Compositions and methods for treating bone injuries

Abstract

The present disclosure relates to compositions and biomimetic scaffolds, and methods used to treat, ameliorate, and promote healing of large segmental bone defects in a subject, comprising implanting said compositions and biomimetic scaffolds in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 239,161, filed August 31, 2021. The contents of this prior application are incorporated herein by reference in their entirety. [Background technology]

[0002] On average, 7.9 million fractures occur annually in the United States alone, with approximately 5-10% of fractures resulting in delayed union, nonunion, minor defects, or large bone defects, presenting significant treatment challenges to surgeons (Zura, et al., JAMA Surg. 2016 Nov 16; 151). In the general population, fracture severity, anatomical location, patient comorbidities, smoking, and use of certain medications can significantly contribute to fracture healing problems (Zura, et al., JAMA Surg. 2016 Nov 16; 151). The cost of treating a tibial, femoral, or humeral nonunion ranges from $31,500 to $34,400 per case, representing a significant burden on annual healthcare costs (Kanakaris NK, Giannoudis PV. Injury. 2007;38 Suppl 2:S77 S84; and Wu, et al Orthopedic Research and Reviews. 2013:5 21-33).

[0003] In addition, over 59,000 military personnel were injured in combat-related operations during service in Iraq and Afghanistan, of which 50% were musculoskeletal injuries, including major segmental bone defects (Belmont Jr. PJ, McCriskin BJ, Sieg RN, Burks R, Schoenfeld AJ. Combat wounds in Iraq and Afghanistan from 2005 to 2009. J Trauma Acute Care Surg. 2012;73:3-12). It is estimated that 78% of musculoskeletal injuries sustained in these conflicts were severe limb injuries (Stansbury LG, Lalliss SJ, Branstetter JG, Bagg MR, Holcomb JB. Amputations in US military personnel in the current conflicts in Afghanistan and Iraq. J Orthop Trauma. 2008;22:43-46). Such bone injuries, especially those associated with high-impact blast injuries due to localized tissue vascularity reduction and destruction of contaminated bone and soft tissue, heal poorly. Even without such complications, bone defects exceeding a critical size have no inherent healing potential. Management of patients with large segmental bone defects is one of the most challenging clinical problems facing both military and civilian surgeons (Pollak AN, Ficke JR, Extremity War Injuries III Session Moderators. Extremity war injuries: challenges in definitive reconstruction. J Am Acad Orthop Surg. 2008;16:628-34). Because these devastating injuries often inevitably result in costly, prolonged, ongoing treatment with significant risks of failure and ultimately amputation, the importance of improving clinicians' ability to properly manage these injuries cannot be overemphasized.Over 2,000 soldiers have required at least one amputation as a result of military activities in Iraq and Afghanistan (OIF / OEF) alone (Fischer HA Guide to USMilitary Casualty Statistics: Operation Freedom's Sentinel, Operation Inherent Resolve, Operation New Dawn, Operation Iraqi Freedom, and Operation Enduring Freedom. Congr Res Serv. 2015;7). Furthermore, persistent non-healing bone defects are associated with delayed return to work or military service, with a corresponding decrease in the individual's quality of life. Thus, there is a clear unmet clinical need, providing a strong motivation for the development of more effective treatment strategies to address these potentially fatal injuries. Summary of the Invention

[0004] The compositions disclosed herein are extracorporeal hematomas comprising: (a) isolated whole blood; (b) sodium citrate; and (c) an extracorporeal hematoma comprising ecarin, oscutin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride; and a bone substitute.

[0005] The compositions disclosed herein are an extracorporeal hematoma comprising (a) platelet-rich plasma, plasma, or plasma having red blood cells, and (b) an extracorporeal hematoma comprising ecarin, oscutin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, and a bone substitute.

[0006] The multi-compartment devices disclosed herein include a first chamber containing isolated whole blood and / or one or more growth factors, one or more bone substitutes, or a combination thereof, and a second chamber containing ecarin or calcium chloride, thrombin, or thrombin and calcium chloride.

[0007] The present specification discloses a biomimetic scaffold comprising a scaffold and an extracorporeal hematoma, the extracorporeal hematoma comprising (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, oscutin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride.

[0008] The present specification discloses a biomimetic scaffold comprising a scaffold and an extracorporeal hematoma, the extracorporeal hematoma comprising (a) platelet rich plasma, plasma, or plasma having red blood cells, and (b) ecarin, oscutin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride.

[0009] The present specification discloses a method of constructing an implant, the method including: a) dimensioning the depot implant to at least one of a shape and a size that facilitates implantation of the depot implant into a bone defect; and b) structuring the depot implant to have a scaffold by introducing (i) isolated whole blood and sodium citrate, or platelet-rich plasma, plasma, or plasma with red blood cells, (ii) ecarin, oscutin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, and (iii) a bone substitute to create the scaffold.

[0010] Disclosed herein is a method of constructing a biomimetic scaffold, the method comprising: a) determining dimensions of the scaffold to at least one of a shape and a size that facilitates implantation of the scaffold into a bone defect; and b) combining the scaffold in a) with (i) isolated whole blood and sodium citrate, or platelet-rich plasma, plasma, or plasma having red blood cells, and (ii) an extracorporeal hematoma containing ecarin, oscutin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride to create the biomimetic scaffold.

[0011] Other features and advantages of the compositions and methods of the invention are set forth in the following description, drawings, and claims. [Brief description of the drawings]

[0012] [Figure 1] Healing results of a 5 mm femoral defect in rats 8 weeks after surgery. ACS-absorbable collagen sponge, BMP2-bone morphogenetic protein 2, MicroCT-microcomputed tomography, WB-whole blood. [Diagram 2] The microstructure of demineralized bone matrix (DBX mixed with extracorporeal hematoma (BH) at ratios of 1:3 and 1:6) is shown. [Diagram 3] Shown is an extracorporeal hematoma delivered within a 3D printed titanium custom spinal cage. [Figure 4] 1 shows a 3D patient-specific titanium femoral implant (cage / scaffold) that can be used as disclosed herein. [Diagram 5] An example of a two-chamber syringe that can be used to deliver components of an extracorporeal hematoma is shown: one chamber can deliver a clotting agent, and the other chamber can deliver whole blood alone or in combination with growth factors and / or a bone substitute. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present disclosure may be understood more readily by reference to the following detailed description of the invention, the figures and examples contained herein.

[0014] Before the present compositions and methods are disclosed and described, it is to be understood that they are not limited to a particular synthesis method unless otherwise specified, or to a particular reagent unless otherwise specified, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein.

[0015] Additionally, unless expressly stated otherwise, it should be understood that in no way is it intended that any method described herein be construed as requiring that its steps be performed in a particular order. Thus, where a method claim does not actually recite the order in which its steps are to be followed, or where the claim or description does not specifically state that the steps are to be limited to a particular order, no order is intended to be inferred in any respect. This holds true over any possible implicit basis for interpretation, including logical questions regarding the placement of steps or operational flow, obvious meanings derived from grammatical construction or punctuation, and the number or type of aspects described herein.

[0016] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation.

[0017] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] As used herein, the term "or" means any one member of a particular list, and also includes any combination of members of that list.

[0019] Throughout the description and claims of this specification, the word "comprise" and variations of that word, such as "comprising" and "comprises," mean "including but not limited to" and are not intended to exclude, for example, other additives, components, integers, or steps. In particular, methods described as including one or more steps or operations are specifically intended to include what is recited in each step (unless that step includes a limiting term such as "consisting of") and each step is not intended to exclude, for example, other additives, components, integers, or steps not recited in that step.

[0020] Ranges may be expressed herein as from "about" or "approximately" one particular value and / or to "about" or "approximately" another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it is understood that by using the antecedent "about" or "approximately," the particular value forms a further aspect. It is further understood that each endpoint of the range is significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, when a value of "10" is disclosed, "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, when 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0021] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0022] As used herein, the term "subject" refers to the target of administration, e.g., a human. Thus, the subject of the disclosed method may be a vertebrate, e.g., a mammal, a fish, a bird, a reptile, or an amphibian. The term "subject" also includes domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). In one aspect, the subject is a mammal. In another aspect, the subject is a human. The term does not imply a particular age or sex. Thus, it is intended to include adult, juvenile, adolescent, and newborn subjects, regardless of gender, as well as fetuses.

[0023] As used herein, the term "patient" refers to a subject suffering from a disease, disorder, or condition. The term "patient" includes human and animal subjects. In some aspects of the disclosed methods, the "patient" has been diagnosed as in need of treatment to heal a bone injury, e.g., prior to the administration step.

[0024] As used herein, the term "treatment" refers to partially or completely alleviating, improving, relieving, delaying the onset of, inhibiting or slowing the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment can be administered to subjects who do not show signs of a disease, disorder, and / or condition and / or to subjects who show only early signs of a disease, disorder, and / or condition, with the aim of reducing the risk of developing pathologies associated with the disease, disorder, and / or condition. For example, the disease, disorder, and / or condition can be a bone injury or fracture.

[0025] Current methods of treating large segmental bone defects. The treatment of skeletal abnormalities has employed many techniques, including autogenous bone grafting, various bone graft substitutes, the Ilizarov technique, macroprosthetic arthroplasty, biological products, and, as a last resort, amputation. However, these existing treatments are characterized by the need for prolonged treatment with multiple surgeries, high costs, high complication rates, and significant risks of treatment failure. For example, autogenous bone grafting remains the treatment of choice when healing large segmental bone defects (Khan SN, Cammisa FP, Sandhu HS, Diwan AD, Girardi FP, Lane JM. J Am Acad Orthop Surg. 2005;13:77-86). Autogenous bone grafting remains the treatment of choice when treating bone defects, however, the supply of sufficient autogenous graft material may be limited, especially in cases of severe trauma and fracture nonunion in civilians and severely injured soldiers, and is associated with significant donor site morbidity. In contrast, bone grafts are available in large quantities, but their use raises concerns about disease transmission and immune reactions (Khan SN, Cammisa FP, Sandhu HS, Diwan AD, Girardi FP, Lane JM. J Am Acad Orthop Surg. 2005;13:77-86), but more importantly, they are composed of dead bone that does not resorb or remodel well. They are also known to accumulate microfractures under load, resulting in a 5-year failure rate of over 30% (Enneking WF, Campanacci DA. J Bone Joint Surg Am. 2001;83-A:971-986; and Wheeler DL, Haynie JL, Berrey H, Scarborough M, Enneking W. Biomed Sci Instrum. 2001;37:251-256). Additionally, various bone substitutes are also developed to aid in the treatment of large segmental bone defects, but these approaches are limited to only marginal improvements in outcomes.Inadequate bone formation, poor mechanical and handling properties, lack of biocompatibility, unpredictable resorption, and associated inflammatory responses remain major limitations for such materials (McKee MD. J Am Acad Orthop Surg. 2006;14:S163-7). For this reason, distraction osteogenesis remains the treatment of choice by military surgeons for bone defects larger than 8 cm. However, the technique can be cumbersome, painful, unreliable, complicated by pin-track infections, and slow healing (Pollak AN, Ficke JR, J Am Acad Orthop Surg. 2008;16:628-34). Above all, the main problem with available treatment options for large segmental bone defects is the inability to rapidly restore function and reduce refractive error.

[0026] Research into the biology of bone formation has led to the discovery of some of the most potent osteoinductive factors, the bone morphogenetic proteins (BMPs) (Urist MR. J Bone Miner Res. 1997;12:343-6). Two members of the BMP family, recombinant human BMP-2 (rhBMP-2, INFUSE®) and recombinant human BMP-7 (rhBMP-7, OP-1®), are approved for clinical use. Although BMPs have shown preclinical efficacy in animal models, clinical efficacy has been disappointing. The modest clinical response to these proteins is related to delivery issues. Furthermore, current therapies using very high physiological doses of BMPs, most of which are rapidly leached from the application site, potentially increasing the incidence and severity of heterotopic / ectopic ossification and many other associated side effects such as antibody formation, implant loss, bone resorption, and even cancer (Carragee EJ, Hurwitz EL, Weiner BK. Spine J. 2011;11:471-491). Therefore, there is a need to develop suitable carriers to significantly minimize the required dose, improve the efficacy of BMPs, and limit the potential side effects of BMPs and the associated costs of treatment.

[0027] One of the most promising alternatives to autologous bone grafting is the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) delivered on absorbable collagen sponges (ACS). However, successful bone healing requires a constantly high physiological dose of rhBMP-2 due to its burst release and short half-life in the body, most of which is rapidly leached out as soon as the collagen sponge is compressed after insertion into the bone defect area. This leads to an increased incidence and severity of heterotopic / ectopic ossification, as well as many other associated side effects such as antibody formation, implant removal, bone resorption, and even cancer. A more suitable carrier is clearly needed to treat these complex bone injuries and improve the efficacy of BMPs by significantly reducing the dose required, thus mitigating their potentially dangerous side effects.

