claim 1, wherein the compression-resistant substrate has any one shape selected from a cylinder, a slab, a sheet, a mesh, a particulate, or any other shape depending on the bone defect.

3. 3. The composition of claim 1, wherein the bone forming osteogenic protein is lyophilized onto a compression resistant substrate.

4. 2. The composition of claim 1, wherein the compression-resistant substrate is selected from hydroxyapatite, tricalcium phosphate, and combinations thereof, and the release of the bone-forming osteogenic protein is observed after 10 days.

5. 2. The composition according to claim 1, wherein the bone morphogenetic protein that effects bone formation is BMP-6 present in an amount of 200 μg per ml of autologous blood.

6. 2. The composition according to claim 1, wherein the bone morphogenetic protein that effects bone formation is BMP-6 present in an amount of 100 μg per ml of autologous blood.

7. The composition of claim 1 , wherein the composition further comprises a blood coagulant.

8. 8. The composition of claim 7, wherein the blood coagulant is selected from pharmacologically acceptable calcium, strontium or magnesium salts either in solution form or as nanoparticles or microspheres in autologous blood, and the blood coagulant is present in the range of 5 to 50 mM.

9. The composition according to claim 8, wherein the bone morphogenetic protein BMP-6, which is responsible for bone formation, is present in an amount ranging from 2 to 200 μg per ml of autologous blood.

10. 9. The composition of claim 8, wherein the bone forming bone morphogenetic protein BMP-6 is present in an amount of 100 μg per ml of autologous blood.

11. The composition of any one of claims 1 to 10, wherein the composition is injectable, extrudable or implantable.

12. A method for preparing an autologous bone graft substitute composition according to any one of claims 1 to 11, said method comprising: （１） a) autologous blood; b) a bone-forming osteogenic protein selected from the group consisting of BMP-6, BMP-2, BMP-7, BMP-4, BMP-5, BMP-8, BMP-9, BMP-12, BMP-13, analogs or heterodimers thereof, and combinations thereof, in the range of 0.002 to 1 mg per ml of autologous blood; and c) a compression-resistant substrate, wherein the compression-resistant substrate is selected from the group consisting of bone autograft, bone graft, hydroxyapatite, tricalcium phosphate, and combinations thereof. and mixing the (2) incubating the components of step (1) for a period of time sufficient to form a coagulum gel having structural and rheological properties that result in sustained release of said osteogenic osteogenic protein over a period of 7-10 days; A preparation method comprising:

13. 13. A method for preparing an autologous bone graft substitute composition according to claim 12, said method comprising: a. mixing the osteogenic protein in aqueous solution and the compression-resistant matrix in a sterile freeze-drying container (10), a mixing step in which the amount of aqueous osteogenic protein solution added to the compression-resistant substrate is optimized to completely wet the compression-resistant substrate; b. freeze-drying the bone-forming osteogenic protein and the compression-resistant substrate; c. adding autologous blood; and d. incubating the lyophilized osteogenic protein, the compression-resistant substrate, and the autologous blood for a time sufficient to form a biomechanically stable blood clot (13) around the lyophilized osteogenic protein and the compression-resistant substrate.

14. 14. The method of claim 12 or 13, wherein the release of the bone-forming osteogenic protein is observed after 10 days for the compression-resistant substrate selected from hydroxyapatite, tricalcium phosphate, and combinations thereof.

15. 13. The method of claim 12, wherein the method further comprises adding a coagulant in step (1), wherein the coagulant is selected from pharmacologically acceptable calcium, strontium, or magnesium salts either in solution form or as nanoparticles or microspheres, and the coagulant is present in the range of 5 to 50 mM.

16. The preparation method according to claim 15, wherein the bone morphogenetic protein that induces bone formation is BMP-6 present in an amount ranging from 2 to 200 μg per ml of autologous blood.

17. The method of claim 16, wherein the bone morphogenetic protein BMP-6 is present in an amount of 100 μg per ml of autologous blood.

18. 16. The method of claim 15, wherein the coagulum gel is allowed to set for 60 to 90 minutes.

19. 12. A composition according to any one of claims 1 to 11 for use in treating bone defects where new bone formation is required, promoting bone growth and / or for the induction during surgery of bone production or regeneration at specific sites in individuals in need of such treatment.

20. 20. The composition of claim 19 for use in treating posterolateral fusion, anterior interbody fusion, adult spinal stenosis, trauma (spinal reconstruction), maxillary cranial reconstruction or high tibial osteotomy.

Images