[0028] In recent years, platelet-rich plasma (PRP), a type of plasma enriched with a high concentration of platelets, has been tested for a wide range of applications, including the treatment of musculoskeletal trauma. PRP is thought to secrete a variety of growth factors and cytokines at supraphysiological concentrations and generate a matrix that supports cells in the form of a fibrin clot. Specifically, the effect of PRP in promoting bone healing (Iqbal J, Pepkowitz SH, Klapper E. Curr Osteoporos Rep. 2011;9:258-263; and Kurikchy MQ, Al-Rawi NH, Ayoub RS, Mohammed SS. Clin Oral Investig. 2013;17:897-904) has been suggested to be due to increased concentrations of factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), bone morphogenetic proteins (BMPs), and insulin-like growth factor 1 (IGF-1) (Soffer E, Ouhayoun JP, Anagnostou F. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 2003.pp.521-528).Nevertheless, experimental (Simman R, Hoffmann A, Bohinc RJ, Peterson WC, Russ AJ. Ann Plast Surg. 2008;61:337-44) and clinical study results (Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998;85:638-46) using PRP in oral and maxillofacial bone graft procedures have been controversial, and to date there is little evidence to suggest that PRP actually improves or accelerates bone healing; in fact, in the majority of cases, bone formation is reduced (Choi BH, Im CJ, Huh JY, Suh JJ, Lee SH. Int J Oral Maxillofac Surg. 2004;33:56-9; and Marden LJ, Fan RSP, Pierce GF, Reddi AH, Hollinger JO. J Clin Invest. 1993;92:2897-2905). This apparent lack of efficacy extends to the long bones and spine. A recent study in sheep following femoral shaft osteotomy followed by distraction osteogenesis failed to show that application of PRP improved new bone formation (Hernandez-Fernandez A, Velez R, Soldado F, Saenz-Rios JC, Barber I, Aguirre-Canyadell M. Injury. 2013;44:901-7).Similarly, rodent studies have shown that PRP has no beneficial effect on bone healing (Pryor ME, Yang J, Polimeni G, Koo K, Hartman MJ, Gross H, et al. J Periodontol. 2005;76:1287-1292; and Ranly DM, Lohmann CH, Andreacchio D, Boyan BD, Schwartz ZJ Bone Joint Surg Am. 2007;89:139-147) or at best has poor regenerative potential (Sanchez AR, Sheridan PJ, Eckert SE, Weaver AL. J Periodontol. 2005;76:1637-1644). Studies of promoting bone healing after spinal fusion also showed no benefit in animals (Li H, Zou X, Xue Q, Egund N, Lind M, Bunger C. Eur Spine J. 2004;13:354-8) or humans (Weiner BK, Walker M. Spine (Phila Pa 1976). 2003;28:1968-1970). The factors responsible for the different results in these studies are unclear, but it has been suggested that proteases present in platelets may degrade growth factors (Thibault L, Beausejour A, De Grandmont MJ, Lemieux R, Leblanc JF. Transfusion. 2006;46:1292-1299), thereby altering the composition of PRP and reducing its experimental and clinical efficacy. Furthermore, activation of PRP requires thrombin, which results in a burst release of growth factors and a decrease in total growth factor concentration. Bovine thrombin is thought to interfere with human clotting proteins by stimulating antibodies against thrombin, which affects the healing process (Oryan A, Alidadi S, Moshiri Expert Opin Biol Ther. 2016;16:213-32). This may also be related to the inability of PRP to form a clot with the appropriate structural properties.

[0029] Based on the poor performance of PRP, a "second generation" platelet concentrate was developed by French maxillofacial surgeon Joseph Choukroun and is called platelet-rich fibrin (PRF) (Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:299-303). It is described as a more natural fibrin matrix that contains blood components favorable for healing and immunity, and has special advantages compared to PRP. For example, the clotting process occurs by a slower spontaneous polymerization compared to the rapid polymerization that occurs when thrombin is added to PRP. Most importantly, PRF does not require anticoagulants or thrombin additives (Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJJ, Mouhyi J, et al. Oral Surgery, Oral Med Oral Pathol Oral Radiol Endodontology. 2006;101). In vitro studies also show that PRF releases growth factors such as PDGF, TGF-β, and BMP in a more sustained manner, lasting up to 28 days (He L, Lin Y, Hu X, Zhang Y, Wu H. Oral Surgery, Oral Med Oral Pathol Oral Radiol Endodontology. 2009;108:707-713). This release profile is significantly different from PRP, which is characterized by a burst release of cytokines and growth factors within 1 day.Nevertheless, no significant improvement was observed when PRF was applied to oral and maxillofacial surgery (Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: 299-303; and Busenlechner D, Huber CD, Vasak C, Dobsak A, Gruber R, Watzek G. Clin Oral Implants Res. 2009; 20: 1078-1083). However, this may be related to an insufficient amount of delivered platelets, as well as inappropriate structural properties, similar to PRP.

[0030] The effect of hematoma formation on fracture healing. The formation of a fracture hematoma occurs within minutes of bone injury and involves a cascade of biological events involving many molecular factors (such as proinflammatory cytokines and growth factors, including osteogenic and angiogenic factors) derived from the periosteum, bone marrow, and surrounding soft tissues, in addition to activated clotting factors from the blood system (Lai BFL, Zou Y, Brooks DE, Kizhakkedathu JN. Biomaterials. Elsevier Ltd; 2010; 31: 5749-5758). At the fracture site, blood vessels constrict to prevent continued blood loss, followed by the coagulation cascade, resulting in the formation of a hematoma or clot between the broken fragments (Schindeler A, McDonald MM, Bokko P, Little DG. Semin Cell Dev Biol. 2008; 19: 459-66). There are two major pathways for the clotting process, intrinsic and extrinsic, with the common pathway being the conversion of clotting factor X to Xa. Thrombin, the last protease in the coagulation cascade, is a typical serine protease of the chymotrypsin family and has both procoagulant and anticoagulant functions (Huntington JA.Thrombin plasticity.Biochim Biophys Acta-Proteins Proteomics.Elsevier BV;2012;1824:246-252). During normal blood clotting, the coagulation cascade is activated by converting the zymogen prothrombin into the serine protease thrombin. Thrombin then converts soluble fibrinogen into insoluble fibrin fibers. Finally, these fibrin fibers contribute to the formation of a mature webbed fibrin clot with the help of clotting factor XIII (Chernysh IN,Nagaswami C,Purohit PK,Weisel JW.Sci Rep. 2012;2: 879). Thus, concentration gradients of fibrinogen, thrombin, and coagulation factor XIII play an important role in regulating the three-dimensional structure of the fibrin clot (Wolberg AS, Campbell RA. Transfus Apher Sci. 2008;38:15-23).The structural parameters of fibrin clots can be characterized by fiber diameter, density, number of branch points, distance between branch points, and pore size (Weisel JW, Litvinov RI. Blood. 2013; 121: 1712-1719). The increase in the diameter of fibrin fibers is inversely proportional to the density of fibrin fibers and directly proportional to the pore size (Eichhorn SJ, Sampson WW. JR Soc Interface. 2005; 2: 309-318; and Kaur S, Sundarrajan S, Rana D, Matsuura T, Ramakrishna SJ Memb Sci. 2012; 392-393: 101-111). Moreover, the diameter and density of the fibers affect the porosity and surface area of ​​the fibrin clot (Pham QP, Sharma U, Antonios G. Mikos. Biomacromolecules. 2006;7:2796-2805) and are involved in stem cell biological functions such as adhesion, proliferation and differentiation (Badami AS, Kreke MR, Thompson MS, Riffle JS, Goldstein AS. Biomaterials. 2006;27:596-606). For example, low thrombin concentrations (<1 nM) generate a porous network of thick fibrin fibers that are highly sensitive to fibrinolysis, whereas high concentrations of thrombin generate thin fibers that form a less permeable fibrin network that is relatively resistant to fibrinolysis (Gabriel DA, Muga K, Boothroyd EM. J Biol Chem. 1992;267:24259-63). Furthermore, while individual thick fibers have higher mechanical strength (stiffness), fibrin clots composed of thick fibers often have lower mechanical strength as a result of a reduced number of fibers (Carlisle CR, Coulais C, Guthold M. Acta Biomater. 2010;6:2997-3003; and Liu W, Thomopoulos S, Xia Y. Adv Healthc Mater. 2012;1:10-25).

[0031] The combination of the scaffold and the disclosed extracorporeal hematoma can be used to successfully and efficiently promote bone regeneration in large segmental defects that do not heal naturally on their own. In some embodiments, the combination can be used with approximately 33 times less rhBMP-2 than the standard dose currently used (11 μg). As used herein, using an in vivo 5 mm femoral defect rat model, rhBMP2 delivered via extracorporeal hematoma (e.g., 0.33 μg) was much more efficient than when delivered via absorbable collagen sponge. Large bone defects were consistently healed with significantly lower doses of rhBMP-2, and the quality of the healed bone was also superior in the extracorporeal hematoma group compared to rhBMP-2 delivered without a segment on absorbable collagen sponge based on callus size and bone morphometric parameters at the end of 8 weeks. The present specification discloses a biomimetic scaffold including a scaffold and an extracorporeal hematoma. The present disclosure discloses biomimetic scaffolds, including scaffolds and extracorporeal hematomas, that can be designed and fabricated to mimic normal fracture hematomas, but more efficiently deliver very low doses of rhBMP-2 with specific concentrations of calcium and thrombin or ecarin. Other bone morphogenetic proteins can also be included in the extracorporeal hematomas described herein. The design and use of biomimetic scaffolds, including scaffolds and extracorporeal hematomas, can be made to mimic hematomas formed upon fracture, at least because the hematoma formed upon injury serves as a scaffold that activates a cascade of biological events. To initiate fracture healing, this cascade involves activation of many molecular factors, including proinflammatory cytokines and growth factors, osteogenic and angiogenic factors, as well as clotting factors from the blood system, from the periosteum, bone marrow, and surrounding soft tissues. Hematomas formed in the biomimetic scaffolds at the fracture site can have a significant impact on how the fracture heals, but removing this hematoma will delay fracture healing.

[0032] Disclosed herein are methods to improve the structural and biological properties of induced clots to promote healing of large segmental bone defects. For example, described herein are biomimetic scaffolds, including ex vivo hematoma scaffolds, generated by constructing fibrin clots that mimic the structural properties of natural fracture hematomas to significantly improve and promote healing of large segmental bone defects in soldiers and civilians.

[0033] Prothrombotic snake venoms. Many snake venom toxins contain proteolytic enzymes that affect hemostasis through the action of clotting thrombin-like enzymes and prothrombin-activating toxins. The conversion of factor X to (activated) Xa is a common connection point between the extrinsic and intrinsic coagulation pathways. Snake venom enzymes have evolved to exploit different steps of the coagulation cascade. These venoms have evolved to exploit the weak interactions that exist between platelets, endothelial cells, plasma proteins and venom proteins that affect the steps of hemostasis in vertebrates (Meier J, Stocker K. Crit Rev Toxicol. 1991;21:171-182). Depending on the snake species, each has specific procoagulant factors. For example, the procoagulant factor oscutarin in the venom of the coastal taipan (Oxyuranus scutellatus) is structurally and functionally similar to mammalian coagulation factor X. Oscutarin is a serine protease belonging to group C prothrombin activator venoms, which, unlike mammalian factor X, does not require the nonenzymatic factor V because it contains a unique factor Va-like molecule (St. Pierre L, Masci PP, Filippovich I, Sorokina N, Marsh N, Miller DJ, et al. Comparative analysis of prothrombin activators from the venom of Australian elapids. Mol Biol Evol. 2005;22:1853-1864). Similarly, ecarin from the venom of the sawtoothed spoonbill (Echis carinatus) is a metalloproteinase that acts without the participation of cofactors such as calcium chloride (CaCl2) or phospholipids. Lacking a cofactor requirement, ecarin can convert both carboxylated and decarboxylated prothrombin to meizothrombin (Hutton R. Blood Rev. 1993;7:176-189).Meizothrombin is an intermediate product of thrombin generation during whole blood clotting and is reported to have limited enzymatic activity in the conversion of fibrinogen to fibrin (Bovill EG, Tracy RP, Hayes TE, Jenny RJ, Bhushan FH, Mann KG. Arterioscler Thromb Vasc Biol. 1995;15:754-758; and Krishnaswamy S, Mann KG, Nesheim ME. J Biol Chem. 1986;261:8977-8984). On the other hand, the serine protease RVV-V isolated from the venom of the Russell's viper (Dabioa russelli) specifically activates factor V in a calcium-independent manner. Another well-characterized protease, RVV-X, also isolated from the venom of the Russell's viper, is a potent and specific activator of factor X. In contrast to RVV-V, RVV-X requires Ca as a cofactor. 2+It is a metalloproteinase that requires factor X but not phospholipids (Takeya H, Nishida S, Miyata T, Kawada SI, Saisaka Y, Morita T, et al. J Biol Chem. 1992; 267: 14109-14117; and Tokunaga F, Nagasawaq K, Miyataq T, Iwanagaqll SJ Biol Chem. 1988; 263: 17471-17481). In comparison, in normal fracture sites, factor X binds to factor V on the platelet membrane, accelerating the generation of thrombin thousands of times, a mechanism that leads to the formation of a mature clot that stabilizes the primary hemostatic plug (Probst A, Spiegel HU. J Investig Surg. 1997; 10: 77-86). The foregoing suggests that procoagulant factors isolated from snake venom should be suitable as more natural coagulants to alter the structural properties of clots. Indeed, snake venom proteins are important in elucidating the complex physiological mechanisms that govern the coagulation cascade and determine platelet function. Furthermore, due to their potency, selectivity and high biological availability, they have helped to elucidate the structural and functional relationships of human coagulation factors and platelet glycoproteins (Hong TT, Huang J, Lucchesi BR. Am J Physiol Heart Circ Physiol. 2006; 290: H959-67; Han SM, Weaver FA, Comerota AJ, Perler BA, Joining MJ Vasc Surg. 2010; 51: 600-9; Sanchez EF, Bush LR, Swenson S, Markland FS. Thromb Res. 1997; 87: 289-302; Swenson S, Bush LR, Markland FS. Arch Biochem Biophys. 2000; 384: 227-37; and Shah AR, Scher L. IDrugs. 2007; 10: 329-35).As a result, several active venom compounds have been identified, isolated, characterized, purified, and are now used for both diagnostic and medical purposes (King GF. Expert Opin Biol Ther. 2011; 11: 1469-84; and Butler MS. Nat Prod Rep. 2008; 25: 475-516). As described herein, a composition containing snake venom clotting enzyme (SVCE) as an alternative clotting agent, as opposed to the addition of thrombin, which has been shown to have side effects when used clinically (Oryan A, Alidadi S, Moshiri A. Expert Opin Biol Ther. 2016; 16: 213-32). Certain SVCEs have been shown to have higher catalytic activity than their mammalian counterparts and are known to be more thermostable and resistant to proteolysis due to the presence of additional disulfide bridges (Kang TS, Georgieva D, Genov N, Murakami MT, Sinha M, Kumar RP, et al. FEBS J. 2011;278:4544-76). Disclosed herein are biomimetic scaffolds and methods that demonstrate the ability of snake venom enzymes to alter hematoma properties and heal large segmental defects using a rat model.

[0034] Clotting factors derived from snake venom have been preferred for several reasons, including that they do not affect other clotting factors, and that their small molecular size means that they are less likely to be recognized and therefore attacked by the body's immune system when used. Furthermore, the molecules selectively bind to targets in the body, minimizing the possibility of unwanted side effects. Ecarin was specifically chosen because it does not require cofactors for activation.

[0035] The present disclosure is important for developing new and improved treatment strategies to promote bone injury healing, improve quality of life, reduce high treatment costs, and reduce amputation rates due to severe trauma and battlefield injuries in civilians and soldiers. The results disclosed herein can provide the requirements for an extracorporeally generated hematoma with properties that enhance the effectiveness of bone healing by mimicking the unique structural and biological properties of naturally healing fracture hematomas. The present disclosure is also important for the development of extracorporeal hematomas as osteoinductive and osteoconductive growth factor reservoirs that can be combined with scaffolds to form biomimetic scaffolds to promote healing of large segmental bone defects, subcritical size defects, and are useful for the treatment of non-healing fractures (delayed union or nonunion). Compositions are disclosed that combine an extracorporeal hematoma with an osteoconductive scaffold that can be designed with suitable mechanical properties for bone repair applications, for example. These scaffolds can be used to regenerate bone over significant distances and control the resulting bone density, structure, and remodeling rate of the scaffold. In some embodiments, the scaffold can be a 3D printed scaffold that can be used to replace current bone grafting techniques and bone graft substitutes.

[0036] Rather than requiring highly specialized equipment in civilian or military clinical settings, rapid product translation and on-demand application are anticipated, simply mixing the required amount of whole blood with a specific concentration of snake venom clotting enzyme in the operating room. The results described herein may also benefit biomaterial researchers by incorporating the compatible structural properties of hematomas into the design of biomaterial scaffolds to improve the ability of poorly performing scaffolds currently used in regenerative medicine to aid in bone repair. Furthermore, procoagulant factors isolated from snake venom could be used as suitable clotting agents to alter the structural properties of blood clots, as well as to stop uncontrollable bleeding in hospitals and on the battlefield. Also disclosed herein are products that can stop bleeding within seconds, are easily portable, have a long shelf life, are resorbable or easily removable, and are inexpensive. These snakes evolved to kill their prey by instantly causing massive coagulopathy, and thus developed highly specific biological agents that turn blood into gelatin. However, if properly isolated and carefully prepared under controlled conditions, the same clotting factors, such as ecarin, could be used instead to save lives. This amazing property allows for immediate control of blood loss, thereby limiting further blood loss in civilian patients and soldiers injured on the battlefield.

[0037] Disclosed herein is a biocompatible snake venom-induced extracorporeal hematoma for healing large bone defects. Also disclosed herein is a method for treating and manipulating the extracorporeal hematoma (thrombus) in specific ways that modify its ultrastructural properties and thereby change its behavior in various clinical situations. Disclosed herein is a method for treating whole blood or blood products with agents that change its structural conformation and alter its biological activity so that it can be used to treat several different medical conditions.

[0038] Disclosed herein are compositions, biomimetic scaffolds, and methods that can be used to improve the regeneration and repair of large bone defects, subcritical size defects, and to improve the treatment of non-healing fractures (delayed union or non-union). Disclosed herein are organized clots (i.e., extracorporeal hematomas that can be combined with scaffolds to form biomimetic scaffolds) that can be used to promote bone healing by acting as a temporary reservoir for the continuous release of important growth factors, and simultaneously aid in cell infiltration, proliferation, and differentiation by providing adequate space. Disclosed herein are biomimetic scaffolds, compositions, and methods to remodel the repair process, including the generation of clots (excorporeal hematomas) that mimic naturally healing fracture hematomas. This concept can be altered using ecarin, a clotting factor derived from snake venom, where the structural properties of the extracorporeal product (excorporeal hematoma) mimic native fracture hematomas that promote and facilitate bone healing when implanted into bone defects in combination with bone substitutes and / or scaffolds. Ecarin, a clotting factor derived from snake venom, can also be used for hemostasis. In some aspects, the composition may be formulated as a powder, liquid, or spray.

[0039] The present specification discloses a composition comprising whole blood, ecarin and BMP-2. In some embodiments, ecarin can be present at a specific concentration. In some embodiments, a low or significantly reduced dose of BMP-2 can be used in the compositions disclosed herein. In some embodiments, the disclosed compositions can be used to promote healing of bone defects. In some embodiments, the disclosed compositions can be formulated as a liquid or gel.

[0040] The type of healing described herein was superior to that provided by a commercially available product sold by Medtronic, which uses rhBMP-2 delivered on an absorbable collagen sponge (Infuse™). The compositions or products described herein require significantly lower doses of BMP-2 (e.g., rhBMP-2) when combined with a scaffold to initiate bone healing. In some embodiments, the dose of BMP-2 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times lower than the commercially available product or composition.

[0041] As disclosed herein, BMP-2 belongs to the family of bone morphogenetic proteins (BMPs). These proteins are bone growth factors that have the ability to induce chondroblastic bone neoplasia. In some embodiments, BMP-2 can be recombinant BMP-2. Methods for producing recombinant BMP-2 are known in the art and can be found, for example, in U.S. Patent No. 7,354,901, which is incorporated herein by reference.

[0042] Also disclosed herein are biomimetic scaffolds that can be prepared on demand by injecting / immersing a 3D printed cage (e.g., implant) into a solution of components that may be used to prepare an extracorporeal hematoma (e.g., specific concentrations of blood products and clotting agents (calcium and thrombin or ecarin, with or without growth factors), allowing to solidify for 15-20 minutes, and then implanting at the desired treatment / injury site. The biomimetic scaffolds, including the scaffolds and extracorporeal hematomas described herein, are currently the only known compositions that can effectively deliver such extremely low doses of rhBMP-2 with such efficiency that they can consistently and reliably initiate the fracture healing cascade to successfully repair critical-sized bone defects. Furthermore, procurement and preparation of the extracorporeal hematoma can be performed reproducibly, intraoperatively, on-demand, very quickly, and at a fraction of the cost.

[0043] composition The compositions disclosed herein include 1) an extracorporeal hematoma comprising (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, and 2) a bone substitute. The compositions disclosed herein also include 1) an extracorporeal hematoma comprising (a) platelet-rich plasma, plasma, or plasma with red blood cells, and (b) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, and 2) a bone substitute. In some embodiments, the extracorporeal hematoma may comprise (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the extracorporeal hematoma may include (a) platelet-rich plasma, plasma, or plasma with red blood cells, and (b) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the extracorporeal hematoma may further include sodium citrate. In some embodiments, the phrase "plasma with red blood cells" refers to plasma that does not contain platelets. In some embodiments, the extracorporeal hematoma includes fibrin fibers having a thickness of at least 150-300 nm ± 10%. In some embodiments, ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride may form one or more fibrin fibers having a thickness of at least 150-300 nm ± 10%. As used herein, the term "extracorporeal" refers to a hematoma that may be formed outside of an organism, e.g., in an external environment. In some embodiments, the extracorporeal hematoma comprises (a) isolated whole blood and sodium citrate-rich plasma, plasma alone, plasma with red blood cells (without platelets), or other blood products, and (b) one or more clotting factors. In some embodiments, the extracorporeal hematoma can comprise whole blood and one or more clotting factors.

[0044] As used herein, the terms "whole blood" and "blood" are used to mean blood that can be taken directly from the body without any of the components being removed, including plasma or platelets. In some aspects, the whole blood or blood can be from a subject or patient who receives any of the compositions described herein or any of the extracorporeal hematomas described herein. In some aspects, the whole blood or blood can be from a donor subject or patient. Whole blood is composed of red blood cells, white blood cells, platelets, and plasma. In some aspects, fibrin gel can be used instead of whole blood.

[0045] Those skilled in the art will understand that blood is a specialized body fluid that delivers vital substances, such as nutrients and oxygen, to cells and transports metabolic waste products from those same cells. In vertebrates, blood is composed of blood cells suspended in plasma. Blood contains various components, such as plasma, red blood cells (irithrocytes), platelets (platelets), and white blood cells (leucocytes). Plasma is the main component of blood, making up about 55% of the blood, and is composed mostly of water, which contains ions, proteins, nutrients, and waste products. Plasma may contain a portion of every protein produced by the body. For example, plasma is composed of water (about 90%) and ions (Na + , K + , Mg +2 , Ca +2 , Cl - , HCO3 - , HPO4 -2 , SO4 -2 ) (Nezafati et al., 2012), proteins (e.g., mainly albumin 55%, globulins, growth factors, enzymes, hormones, antibodies), clotting factors (Factors I-XIII) (labtestsonline.org.au), sugars (glucose), lipids (cholesterol), minerals (sodium, calcium, magnesium, potassium, iron, zinc, copper, selenium) (Harrington et al., 2014), waste products, and a mixture of dissolved gases. Red blood cells (irithrocytes) are responsible for transporting oxygen and carbon dioxide. They are about 7-8 μm in size, contain neither mitochondria nor nuclei even at maturity, and have an average life span of 120 days. Women have about 3.6-5.0 million cells / mm 3men have approximately 4.2 to 5.4 million red blood cells per mm 3 There are red blood cells. Platelets are involved in blood clotting. The normal platelet count is about 150,000 to 450,000 / mm 3 White blood cells (WBCs, leukocytes) are part of the immune system and function in the immune response. About 1% of cells are present in blood. They are larger in size than red blood cells and contain normal nuclei and mitochondria. A normal white blood cell count is about 5,000-10,000 / mm 3 White blood cells are divided into five main types, which can be further divided into two distinct groups: granulocytes (60-70% of WBCs or 3,000-7,000 / mm3) and neutrophils (60-70% of WBCs or 3,000-7,000 / mm3). 3 ), eosinophils (1–3% of WBC or 50–400 / mm 3 ), and basophils (0.3–0.5% of WBCs or 25–200 / mm 3 )), agranulocytes (lymphocytes (20-30% of WBC or 1,000-4,000 / mm 3 ), and monocytes (3–8% of WBCs or 100–600 / mm 3 ).

[0046] As used herein, the term "platelet-rich plasma" (also known as autologous conditioned plasma) refers to a concentrated form of platelet-rich plasma protein derived from whole blood. For example, whole blood can be centrifuged to remove red blood cells. In some aspects, the terms "blood plasma alone," "plasma alone," or "plasma" can refer to the yellowish liquid component derived from whole blood that typically holds the blood cells in whole blood in suspension. For example, plasma can be separated from whole blood by centrifuging the blood until the blood cells fall to the bottom of the tube, and the plasma can then be removed from the top of the tube. In some aspects, the term "plasma with red blood cells" can refer to "plasma alone" to which red blood cells have been added. For example, red blood cells can be obtained by centrifuging whole blood until it falls to the bottom of the tube, and are collected after removing the plasma, white blood cells, and platelets from the top of the tube.

[0047] In some embodiments, the composition may include one or more bone substitutes. In some embodiments, the one or more bone substitutes may be derived from a biological product, may be a synthetic bone substitute, or a combination thereof. Examples of bone substitutes derived from biological products include, but are not limited to, bone marrow aspirate concentrate (BMAC) containing demineralized bone matrix (DBM), bone morphogenetic protein (BMP), hydroxyapatite (HA), and coral, allogeneic cancellous bone chips, or bone grafts from long bones harvested using a reamer irrigator aspirator (RIA). In some embodiments, the one or more bone substitutes may be derived from a biological product, and the biological product may be bone marrow aspirate concentrate (BMAC) containing bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), demineralized bone matrix (DBM), hydroxyapatite (HA), coral, allogeneic cancellous bone chips, or bone grafts from long bones harvested using a reamer irrigator aspirator (RIA). In some embodiments, the bone substitute may be a synthetic bone substitute. Examples of synthetic bone substitutes include, but are not limited to, calcium sulfate, calcium phosphate cement, beta-tricalcium phosphate (TCP) ceramics, biphasic calcium phosphate (hydroxyapatite (HA) and beta-TCP ceramics), bioactive glass, and polymer-based bone substitutes. Further examples of synthetic bone substitutes include, but are not limited to, Calcigen® S calcium sulfate bone void filler, STIMULAN® beads, HydroSet injectable bone substitute (calcium phosphate), Ossilix calcium phosphate cement, Syntoss synthetic beta-tricalcium phosphate bone graft material, CERASORB® tricalcium phosphate bone graft, GL1894P / -20 58S bioactive glass, UniGraft bioactive glass 200-600um, BonAlive (BonAlive Biomaterials Ltd, Finland), Cerament (bone void filler), and Cerament G (Bonesupport Holding AB, Lund Sweden).Examples of polymers include, but are not limited to, collagen, gelatin, chitosan, and synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polycaprolactone (PCL)-GalaFlex P4HB biopolymer. In some embodiments, the bone substitute is available in a variety of forms, including, but not limited to, dry, moldable, or injectable forms, and pastes, powders, putties, granules, gels, sponges, or strips. In some embodiments, the bone substitute can be a commercially available product. In some embodiments, the bone substitute can be demineralized bone matrix (DBX, MTF Biologics, Edison, NJ), RegenaVate DBM, Puros DBM, StaGraft DBM, or FiberStack DBM (Zimmer Biomet, Warsaw, IN). In some embodiments, the DBM can be allograft cancellous bone or cortical bone that has been demineralized to generate a collagen and non-collagenous protein product. Examples of DBM include, but are not limited to, Grafton DBM (Osteotech, Inc, Eatontown, New Jersey), Allosource (Denver, Colorado), Dynagraft II (Integra LifeSciences, Plainsboro, New Jersey), DBX (Musculoskeletal Transplant Foundation and Synthes, Paoli, Pennsylvania), and Osteofil (Medtronic Sofamor Danek, Minneapolis, Minnesota). Examples of corals include, but are not limited to, Animalia, Coelenterata, Scleractinia, Corallinae, Porites species, and Gonioporus species, each of which may be used in the development of a coralline hydroxyapatite (CHA) bone substitute. In some embodiments, the bone substitute is not a BMP, rhBMP-2, or BMP-2.

[0048] In some embodiments, the ratio of extracorporeal hematoma to bone substitute can be 1000:1 to 1:1000, or any ratio therebetween. In some embodiments, the ratio of extracorporeal hematoma to bone substitute can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the ratio of extracorporeal hematoma to bone substitute can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0049] In some embodiments, the composition, the extracorporeal hematoma, the bone substitute, or the biomimetic scaffold may include one or more growth factors. In some embodiments, the one or more growth factors may be one or more bone morphogenetic proteins. Examples of BMPs include, but are not limited to, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, and BMP-14 (also known as GDF5). Any BMP, including BMP-1 through BMP-18, is contemplated. In some embodiments, the one or more growth factors may be platelet-derived growth factors. In some embodiments, the one or more growth factors may be vascular endothelial growth factors. In some embodiments, the one or more growth factors may be fibroblast growth factor 2. In some embodiments, the one or more growth factors may be one or more of bone morphogenetic proteins, platelet-derived growth factors, vascular endothelial growth factors, fibroblast growth factor 2, or combinations thereof. In some embodiments, the composition, the extracorporeal hematoma, the bone substitute, or the biomimetic scaffold may further include BMP-2. In some embodiments, the one or more growth factors can be BMP-2. In some embodiments, the one or more growth factors is not a BMP, rhBMP-2, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, or BMP-14.

[0050] In some embodiments, the whole blood may include viable cells. In some embodiments, about 50%-70% of the viable cells of the whole blood remain viable after formation of the extracorporeal hematoma. In some embodiments, at least 50% of the viable cells of the whole blood remain viable after formation of the hematoma. In some embodiments, at least 60% of the viable cells of the whole blood remain viable after formation of the hematoma. In some embodiments, at least 70% of the viable cells of the whole blood remain viable after formation of the extracorporeal hematoma. In some embodiments, at least 80% of the viable cells of the whole blood remain viable after formation of the extracorporeal hematoma. In some embodiments, at least 90% of the viable cells of the whole blood remain viable after formation of the extracorporeal hematoma. In some embodiments, more than 90% of the viable cells of the whole blood remain viable after formation of the extracorporeal hematoma.

[0051] In some embodiments, whole blood may contain one or more biological factors. In some embodiments, the term "biological factor" or "other biological factor" refers to the plasma components of whole blood except water. Examples of other biological factors include, but are not limited to, ions, proteins, clotting factors, sugars, lipids, minerals, etc.

[0052] In some embodiments, the one or more biological factors present in whole blood may be endogenous biological factors. Platelets are present in whole blood. Many growth factors are present in platelets. Growth factors within platelet α-granules of platelet-rich plasma have been shown to contain mitogenic and chemotactic growth factors along with inactive forms of related healing molecules important for wound healing, including, but not limited to, platelet-derived growth factor (PDGF), transforming growth factors β1, β2, β3 (TGF-β1, TGF-β2, TGF-β3, platelet-derived angiogenic factor (PDAF), insulin-like growth factor 1 (IGF-1), platelet factor 4 (PF-4), epidermal growth factor (EGF), epidermal growth factor (ECGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and other cytokines. In addition, plasma fluid contains numerous biologically important growth factors, such as the growth factors IGF-I and hepatocyte growth factor (HGF). They also contain active proteins. During normal wound healing, trapped platelets are activated and degranulated, resulting in the release of alpha granule contents. Examples of growth factors present in platelets include, but are not limited to, platelet-derived growth factor, transforming growth factor beta 1, beta 2, beta 3, platelet-derived angiogenic factor, insulin-like growth factor 1, platelet factor 4, epidermal growth factor, epidermal growth factor, vascular endothelial growth factor, basic fibroblast growth factor, and other cytokines, as well as platelet-derived endothelial growth factor (PDEGF), interleukin 1, osteocalcin, and osteonectin. Growth factors present in plasma fluids include, but are not limited to, insulin-like growth factor 1 and hepatocyte growth factor.

[0053] In some aspects, the extracorporeal hematoma may include whole blood, ecarin, and sodium citrate. In some aspects, the extracorporeal hematoma may include whole blood, calcium chloride, and sodium citrate. In some aspects, the extracorporeal hematoma may include platelet-rich plasma and ecarin. In some aspects, the extracorporeal hematoma may include platelet-rich plasma and calcium chloride. In some aspects, the extracorporeal hematoma may include whole blood, calcium chloride, or oscutaline and calcium chloride, and sodium citrate. In some aspects, (a) a combination of one of isolated whole blood and sodium citrate, platelet-rich plasma, or plasma with red blood cells can be combined with (b) one of ecarin, oscutaline and calcium chloride, or calcium chloride. In some aspects, (a) a combination of one of isolated whole blood and sodium citrate, platelet-rich plasma, or plasma with red blood cells can be combined with (b) one of thrombin, or thrombin and calcium chloride. In some embodiments, any of the extracorporeal hematoma combinations described herein may further include one or more antibiotics.

[0054] In some embodiments, the concentration of calcium chloride present in the extracorporeal hematoma can range from 1 mM to 20 mM. In some embodiments, the concentration of calcium chloride can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mM, or any number therebetween. In some embodiments, the concentration of calcium chloride can be about 10 mM.

[0055] In some embodiments, the concentration of thrombin may range from 0.1 to 1 U / mL. In some embodiments, the concentration of thrombin present in the extracorporeal hematoma may be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 U / mL, or any number therebetween, or more. In some embodiments, the concentration of thrombin present in the extracorporeal hematoma may be 0.5 U / mL.

[0056] In some embodiments, the concentration of ecarin present in the extracorporeal hematoma may be at least 0.05 U / mL. In some embodiments, the concentration of ecarin present in the extracorporeal hematoma may be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 U / mL, or any number therebetween, or more. In some embodiments, the concentration of ecarin present in the extracorporeal hematoma may be 0.3 U / mL. In some embodiments, the concentration of ecarin present in the extracorporeal hematoma may be 0.6 U / mL. In some embodiments, the concentration of ecarin present in the extracorporeal hematoma may be 0.75 U / mL. In some embodiments, the concentration of ecarin present in the extracorporeal hematoma may be any value (rational or non-rational) between 0 and 2.

[0057] In some embodiments, the extracorporeal hematoma, bone substitute, biomimetic scaffold or composition described herein may further comprise BMP-2. In some embodiments, the BMP-2 may be recombinant BMP-2. In some embodiments, the recombinant BMP-2 may comprise human BMP-2. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold may be at least 0.01 mg. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold may be 0.01 to 5 mg. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold can be 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mg, or any number therebetween. In some embodiments, recombinant BMP-2 can be used at a dose of about 0.01 mg to about 12 mg. In some embodiments, recombinant BMP-2 may be used at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 mg, or any dose therebetween. In some embodiments, recombinant BMP-2 may be used at a dose of more than 12.0 mg. In some embodiments, the dose of BMP-2 may be about 1 mg to 5 mg. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold may be at least 0.01 μg. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold may be 0.01 to 5 μg. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold may be 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 μg, or any number therebetween. In some embodiments, recombinant BMP-2 may be used at a dose of about 0.01 μg to about 12 μg.In some embodiments, recombinant BMP-2 may be used at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 μg, or any dose therebetween. In some embodiments, recombinant BMP-2 may be used at a dose of more than 12.0 μg. In some embodiments, the dose of BMP-2 may be about 1 μg to 5 μg. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold may be 0.3 to 0.4 μg. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold can be lower than the standard dose. In some embodiments, the dose of BMP-2 present in the extracorporeal hematoma, bone substitute, composition, or biomimetic scaffold can be 10-50 times lower than the standard dose or the lowest effective dose of BMP-2 / ACS.

[0058] In some embodiments, the amount of ecarin present in the extracorporeal hematoma may be at least 0.05 U / mL and the amount of BMP-2 present in the extracorporeal hematoma may be at least 0.01 mg.

[0059] In some embodiments, the amount of ecarin present in the extracorporeal hematoma may be at least 0.05 U / mL and the amount of BMP-2 present in the extracorporeal hematoma may be at least 0.01 μg.

[0060] In some embodiments, the concentration of sodium citrate can be about 3.2-4 mg / ml. In some embodiments, the solution is about 3.2-4% (weight / volume) sodium citrate, and one part of the solution can be mixed with nine parts whole blood.

[0061] In some embodiments, the extracorporeal hematoma or compositions described herein may further comprise one or more therapeutic agents. In some embodiments, the therapeutic agent may be a growth factor. In some embodiments, the therapeutic agent may be BMP-2. In some embodiments, the therapeutic agent may be recombinant BMP-2. In some embodiments, the therapeutic agent may be a stem cell or pre-differentiated stem cell, including, but not limited to, mesenchymal stem cells, adipose stem cells, and induced pluripotent stem cells. In some embodiments, the therapeutic agent may be ecarin.

[0062] In some aspects, the extracorporeal hematoma, biomimetic scaffold or composition disclosed herein may be formulated as a liquid or gel. In some aspects, the extracorporeal hematoma, biomimetic scaffold or composition disclosed herein may be formulated as a paste or putty. In some aspects, the extracorporeal hematoma may be formulated in a lyophilized or powder form. The lyophilized or powder form may make the extracorporeal hematoma more stable for storage. In some aspects, the extracorporeal hematoma, biomimetic scaffold or composition disclosed herein may be formulated as a liquid, gel, powder, granule, paste or putty. In some aspects, the composition may be formulated in a lyophilized or powder form. The lyophilized or powder form may make the extracorporeal hematoma, biomimetic scaffold or composition disclosed herein more stable for storage. In some aspects, the growth factors (such as BMPs), clotting factors (such as ecarin, calcium chloride, etc.), and sodium citrate are available in lyophilized or powder form. In some embodiments, the compounds used to create the extracorporeal hematoma disclosed herein can be dissolved in sterile distilled water before mixing with whole blood or other blood products (e.g., PRP, plasma, etc.). The diluent is sterile distilled water. No additional components are required for preparation or storage. Whole blood (or other blood products) can be drawn from the patient prior to (e.g., immediately before) surgery and citrated to prevent clotting. In some embodiments, donor blood can be used for patients with blood diseases, including, but not limited to, anemia, hemophilia, leukemia, HIV, etc. The remaining components of the extracorporeal hematoma do not require additional stabilizers for storage. For example, BMP-2 is commercially available in a bottle and ready to use, and CaCl2 is available in powder form, and in some cases already dissolved in sterile distilled water (it is very stable after dissolution). Both BMP-2 and CaCl2 can be stored at room temperature. Ecarin is available in a lyophilized form (freeze-dried) stored at -20°C and can be dissolved in sterile distilled water before use. The extracorporeal hematoma can be prepared relatively simply by using the components described herein in volumes based on the volume of the defect to be filled, so that after the components are prepared, they can be mixed together in the tube / mold.Typically, the extracorporeal hematoma forms in about 30-45 minutes and can then be inserted (or implanted) into the bone defect. In some embodiments, the extracorporeal hematomas described herein can be stored using a "smart storage system" that uses a radio frequency identification-based system (e.g., Smartstorage™) that includes a near real-time tissue tracking system, which can streamline inventory management, such as keeping accurate usage history and temperature logs.

[0063] scaffold Disclosed herein is a biomimetic scaffold. The biomimetic scaffold may include any of the scaffolds described herein and any of the extracorporeal hematomas described herein.

[0064] The present disclosure discloses a biomimetic scaffold comprising a scaffold and an extracorporeal hematoma, the extracorporeal hematoma comprising (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. The present disclosure discloses a biomimetic scaffold comprising a scaffold and an extracorporeal hematoma, the extracorporeal hematoma comprising (a) platelet-rich plasma, plasma, or plasma with red blood cells, and (b) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the extracorporeal hematoma comprises fibrin fibers having a thickness of at least 150-300 nm±10%. In some embodiments, the biomimetic scaffold further comprises one or more bone substitutes. In some embodiments, the extracorporeal hematoma of the biomimetic scaffold comprises one or more bone substitutes.

[0065] In some embodiments, the extracorporeal hematoma described herein can be further combined with a carrier such as a scaffold. For example, the carrier can be a biodegradable biomaterial scaffold (e.g., silk fibroin scaffold, poly(lactide-co-glycolide (PLGA)), or other similar resorbable products or materials). Such a carrier can be used to provide additional mechanical support for the extracorporeal hematoma. Examples of scaffolds used in the disclosed biomimetic scaffolds include, but are not limited to, biocompatible scaffolds, osteoconductive scaffolds, collagen, absorbable collagen scaffolds (collagen type 1 bovine or porcine), collagen bovine membrane, or collagen from other mammals or non-mammals (such as marine), chitin, bioabsorbable polymers such as PLA, or nonabsorbable polymers such as PEEK, or other biocompatible metal alloys such as titanium or resorbable magnesium (such as magnesium calcium alloy) or 3D printed scaffolds.

[0066] In some aspects, the scaffolds that can be combined with the extracorporeal hematomas described herein can be biocompatible and biodegradable. For example, the scaffolds need to induce a negligible immune response to prevent a severe inflammatory response that could reduce healing or cause rejection by the body. In some aspects, the scaffolds can be amended to allow the cells to adhere to them, so that the cells can function normally, migrate to the surface, and eventually grow through the scaffold. The scaffolds disclosed herein can be biodegradable to allow the cells to generate their own extracellular matrix. The by-products of this degradation are also non-toxic and are excreted from the body without interference from other organs.

[0067] In some aspects, the scaffold can have mechanical properties that match the anatomical site where it is to be implanted and can be strong enough to allow for surgical handling during implantation. Additionally, the scaffold can be strong enough to allow for cell infiltration and vascularization. When designing an appropriate scaffold, it is important to consider the structure of the scaffold. For example, the scaffold can have an interconnected pore structure and high porosity to ensure cell penetration and proper diffusion of nutrients to the cells. The porous interconnected structure is important to allow diffusion of waste products from the scaffold, and the products of scaffold degradation must be able to exit the body without interfering with other organs or surrounding tissues.

[0068] In some embodiments, the scaffold can be a 3D printed scaffold. In some embodiments, the 3D printed scaffold can be custom produced.

[0069] Three-dimensional scaffolds can be fabricated using a 3D printing technique called robotic deposition or direct write (DW) technology, which uses a computer-controlled printing process and colloidal inks to form three-dimensional structures. These structures can be formed on autogenous components or custom-formed to fill individual bone defects from tomographic data (X-ray, ultrasound, or MRI).

[0070] The ink production and printing system itself uses rheology-controlled water-based inks that become solid as they exit the print nozzle. These inks consist of finely controlled ceramic particles in a water-based slurry that contains organic chemicals that control the handling properties of the colloidal ink. This allows 3D lattice-like structures to be printed layer by layer, with or without sagging unsupported structural elements.

[0071] With this system, a first layer of elements can be printed by forcing ink through a small (approximately 50-400 μm in diameter) nozzle onto a support plate using the x and y coordinate control system of an xyz-controlled gantry system. The nozzle is then moved to a position slightly less than one nozzle diameter using the Z control system. The next layer is then printed on top of the first layer. This continues layer by layer until the entire 3D structure is completed.

[0072] The entire structure can be printed in an oil bath to prevent drying. The system has three nozzles and ink reservoirs, so up to three materials can be used to print a single structure. Temporary inks (inks consisting only of materials that burn off during firing) can also be used as part of the printing process. These can be used to print support structures for complex parts that require temporary support.

[0073] The resulting structure is then removed from the oil bath, dried, and fired in a programmable furnace to produce the final ceramic structure. Currently, firing occurs at about 1100°C for about four hours, which essentially burns off the organic components and sinters the ceramic particles together into a solid structure. This can result in a small amount of predictable shrinkage, which can be accounted for in the printing process to produce precise, predictable structures.

[0074] Printing nozzles are typically cylindrical and capable of producing cylindrical rod printed structures, however, nozzles can be fabricated with shapes that produce non-cylindrical structures or structures with surface stripes sized to control cell migration, growth, and differentiation.

[0075] In some aspects, various biomaterials can be used to make the scaffolds disclosed herein. In some aspects, the biomaterials can be ceramics, synthetic polymers and / or natural polymers, or combinations thereof. Examples of ceramics include, but are not limited to, hydroxyapatite (HA) and tricalcium phosphate (TCP). Examples of synthetic polymers include, but are not limited to, polystyrene, poly-1-lactic acid (PLLA), polyglycolic acid (PGA) and poly-dl-lactic-co-glycolic acid (PLGA). Examples of natural polymers include, but are not limited to, collagen, various proteoglycans, alginate-based matrices, and chitosan. The advantage of using natural polymers is that they are biologically active and usually promote excellent cell attachment and proliferation. In addition, they are also biodegradable, so that the host cells can generate their own extracellular matrix over time to replace the degraded scaffold. In some aspects, the scaffolds can be made with a combination of biomaterials. In some aspects, collagen can be combined with polysaccharides (such as glycosaminoglycans). In some aspects, the scaffolds may be prepared using chemical crosslinking methods.

[0076] In some aspects, the composition comprising the scaffold and the extracorporeal hematoma or the biomimetic scaffold may be formulated for local administration. In some aspects, the composition disclosed herein (e.g., in liquid form) or the extracorporeal hematoma (e.g., in gel form) may be administered locally, surgically implanted, or injected percutaneously in combination with any of the carriers or scaffolds disclosed herein. In some aspects, the liquid formulation may be delivered to the scaffold via a syringe. In some aspects, the gel formulation may be implanted into the bone defect site. The gel formulation may be prepared using an external mold that corresponds to the size and shape of the bone for implantation into the bone defect site. In some aspects, the formulation may be an intermediate form between a liquid and a gel. In some aspects, the intermediate formulation may be applied to a solid scaffold or carrier to fill gaps (e.g., large gaps) that may exist in the solid scaffold itself while also independently providing mechanical support. Examples of solid scaffolds include, but are not limited to, titanium cages or other porous metal implants. Such scaffolds may be used to reconstruct skeletal defects or achieve spinal fusion. Considering that PEEK itself is biologically inert and has essentially no bone healing capabilities, the formulations disclosed herein may be used to enhance healing when PEEK spinal cages are used for interbody spinal fusion. Alternatively, any of the formulations disclosed herein may be injected or applied locally to a resorbable scaffold so that it may be used to reconstruct partial or segmental skeletal defects. When used in combination with a metal porous implant or resorbable scaffold, this includes bone defects associated with wedge opening osteotomies (of the femur, tibia, or other long bones), joint distraction fusion sites, and arthroplasty. Additionally, any of the biomimetic scaffolds, compositions, and extracorporeal hematoma formulations disclosed herein may be applied in the same manner to other arthrodesis sites with bone defects, such as the ankle, knee, wrist, shoulder, hip, or other small joints, including but not limited to the Lisfranc joint, hand, wrist, or foot, and include applications to fill bone defects created when harvesting bone grafts for implantation in secondary anatomical locations.

[0077] device The present specification discloses a multi-compartment device for delivering any of the compositions and / or extracorporeal hematomas described herein. In some embodiments, the multi-compartment device may include two or more chambers, or may use two or more syringes to deliver the extracorporeal hematoma components and the bone substitute separately. In some embodiments, the first chamber or syringe may include a clotting agent (e.g., calcium and thrombin, or ecarin) at a predetermined concentration. In some embodiments, the second chamber or syringe may include only whole blood. In some embodiments, the second chamber or syringe may include whole blood in combination with exogenous growth factors (e.g., BMP2, PDGF, VEGF). In some embodiments, the second chamber or syringe may include whole blood in combination with a bone substitute (e.g., DBM, allogeneic cancellous bone chips). In some embodiments, the second chamber or syringe may include whole blood in combination with an exogenous growth factor (e.g., BMP2, PDGF, VEGF) bone substitute. In some embodiments, the third chamber or syringe may include exogenous growth factors and additional bone substitutes. In some embodiments, the third chamber or syringe may include exogenous growth factors. In some embodiments, the third chamber or syringe may include one or more bone substitutes. In some embodiments, the fourth chamber or syringe may include one or more bone substitutes.

[0078] method The present specification discloses a method for promoting bone healing. The present specification also discloses a method for preparing a bone substitute material. Furthermore, the present specification discloses a method for preparing an implant or a biomimetic scaffold or any of the compositions described herein. In some aspects, the composition comprising an extracorporeal hematoma can also function as a scaffold. In some aspects, the composition comprising an extracorporeal hematoma can be combined with a scaffold to form or create a biomimetic scaffold. In some aspects, the methods disclosed herein can be combined. The present specification discloses a method for promoting bone healing, preparing a bone substitute material, preparing an implant, a composition comprising an extracorporeal hematoma, a biomimetic scaffold, or a combination thereof.

[0079] The present disclosure discloses a method for promoting bone healing or preparing a bone substitute material or implant. In some embodiments, the method comprises administering to a subject in need of treatment a therapeutically effective amount of any of the compositions disclosed herein. In some embodiments, the method can comprise administering to a subject in need of treatment a therapeutically effective amount of a composition comprising an extracorporeal hematoma and one or more bone substitutes disclosed herein. In some embodiments, the method comprises implanting any of the biomimetic scaffolds described herein into a target site of the subject. In some embodiments, the biomimetic scaffold can further comprise one or more bone substitutes.

[0080] In some embodiments, the extracorporeal hematoma may include (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the extracorporeal hematoma may include (a) platelet-rich plasma, plasma, or plasma with red blood cells, and (b) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the extracorporeal hematoma may further include sodium citrate. In some embodiments, the extracorporeal hematoma includes fibrin fibers having a thickness of at least 150-300 nm ± 10%. In some embodiments, one or more fibrin fibers having a thickness of at least 150-300 nm ± 10% may be formed by ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some aspects, the extracorporeal hematoma comprises (a) isolated whole blood and sodium citrate platelet-rich plasma, plasma alone, plasma with red blood cells (without platelets), or other blood products, and (b) one or more clotting factors. In some aspects, the extracorporeal hematoma may comprise whole blood and one or more clotting factors. In some aspects, the whole blood may comprise one or more viable cells. In some aspects, the whole blood may comprise one or more biological factors. In some aspects, the extracorporeal hematoma may comprise whole blood, ecarin, and sodium citrate. In some aspects, the extracorporeal hematoma may comprise whole blood, calcium chloride, and sodium citrate. In some aspects, the extracorporeal hematoma may comprise platelet-rich plasma and ecarin. In some aspects, the extracorporeal hematoma may comprise platelet-rich plasma and calcium chloride. In some aspects, the extracorporeal hematoma may comprise plasma and ecarin. In some aspects, the extracorporeal hematoma may comprise plasma and calcium chloride. In some aspects, the extracorporeal hematoma may include plasma with red blood cells and ecarin. In some aspects, the extracorporeal hematoma may include plasma with red blood cells and calcium chloride. In some aspects, the extracorporeal hematoma may include plasma containing oscutaline and calcium chloride. In some aspects, the extracorporeal hematoma may include plasma containing thrombin and calcium chloride.In some embodiments, the extracorporeal hematoma may further comprise bone morphogenetic protein 2 (BMP-2). In some embodiments, the BMP-2 may be recombinant BMP-2. In some embodiments, the recombinant BMP-2 may comprise human BMP-2. In some embodiments, the composition may further comprise one or more growth factors, one or more platelets, and one or more cells. In some embodiments, the composition may be formulated as a clot or scaffold. In some embodiments, the scaffold may be chemotactic. In some embodiments, the scaffold may attract endogenous growth factors that aid in bone healing.

[0081] In some embodiments, the one or more bone substitutes may be derived from a biological product, may be a synthetic bone substitute, or a combination thereof. Examples of bone substitutes derived from biological products include, but are not limited to, bone marrow aspirate concentrate (BMAC) containing demineralized bone matrix (DBM), bone morphogenetic protein (BMP), hydroxyapatite (HA), and coral, allogeneic cancellous bone chips, or bone grafts from long bones harvested using a reamer irrigator aspirator (RIA). In some embodiments, the one or more bone substitutes may be derived from a biological product, and the biological product may be bone marrow aspirate concentrate (BMAC) containing bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), demineralized bone matrix (DBM), hydroxyapatite (HA), coral, allogeneic cancellous bone chips, or bone grafts from long bones harvested using a reamer irrigator aspirator (RIA). In some embodiments, the bone substitute may be a synthetic bone substitute. Examples of synthetic bone substitutes include, but are not limited to, calcium sulfate, calcium phosphate cement, beta-tricalcium phosphate (TCP) ceramics, biphasic calcium phosphate (hydroxyapatite (HA) and beta-TCP ceramics), bioactive glass, and polymer-based bone substitutes. Further examples of synthetic bone substitutes include, but are not limited to, Calcigen® S calcium sulfate bone void filler, STIMULAN® beads, HydroSet injectable bone substitute (calcium phosphate), Ossilix calcium phosphate cement, Syntoss synthetic beta-tricalcium phosphate bone graft material, CERASORB® tricalcium phosphate bone graft, GL1894P / -20 58S bioactive glass, UniGraft bioactive glass 200-600um, BonAlive (BonAlive Biomaterials Ltd, Finland), Cerament (bone void filler), and Cerament G (Bonesupport Holding AB, Lund Sweden).Examples of polymers include, but are not limited to, collagen, gelatin, chitosan, and synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polycaprolactone (PCL)-GalaFlex P4HB biopolymer. In some embodiments, the bone substitute is available in a variety of forms, including, but not limited to, dry, moldable, or injectable forms, and pastes, powders, putties, granules, gels, sponges, or strips. In some embodiments, the bone substitute can be a commercially available product. In some embodiments, the bone substitute can be demineralized bone matrix (DBX, MTF Biologics, Edison, NJ), RegenaVate DBM, Puros DBM, StaGraft DBM, or FiberStack DBM (Zimmer Biomet, Warsaw, IN). In some embodiments, the DBM can be allograft cancellous bone or cortical bone that has been demineralized to generate a collagen and non-collagenous protein product. Examples of DBM include, but are not limited to, Grafton DBM (Osteotech, Inc, Eatontown, New Jersey), Allosource (Denver, Colorado), Dynagraft II (Integra LifeSciences, Plainsboro, New Jersey), DBX (Musculoskeletal Transplant Foundation and Synthes, Paoli, Pennsylvania), and Osteofil (Medtronic Sofamor Danek, Minneapolis, Minnesota). Examples of corals include, but are not limited to, Animalia, Coelenterata, Scleractinia, Corallinae, Porites species, and Gonioporus species, each of which may be used in the development of a coralline hydroxyapatite (CHA) bone substitute. In some embodiments, the bone substitute is not a BMP, rhBMP-2, or BMP-2.

[0082] In some aspects, the subject may be a human. In some aspects, the subject has a skeletal abnormality. In some aspects, the skeletal abnormality may be a large segmental bone defect. In some aspects, the skeletal abnormality may be a small segmental bone defect. In some aspects, the skeletal abnormality may be independent of the size or volume of the defect, whether the defect is complete or incomplete. In some aspects, the subject has a dental bone defect.

[0083] In some embodiments, the subject has one or more bone fractures, hi some embodiments, the subject has one or more bone injuries.

[0084] In some embodiments, the composition may be formulated as a clot or scaffold. In some embodiments, the composition may be formulated for local administration and combined with any of the scaffolds disclosed herein. In some embodiments, the composition may be administered locally via a carrier or scaffold. In some embodiments, the composition may be administered locally without a carrier or scaffold. In some embodiments, the composition may be implanted or delivered transdermally. In some embodiments, the composition may be implanted. In some embodiments, the composition may be implanted directly or indirectly. In some embodiments, the composition may be delivered by a surgeon or by any autonomous or semi-autonomous delivery device operating on behalf of a human or a robotic / semi-autonomous agent. In some embodiments, the composition may be delivered transdermally.

[0085] Disclosed herein is a method of constructing an implant. In some embodiments, the method includes a) dimensioning the depot implant to at least one of a shape and a size that facilitates implantation of the depot implant into the bone defect; and b) structuring the depot implant to have a scaffold by introducing (i) isolated whole blood and sodium citrate, or platelet-rich plasma, plasma, or plasma with red blood cells, (ii) ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, and (iii) a bone substitute to create the scaffold. In some embodiments, the scaffold may have a porosity of 55-75%. In some embodiments, the scaffold comprises fibrin fibers having a thickness of at least 150-300 nm±10%. In some embodiments, the shape of the depot implant is cylindrical or spherical. In some embodiments, the scaffold may be constructed as a blood clot. In some embodiments, one or more growth factors may be introduced into the scaffold. In some embodiments, the one or more growth factors can be bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4, BMP-6, BMP-9, BMP-14, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), or combinations thereof. In some embodiments, BMP-2 can be introduced into the scaffold. In some embodiments, the amount of ecarin present in the scaffold can be at least 0.05 U / mL and the amount of BMP-2 present in the scaffold can be at least 0.01 mg. In some embodiments, the ratio of extracorporeal hematoma to bone substitute is 1000:1 to 1:1000. In some embodiments, the scaffold can resemble the size and shape of a particular bone defect. In some embodiments, the scaffold can be chemotactic. In some embodiments, the scaffold can include live blood cells and appropriate biological factors. In some embodiments, the bone substitute can be a demineralized bone matrix. In some embodiments, the bone substitute can be derived from a biological product, a synthetic bone substitute, or a combination thereof. In some embodiments, the biological product can be demineralized bone matrix, hydroxyapatite, or coral.In some embodiments, the synthetic bone substitute can be calcium sulfate, calcium phosphate cement, β-tricalcium phosphate ceramic, bioactive glass, or a polymer. In some embodiments, the one or more growth factors is not BMP, rhBMP-2, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, or BMP-14.

[0086] Also disclosed herein is a method of constructing a biomimetic scaffold. In some embodiments, the method includes a) dimensioning the scaffold to at least one of a shape and a size that facilitates implantation of the scaffold into the bone defect; and b) combining the scaffold in a) with (i) isolated whole blood and sodium citrate, or platelet-rich plasma, plasma, or plasma with red blood cells, and (ii) an extracorporeal hematoma containing ecarin, oscutin, calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride to create a biomimetic scaffold. In some embodiments, the scaffold may have a porosity of 55-75%. In some embodiments, the scaffold comprises fibrin fibers having a thickness of at least 150-300 nm±10%. In some embodiments, the shape of the scaffold is cylindrical or spherical. In some embodiments, the shape of the scaffold may be any other geometric shape or shapes that can theoretically occupy a discrete subset or volume in Euclidean space. In some embodiments, the scaffold may be collagen, chitin, a bioabsorbable polymer, a nonabsorbable polymer such as PEEK, or titanium or a metal alloy. In some embodiments, one or more growth factors may be introduced into the scaffold or extracorporeal hematoma. In some embodiments, the one or more growth factors may be bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4, BMP-6, BMP-9, BMP-14, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), or a combination thereof. In some embodiments, BMP-2 may be introduced into the scaffold or extracorporeal hematoma. In some embodiments, the amount of ecarin present in the scaffold or extracorporeal hematoma may be at least 0.05 U / mL, and the amount of BMP-2 present in the scaffold or extracorporeal hematoma may be at least 0.01 mg. In some embodiments, the ratio of extracorporeal hematoma to bone substitute is 1000:1 to 1:1000. In some aspects, the extracorporeal hematoma can include viable blood cells and appropriate biological factors. In some aspects, the bone substitute can be a demineralized bone matrix. In some aspects, the bone substitute can be derived from a biological product, a synthetic bone substitute, or a combination thereof.In some embodiments, the biological product may be demineralized bone matrix, hydroxyapatite, or coral. In some embodiments, the synthetic bone substitute may be calcium sulfate, calcium phosphate cement, β-tricalcium phosphate ceramic, bioactive glass, or polymer. In some embodiments, the scaffold may mimic the size and shape of a particular bone defect. In some embodiments, the scaffold may be chemotactic. Additionally, the scaffold may be biodegradable so as to degrade without the need for surgical removal. In some embodiments, the one or more growth factors are not BMP, rhBMP-2, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, or BMP-14.

[0087] In some embodiments, the composition may be implanted as all or part of a biomimetic scaffold. In some embodiments, the composition may be injected into the carrier or scaffold using a syringe. In some embodiments, the amount of ecarin present in the composition may be at least 0.05 U / mL, and the amount of BMP-2 present in the composition may be at least 0.01-5 mg, or any amount therebetween. In some embodiments, the amount of ecarin present in the composition may be at least 0.05 U / mL, and the amount of BMP-2 present in the composition may be at least 0.01-1 μg, or any amount therebetween. In some embodiments, the ratio of extracorporeal hematoma to bone substitute may be 1000:1-1:1000, or any ratio therebetween.

[0088] In some aspects, the treatment plan may be a standard treatment plan for treating any bone defect. Briefly, the defect wound is debrided and fixed with an internal plate, an external fixator, or an intramedullary nail. The composition, the composition and scaffold, and the biomimetic scaffold and extracorporeal hematoma described herein may be inserted as a unit into the skeletal defect before closing the wound. In some aspects, the components of the composition, the components of the composition and scaffold, and the biomimetic scaffold and the extracorporeal hematoma described herein may be inserted separately into the skeletal defect before closing the wound. For example, the multi-compartment device may include two or more chambers or use two or more syringes to deliver the components of the extracorporeal hematoma and the bone substitute separately. In some aspects, the first chamber or syringe may include a predetermined concentration of a clotting agent (e.g., calcium and thrombin, or ecarin). In some aspects, the second chamber or syringe may include only whole blood. In some embodiments, the second chamber or syringe may contain whole blood in combination with exogenous growth factors (e.g., BMP2, PDGF, VEGF). In some embodiments, the second chamber or syringe may contain whole blood in combination with a bone substitute (e.g., DBM, allogeneic cancellous bone chips). In some embodiments, the second chamber or syringe may contain whole blood in combination with an exogenous growth factor (e.g., BMP2, PDGF, VEGF) bone substitute. In some embodiments, the third chamber or syringe may contain exogenous growth factors and additional bone substitute. In some embodiments, the third chamber or syringe may contain exogenous growth factors. In some embodiments, the third chamber or syringe may contain one or more bone substitutes. In some embodiments, the fourth chamber or syringe may contain one or more bone substitutes. In some embodiments, the one or more growth factors is not a BMP, rhBMP-2, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, or BMP-14.

[0089] As long as no infection is present and the defect is ready for definitive treatment, the treatment plan can remain consistent. The compositions or biomimetic scaffolds (or implants) disclosed herein can be inserted into the bone area by entering the body through the skin or through a body cavity or anatomical opening, minimizing further damage to nearby structures. The selection of the type, including size and shape, of the composition, biomimetic scaffold, scaffold or implant includes, but is not limited to, the shape and / or size of the bone into which the composition, biomimetic scaffold, scaffold or implant is to be implanted, the percentage of bone density (i.e., the porosity of the remaining bone), and / or the desired rate and distribution of diffusion of the scaffold or implant into the bone, or a combination of such factors. In some aspects, the shape of the composition, biomimetic scaffold, scaffold or implant can be constructed to match the shape of the bone or vertebral body, thus allowing for a more uniform distribution of the composition, biomimetic scaffold, implant or extracorporeal hematoma, or the components present in the composition, scaffold, biomimetic scaffold, implant or extracorporeal hematoma. Application of the composition, biomimetic scaffold or implant can be performed at the time of surgery or in any other suitable manner.

[0090] In some embodiments, the shape of the depot implant or scaffold can be spherical or cylindrical. In some embodiments, the shape of the depot implant or scaffold can be spherical or any other patient-specific geometry, form, or shape as determined by clinical exigencies. In some embodiments, the cylindrical shape can be at least 5 mm to about 30 cm (or more) in length. In some embodiments, the cylindrical shape can be at least 1 mm to about 60 mm (or more) in diameter. In some embodiments, the cylindrical shape can be straight and / or curved. In some embodiments, the cylindrical shape can be a straight rod or a curved rod. The shape of the cylinder or rod can be any shape with a longitudinal axis that can be longer along one direction than the other. The cross-sectional shape of the depot across the longitudinal axis can be any shape. In some embodiments, the cross-sectional shape can be elliptical, circular, trilobal, or other shape. In some embodiments, the depot or scaffold can be straight or curved in such longitudinal direction. The end faces of the depot or scaffold may be shaped to be either planar, rounded, or convoluted.

[0091] The dimensions of the implant depot or scaffold, or extracorporeal hematoma, may depend on the size of the bone defect and the anatomical site to be treated. In some embodiments, the scaffold may be about 20% longer than the actual size of the defect, so that it fits tightly and completely fills the volume of the missing bone. For example, if the size of the bone defect is 3 cm and it is the femoral shaft of an adult, the implant depot or scaffold or extracorporeal hematoma should be constructed with dimensions of, for example, about 3-4 cm in diameter and 3.6 cm in length. In some embodiments, the implant depot, scaffold, or extracorporeal hematoma can resemble the size and shape of a particular bone defect. In some embodiments, the implant depot or scaffold may be chemotactic.

[0092] Also disclosed herein is a method of using any of the compositions described herein and combining said compositions with any of the scaffolds described herein to initiate or promote bone healing. Also disclosed herein is a method of reconstructing segmental bone defects using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein is a method of reconstructing segmental bone defects resulting from tumors, trauma, or infection using any of the biomimetic scaffolds and compositions described herein using ecarin to create a biomimetic scaffold, or the biomimetic scaffold delivers catalytic amounts of BMPs or one or more bone substitutes to initiate the normal fracture healing cascade, followed by local hyperactivation of endogenous growth factors.

[0093] Also disclosed herein are methods of treating at-risk fractures (e.g., in osteoporotic, diabetic, elderly, or smokers) using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of using ecarin to deliver catalytic amounts of BMPs or one more bone substitute to initiate the normal fracture healing cascade, followed by local hyperactivation of endogenous growth factors, and treating at-risk fractures using any of the biomimetic scaffolds and compositions described herein.

[0094] Also disclosed herein is a method of treating an atypical femoral fracture using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein is a method of treating an atypical femoral fracture percutaneously using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the normal fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0095] Also disclosed herein is a method of treating a minimally displaced femoral neck fracture using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein is a method of treating a minimally displaced femoral neck fracture percutaneously using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the normal fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0096] Also disclosed herein is a method of treating osteoporotic insufficiency fractures (e.g., pelvis, spine) using any of the biomimetic scaffolds and compositions described herein. Also disclosed herein is a method of treating osteoporotic insufficiency fractures (e.g., pelvis, spine) percutaneously using any of the biomimetic scaffolds and compositions described herein by creating an extracorporeal hematoma using ecarin, which initiates the normal fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0097] Also disclosed herein is a method of augmenting spinal fusion using any of the compositions described herein in combination with a scaffold such as a vertebral cage (either ceramic, PEEK, or metal alloy). Also disclosed herein is a method of augmenting spinal fusion using any of the compositions described herein in combination with a scaffold such as a vertebral cage (either ceramic, PEEK, or metal alloy) that allows immediate full weight bearing, provides more stable fixation, and promotes post-operative recovery, using ecarin to induce local formation of an extracorporeal hematoma that is embedded on a substrate that constitutes the cage (scaffold) that initiates the bone healing cascade by delivering catalytic amounts of BMP or a bone substitute that then hyperactivates endogenous growth factors locally.

[0098] Also disclosed herein is a method of treating delayed union of a long bone fracture (percutaneous or open) using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein is a method of treating delayed union of a long bone fracture (percutaneous or open) using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0099] Also disclosed herein are methods of treating non-union of long bone fractures (percutaneous or open) using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of treating non-union of established long bone fractures (percutaneous or open) using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0100] Also disclosed herein are methods of improving (e.g., accelerating) healing of long bone fractures using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of improving (e.g., accelerating) healing of long bone fractures in selected candidates (such as high performance athletes) using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin to promote more rapid recovery, which initiates the fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0101] Also disclosed herein are methods of promoting healing of long bone fractures in selected veterinary candidates, such as thoroughbred race horses, using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of promoting healing of long bone fractures in selected veterinary candidates, such as thoroughbred race horses, using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin to promote more rapid recovery, which initiates the fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0102] Also disclosed herein are methods for promoting more rapid and predictable tooth and maxillofacial reconstruction using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods for promoting more rapid and predictable tooth and maxillofacial reconstruction using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the osteogenic cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0103] Also disclosed herein are methods of reversing conditions leading to idiopathic bone resorption of the jaw using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of creating an extracorporeal hematoma and locally regenerating bone using ecarin, and of reversing conditions leading to idiopathic bone resorption of the jaw using any of the compositions described herein.

[0104] Also disclosed herein are methods of treating and / or reversing conditions leading to idiopathic osteonecrosis using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of using ecarin to create an extracorporeal hematoma delivered percutaneously or openly, and using any of the compositions and biomimetic scaffolds described herein to reverse conditions leading to idiopathic osteonecrosis (such as Kienböck's disease, avascular necrosis of the femoral head, and osteonecrosis at various other anatomical sites, including but not limited to the femoral condyle).

[0105] Also disclosed herein are methods of treating and / or reversing conditions that cause idiopathic avascular necrosis of the femoral head in patients with a collapsed femoral head using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of using ecarin to create an extracorporeal hematoma delivered in an open procedure following surgical hip dislocation, and of treating and / or reversing conditions that cause idiopathic avascular necrosis of the femoral head in patients with a collapsed femoral head using any of the compositions and biomimetic scaffolds described herein.

[0106] Also disclosed herein are methods of treating osteonecrosis resulting from chemotherapy, alcoholism, smoking, or other exogenous factors using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of treating osteonecrosis resulting from chemotherapy, alcoholism, smoking, or other exogenous factors using any of the compositions and biomimetic scaffolds described herein to create an extracorporeal hematoma delivered percutaneously or openly using ecarin.

[0107] Also disclosed herein is a method of enhancing standard fusion procedures using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein is a method of enhancing any standard fusion procedure (e.g., limited fusion of the hip, knee, ankle, wrist, elbow, shoulder, subtalar joint, carpal bone, midfoot, etc., fusion of small joints such as the big toe, thumb, and little toe (either toe or finger)) using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the bone formation cascade by delivering catalytic amounts of BMP or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0108] Also disclosed herein are methods of promoting healing of scaphoid hip fractures using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of promoting healing of scaphoid hip fractures and promoting faster recovery using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0109] Also disclosed herein is a method for reconstructing complex skeletal defects using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein is a method for reconstructing complex skeletal defects of the skull, whether due to trauma, tumor, or infection, using any of the compositions and biomimetic scaffolds described herein by creating an extracorporeal hematoma using ecarin, which initiates the bone formation cascade by delivering catalytic amounts of BMP or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0110] Also disclosed herein are methods of promoting healing of sternotomy using any of the compositions and biomimetic scaffolds described herein. Also disclosed herein are methods of promoting faster recovery and promoting healing of sternotomy associated with open chest surgery using any of the compositions and biomimetic scaffolds described herein by using ecarin to create an extracorporeal hematoma that initiates the fracture healing cascade by delivering catalytic amounts of BMPs or one or more bone substitutes, followed by local hyperactivation of endogenous growth factors.

[0111] Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein in arthroplasty components having specially adapted bone ingrowth surfaces augmented with ecarin to induce localized formation of extracorporeal hematomas embedded on structural substrates that more rapidly initiate the bone healing cascade.

[0112] The present specification also discloses methods of using any of the compositions and biomimetic scaffolds described herein in conjunction with osseointegration stems and components with specially adapted bone ingrowth surfaces enhanced with ecarin to induce the local formation of extracorporeal hematomas embedded on structural substrates that more rapidly initiate the bone healing cascade.

[0113] In some embodiments, the one or more bone substitutes is not a BMP, rhBMP-2, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, or BMP-14.

[0114] As used herein, the term "biomimetic hematoma" may be used to refer to an "extracorporeal hematoma."

[0115] Also disclosed herein are methods of reducing or controlling bleeding using any of the compositions described herein.

[0116] Also disclosed herein are methods of managing extensive venous bleeding / oozing during surgery using any of the compositions described herein. In some embodiments, in the methods disclosed herein, any of the compositions described herein can be formulated to be sprayed locally as an aqueous aerosol (using an atomizer to distribute ecarin to the affected area).

[0117] Also disclosed herein are methods of managing or stopping bleeding from individual injured vessels (e.g., hemorrhagic subjects) during surgery or in emergency injury situations using any of the compositions described herein. In some embodiments, in the methods disclosed herein, any of the compositions described herein can be administered on beads (e.g., magnetic beads). In some embodiments, in the methods disclosed herein, any of the compositions described herein can be applied to the end of the vessel as a clamp / clamshell, which can simultaneously clamp off and deliver ecarin locally, limiting application to a specific injured vessel end. The clamp or clamping element can clamp down on adjacent injured vessels, eliminating or minimizing the risk of systemic administration of the composition.

[0118] Also disclosed herein are methods of using any of the compositions described herein as selective embolization. In some embodiments, the methods disclosed herein involve delivery of any of the compositions described herein to one or more target vessels via an interventional radiologist to manage or stop intrapelvic / intraperitoneal / esophageal / intracranial bleeding using a long catheter directed to radiography, thereby allowing selective and highly specific administration of ecarin limited to the individual pathology indicated (similar to methods performed using angiography coils, for example).

[0119] Also disclosed herein are methods of treating dysfunctional uterine bleeding using any of the compositions described herein. In some aspects, the methods disclosed herein can be used to introduce or place any of the compositions described herein into the uterus of an affected woman. In some aspects, ecarin can be formulated to be delivered as part of a biodegradable collagen bead(s).

[0120] Also disclosed herein are methods of treating hemophilia-associated osteoarthritis using any of the compositions described herein. In some aspects, ecarin may be formulated to be delivered as part of a biodegradable collagen bead(s).

[0121] Also disclosed herein are methods of treating osteoarthritis associated with anticoagulant (e.g., warfarin, coumadin, etc.) overdose using any of the compositions described herein. In some aspects, ecarin may be formulated to be delivered as part of a biodegradable collagen bead(s).

[0122] Also disclosed herein are methods of treating spontaneous muscle bleeding associated with an overdose of anticoagulants (e.g., warfarin, coumadin, etc.) using any of the compositions described herein. In some aspects, any of the compositions described herein may be used as selective embolization in the methods disclosed herein.

[0123] Also disclosed herein are methods of treating spontaneous muscle bleeding associated with hemophilia using any of the compositions described herein. In some aspects, any of the compositions described herein may be used as selective embolization in the methods disclosed herein.

[0124] Also disclosed herein is a method for treating postoperative osteoarthritis in any elective total knee replacement surgery using any of the compositions described herein. In some embodiments, ecarin can be formulated to be delivered as part of a biodegradable collagen bead(s) or nanoparticle(s). In some embodiments, the biodegradable collagen bead(s) or nanoparticle(s) can be delivered or dispersed liberally into the joint before closing the wound.

[0125] Also disclosed herein is a method of treating nosebleeds using any of the compositions described herein. In some aspects, ecarin can be formulated to be delivered as part of a biodegradable collagen bead(s). In some aspects, the biodegradable collagen bead(s) can be embedded in a fabric packaging material or enclosed in a fabric sheath to limit distribution and provide localized containment. In some aspects, ecarin can be delivered in the form of a nose pack, formulated to be part of a biodegradable collagen bead(s) that is embedded in a fabric packaging material or enclosed in a fabric sheath.

[0126] Also disclosed are methods of treating retinal hemorrhage using any of the compositions described herein. In some embodiments, any of the compositions described herein may be used as selective embolization in the methods disclosed herein. In some embodiments, ecarin may be formulated to be delivered as part of a biodegradable collagen bead(s) or nanoparticle(s). In some embodiments, the use of a biodegradable collagen bead or nanoparticle formulation can create a Velcro-type effect by creating a self-adhesive structure that minimizes the risk of recurrence and actively addresses retinal detachment.

[0127] In some embodiments, "bleeding" can be hemorrhage. In some embodiments, blood can leak out of the circulatory system from an abnormal damaged blood vessel. In some embodiments, bleeding can be internal or external bleeding.

[0128] manufactured goods The compositions and biomimetic scaffolds described herein can be packaged in a suitable container with a label for use as a therapy to treat, for example, a bone defect or any of the methods disclosed herein. In some aspects, the compositions comprising the extracorporeal hematoma described herein can be packaged in a suitable container with a label for use as a therapy to treat, for example, a bone defect or any of the methods disclosed herein, and can be packaged separately from the scaffold portion of the biomimetic scaffold. Thus, packaged articles (e.g., scaffolds, sterile containers containing compositions comprising any of the individual components of the compositions or extracorporeal hematoma described herein and packaged for storage, shipping, or sale in a concentrated or ready-to-use concentration) and kits include at least isolated whole blood and sodium citrate, or platelet-rich plasma, plasma, or plasma with red blood cells, and ecarin, oscutarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride described herein, and instructions for use within the scope of the present disclosure. The articles of manufacture can include a container (e.g., a vial, jar, bottle, bag, etc.) containing the biomimetic scaffold or composition described herein or the extracorporeal hematoma. Additionally, the article of manufacture may further include, for example, packaging materials, instructions for use, a syringe, buffer, or other control reagents for treating or monitoring a condition requiring prevention or treatment. The article of manufacture may include instructions (e.g., a printed label or insert, or other media (e.g., audiotape or videotape) explaining how to use the product. The instructions may be associated with (e.g., affixed to) the container and may describe how (e.g., frequency and route of administration) the biomimetic scaffold, composition therein, or extracorporeal hematoma should be administered, its indications, and other uses. The biomimetic scaffold or composition or extracorporeal hematoma may be ready for administration (e.g., present in an appropriate dosage unit) and may include a pharma- ceutically acceptable adjuvant, carrier, or other diluent. Alternatively, the compound may be provided in a concentrated form with a diluent, and instructions for dilution may also be included. EXAMPLES

[0129] In Example 1, a resorbable collagen scaffold was combined with clotted whole blood and rhBMP-2 to successfully repair large bone defects in rats. One of the most promising alternatives to autologous bone grafting is the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) delivered on absorbable collagen sponges (ACS). However, successful bone healing requires a constantly high physiological dose of rhBMP-2 due to its burst release and short half-life in the body, most of which is rapidly leached out as soon as the collagen sponge is compressed after insertion into the bone defect area. This leads to an increased incidence and severity of heterotopic / ectopic ossification, as well as many other associated side effects such as antibody formation, implant removal, bone resorption, and even cancer. A more suitable carrier is clearly needed to treat these complex bone injuries and improve the efficacy of BMPs by significantly reducing the dose required, thus mitigating their potentially dangerous side effects.

[0130] The hematoma that forms at the fracture site acts as an endogenous scaffold that activates a cascade of biological events that incorporate molecular factors derived from the surrounding tissues. Studies show that the hematoma that forms at the fracture site has a profound effect on how the fracture heals, and removal of this hematoma delays fracture healing. To enhance the efficacy of ACS, we combined ACS with an extracorporeal hematoma that mimics the unique structural and biological properties of an endogenous fracture hematoma. In a 5 mm large critical size defect model in rat femurs, we tested whether an extracorporeal hematoma created by combining ACS with a mixture of whole blood, coagulant, and a low dose of rhBMP-2 significantly enhanced bone regenerative capacity and reduced the required rhBMP-2 dose compared to the use of ACS and BMP-2 alone.

[0131] Materials and Methods. Preparation of ACS / whole blood biomimetic scaffolds. Blood was collected from anesthetized male donor Fisher 344 rats by peripheral bleeding at the time of euthanasia. Blood was collected by cardiac puncture using a 21-gauge needle. Nine parts of blood were mixed with one part of 3.2% trisodium citrate solution to prevent clotting. To create cylindrical biomimetic scaffolds, 0.55 μg of rhBMP-2, 10 mM calcium chloride, and 5 U / mL of thrombin (ex vivo hematoma) were pipetted into the bottom of a flat-bottom well of a 96-well plate before adding the citrated blood. The solution was mixed by pipetting up and down and quickly transferred to another well containing cylindrical dried ACS. The biomimetic scaffolds were incubated at room temperature for 15 minutes before being implanted into a 5 mm femoral defect in rats. ACS without whole blood was used as the control group.

[0132] Results: Bone defects treated with ACS / WB / BMP2 were fully bridged 4 weeks after surgery. After 8 weeks, healing of the bone defects was complete, with the cortical bone almost completely restored to its original shape, although a small amount of intramedullary trabecular bone remained, indicating ongoing healing and remodeling. In contrast, bone defects treated with ACS / BMP2 did not heal within 8 weeks and consistently led to nonunions (Figure 1).

[0133] Conclusion. The data show that by adding a mixture of whole blood and clotting agent to the ACS at a specific concentration (mimicking a natural fracture hematoma, ex vivo hematoma), the biomimetic scaffold can deliver rhBMP-2 with much higher efficiency compared to the ACS alone. Furthermore, this healing was achieved using a significantly lower dose of rhBMP-2, whereas the same dose delivered to the ACS was not even able to initiate healing of the bone defect. The superior healing is likely due to the bioscaffold more closely resembling a congenital fracture hematoma, an endogenous reservoir of important growth factors such as VEGF, FGF, and TGF, and thus binding rhBMP-2 more efficiently than the ACS alone. Furthermore, an added benefit of adding an ex vivo hematoma to the ACS is that the delivered rhBMP-2 is safely contained within the scaffold, preventing heterotopic / heterotopic ossification and other adverse side effects. The use of this biomimetic scaffold allows for the delivery of rhBMP-2 at a significantly reduced dose without compromising the healing outcome.

[0134] Example 2: Extracorporeal hematoma combined with various bone substitutes. The extracorporeal hematoma can be mixed with any of the bone substitutes derived from currently available biological products on the market, such as DBX, DBM, coral, allogeneic cancellous bone chips, or bone marrow aspirate concentrate (BMAC), including bone grafts from long bones harvested using a reamer irrigator aspirator (RIA).The extracorporeal hematoma can also be mixed with synthetic bone substitutes, such as calcium sulfate, calcium phosphate cement, beta-tricalcium (TCP) phosphate ceramics, biphasic calcium phosphates (hydroxyapatite (HA) and beta-TCP ceramics), bioactive glass, and polymer-based bone substitutes.

[0135] Demineralized bone matrix (DBM) is a substitute for cancellous and cortical bone and is available in dry, moldable, or injectable forms and can exist as pastes, powders, putties, granules, gels, sponges, or strips. DBM is prepared from bone grafts by demineralization and provides a framework for cell and matrix protein attachment, and also contains osteogenic substances that induce new bone growth. The osteogenic components of DBM are a mixture of growth factors, including many in the transforming growth factor beta family (TGF-B), such as BMP-2. DBM offers little biomechanical strength, but functions as an osteoconductive and, to a lesser extent, osteoinductive material. In orthopedics, DBM has been used as an extender of autogenous bone grafts and has been excellent when used as an additive (extender) when there is limited availability of autogenous bone graft, but as a standalone product it is not capable of initiating bone formation. This is especially true for large bone defects due to processing, donor selection, terminal sterilization, and additives (e.g., hyaluronic acid, pulonic acid, glycerin, gelatin, etc.) that result in products with variable bioactivity and therefore unpredictable biological osteoinductivity and clinical outcomes. The same is true for many other bone substitutes currently available on the market.

[0136] In contrast, extracorporeal hematomas were developed to mimic healing fracture hematomas and effectively and efficiently deliver growth factors such as rhBMP-2 to large segmental bone defects that do not heal naturally. The main concept is to use autologous blood in combination with a specific concentration of a coagulant to mimic an endogenous fracture hematoma, since the hematoma formed at the time of injury contains specific microstructural characteristics in the body that activate a cascade of biological events to initiate bone healing. This blood clot (hematoma) usually contains the components necessary to initiate the bone healing cascade, including blood cells such as red blood cells, white blood cells, and platelets. In fact, platelets play a key role in bone healing by releasing and activating important angiogenic and osteogenic growth factors. Experimental results using extracorporeal hematomas as a delivery vehicle showed that this model was able to heal critical-sized defects in rats using a fraction of the BMP-2 dose required for healing. In contrast, absorbable collagen sponges were completely unable to initiate the healing process even with such small amounts. Furthermore, bone quality was significantly better than when BMP-2 was administered on absorbable collagen sponges. Based on these results, we tested the extracorporeal hematoma combined with one or more bone substitutes, such as DBM, to see whether such a combination would enhance the efficacy of the hematoma and thus render the regeneration and healing of bone defects more efficient and robust. For example, Figure 2 shows the structure of one of the demineralized bone matrix products (DBX), clearly showing that the solid material lacks porosity, which is important for cell recruitment and infiltration. However, when DBM is mixed with the extracorporeal hematoma, the resulting scaffold has a microstructure that mimics the endogenous healing fracture hematoma. Moreover, as a combination product, the extracorporeal hematoma contains important components that promote fracture healing. Furthermore, the addition of DBM increases the amount of growth factors available at the bone defect site, initiating bone regeneration efficiently and robustly. Thus, a structurally well-organized extracorporeal hematoma containing DBM can also act as a temporary reservoir for the continuous release of growth factors and provide an appropriate environment to promote cell infiltration, proliferation, and differentiation from the surrounding tissues.

[0137] Example 3, Extracorporeal hematoma combined with a scaffold. Patient-specific 3D printed titanium cages are used to treat large segmental bone loss and spinal fusions (Figures 3 and 4). These titanium cages are used to address segmental bone defects and spinal pathologies that are greater than 6 cm in length. These metal implants or scaffolds are not osteoinductive and therefore are unable to initiate bone regeneration and incorporation within the pores of these titanium scaffolds. To initiate bone healing and regeneration, orthopedic surgeons currently increase the regenerative potential by adding autologous bone grafts, bone substitutes, and growth factors, however, none of these products are able to initiate reliable, consistent, and robust bone regeneration, especially in long bones. Furthermore, these particular options unfortunately come with many drawbacks. For example, autologous bone grafts are often not readily available in the required quantities and there are issues of potential donor site morbidity. Furthermore, these grafts are not user-friendly as they require the surgeon to manually push them into the cavities / pores of the cage / scaffold (Figure 4), which is time-consuming and makes it nearly impossible to achieve uniform graft incorporation within the titanium cage / scaffold, for example in the center of the cage. Growth factors also need to be delivered in high concentrations, which are shown to be associated with many adverse side effects such as ectopic bone formation. Moreover, their uniform incorporation within the cage / scaffold is not a simple procedure, and growth factors often need to be used in combination with a bone substitute. Naturally, the bone substitute itself also comes with various drawbacks very similar to those of autogenous bone grafts and growth factors, the main issue being the need to manually push them into the cavities / pores of the cage / scaffold, in addition to unpredictable biological osteoinductivity and clinical outcomes.

[0138] To overcome this issue, extracorporeal hematomas, even in combination with growth factors and bone substitutes such as DBM, promote a more uniform incorporation within the titanium cage / scaffold because the extracorporeal hematoma starts as a liquid (Figure 3) and is fully permeated, simultaneously promoting consistent bone regeneration. This treatment strategy works well because the bone substitute is delivered within the extracorporeal hematoma itself, mimicking the microstructural properties of an endogenous healing fracture hematoma. This increases the amount of growth factors required to efficiently and reliably initiate bone regeneration. Furthermore, a structurally well-organized scaffold (e.g., extracorporeal hematoma) also serves as a temporary reservoir for the continuous release of growth factors, providing a highly favorable environment that promotes cell infiltration, proliferation, and differentiation from the surrounding tissue.

[0139] Example 4. Percutaneous delivery of extracorporeal hematoma for the treatment of delayed union, nonunion, and bone defects. A mixture of extracorporeal hematoma and either bone substitutes (e.g., DBM, bone chips) and / or growth factors (e.g., BMP-2, PDGF) can be delivered percutaneously to promote healing of delayed bone unions, nonunions, and bone defects.

[0140] The method is as follows: Similar to arthroscopic or endoscopic surgery, the surgeon inserts a thin tube (plastic or metal) as a cannula to allow the introduction of a fiber optic video camera through a small incision the size of a buttonhole. Images of the interior of the delayed / nonunion or bone defect can be transmitted to a high resolution video monitor. Another small incision is made to introduce a second small tube (cannula) through which a rotating blade is inserted and gradually unfolds like an umbrella as it enters the nonunion site, the position of which can be monitored by fluoroscopy. It extends approximately 5-10 mm from the center of the nonunion to allow limited debridement to remove fibrotic tissue within the nonunion site and prepare the space for injection of the extracorporeal hematoma containing the scaffold. This step not only creates a cavity for the delivery of biological products, but also removes dense avascular fibrotic tissue that is known to impede successful bone healing. An optional third small incision can be made to insert another cannula / tube to remove and clean debris generated during debridement of the fibrotic nonunion tissue. Once the nonunion site is prepared for the delivery of the extracorporeal hematoma, for example, a syringe with two chambers can be used (Figure 5). A long needle attached to the two-chamber syringe can be inserted through the same tubing used to remove the fibrous tissue of the nonunion. One of the chambers contains a clotting agent (calcium and thrombin, or ecarin), while the second chamber contains whole blood alone or in combination with exogenous growth factors (e.g., BMP2, PDGF, VEGF) and / or bone substitutes (e.g., DBM, allogeneic cancellous bone chips). During application, when mixed as it enters the needle, the interaction of these components forms an extracorporeal hematoma, initiating the bone healing process. After the extracorporeal hematoma has been delivered to the nonunion site, the cannula is removed and the incision is closed with sutures. This procedure reduces the patient's pain, limits the risk of infection and other complications, minimizes the operative time, and shortens the time it takes for the patient to recover and return to normal activities.

[0141] Example 5. Structural and biological characteristics of hematomas. Approaches to healing large segmental bone defects. It is well established that hematomas formed at the fracture site have a significant impact on how the fracture heals. For example, studies show that removal of the hematoma slows fracture healing (Schell H, Peters A, Duda GN. Removal of fracture hematoma and replacement with fresh hematoma delays bone healing in sheep. Bone. 2012). Furthermore, some reports suggest that structural properties of the formed fibrin clot, such as the porosity and thickness of the fibrin fibers, influence bone repair (Wang X, Friis TE, Masci PP, Crawford RW, Liao W, Xiao Y. Alteration of blood clot structures by interleukin-1 beta in association with bone defects healing. Sci Rep. Nature Publishing Group; 2016; 6: 35645; and Wang X, Friis T, Glatt V, Crawford R, Xiao YJ Tissue Eng Regen Med. 2017; 11: 2864-2875.). The results described herein demonstrate that in vivo hematomas isolated from 0.5 mm rat femoral defects (normally healing fractures) had 35% thinner fibrin fibers (209 ± 20 nm) compared to 5 mm segmental bone defects (non-healing without intervention, 320 ± 64 nm) 3 days after surgery. Furthermore, a less porous network was also observed in 5 mm defects compared to 0.5 mm (42.56% vs. 50.03%), resulting in a difference of 16% between the groups.To investigate whether there were differences in biological properties between hematomas formed in large bone defects (5 mm) and normally healing fractures (0.5 mm), an in vivo study was performed using RNA sequencing analysis.

[0142] Major differences were found in genes mediating the inflammatory response (e.g., Il1b-produced by activated macrophages, Sdf1-expressed in areas of inflammatory bone destruction and mediating an inhibitory effect on osteoclast formation), which were mainly upregulated in 5 mm vs. 0.5 mm defects. Genes important for structural components of the extracellular matrix (ECM) (e.g., Col1a1, Col2a1, Col3a1), ECM proteases (e.g., Mmp2) and their inhibitors (e.g., Timp1) were mostly downregulated. In contrast, genes regulating cell adhesion molecules (e.g., Thbs1) were upregulated. Analysis of genes involved in the process of angiogenesis shows that angiogenin (Ang), a potent stimulator of new blood vessel formation, is downregulated, while endothelin (Edn1), an angiogenic cytokine that is a potent vasoconstrictor that also stimulates cells of the osteoblast lineage, is upregulated. The expression of genes related to bone formation demonstrated a significant portion of downregulated genes involved in osteoblast differentiation (e.g., Bglap, also known as osteocalcin - secreted by osteoblasts to initiate bone repair) and bone formation (e.g., Bmp7 - plays a key role in bone transformation).

[0143] These results are the first to demonstrate important differences in gene expression between normally healing fractures and large bone defects during the early stages of bone healing. The most striking differences were found in gene sets involved in the inflammatory response, a key event after fracture. The upregulation of inflammatory genes in 5 mm vs. 0.5 mm defects suggests that large bone defects induce a stronger inflammatory response than normal fractures, leading to increased recruitment of macrophages, fibroblasts, MSCs, and osteoprogenitor cells. The invaded inflammatory cells also produce proangiogenic cytokines, which explains the upregulation observed when comparing 5 mm vs. 0.5 mm defects. At the same time, the downregulation of many genes important for skeletal development, bone mineral metabolism, and ECM formation suggests that the osteogenic response is reduced in large bone defects.

[0144] In vivo studies were also performed to determine whether SVCE, ecarin, could be used to generate in vitro clots with specific structural properties and whether ecarin was toxic to stem cells. The results convincingly showed that the structural properties of the clots changed with the concentration of ecarin. For example, higher concentrations of ecarin resulted in thinner fibrin fibers with an average thickness of 93 ± 3 nm, whereas at the lowest concentration, the fibrin fibers were 173 ± 9 nm thick. The cell proliferation rate from 1 to 7 days decreased with increasing concentrations of ecarin. For example, there was a 14.8 ± 2.6-fold increase without ecarin, compared with a 4.3 ± 0.7-fold increase at the highest concentration and a 13.7 ± 3.1-fold increase at the lowest concentration. Cells cultured within the in vitro hematoma showed stable cell numbers over 7 days. Thus, these results suggest that ecarin does not cause toxicity but may decrease cell proliferation rates at higher concentrations.

[0145] Based on these observations, additional experiments will be performed to determine the structural and biological properties of hematomas (which heal) formed in normal fractures and compare them to large bone defects that are non-healing without intervention (see Examples below). For example, well-organized fibrin clots will be studied for their ability to promote bone healing by acting as a temporary reservoir for the continuous release of growth factors and providing an appropriate space to support cell infiltration, proliferation, and differentiation. Thus, a major opportunity to enhance the repair process lies in the generation of clots that mimic the intrinsic properties of healing fracture hematomas. The structural properties of hematomas generated in vitro can be modified using SVCE, thereby mimicking endogenous fracture hematomas, and when implanted into segmental bone defects, bone healing is enhanced and accelerated. Various snake venoms are used successfully in patients with heart disease, cancer, and stroke, as well as for diagnostic purposes in diseases such as lupus. These experiments will therefore also be the first to examine the use of the snake venom enzyme ecarin as an alternative clotting agent compared to the addition of bovine thrombin, which has been shown to have side effects when used clinically (Diesen DL, Lawson JH. Vascular. 2008;16:S29-36; Ofosu FA, Crean S, Reynolds MW. Clin Ther. 2009;31:679-691; and Sands JJ, Nudo SA, Ashford RG, Moore KD, Ortel TL. Am J Kidney Dis. 2000;35:796-801). This approach will be considered as an improved therapeutic strategy, as it is a biomimetic scaffold that can be used to more reliably promote bone healing by either completely eliminating the need for growth factors such as BMPs or by drastically minimizing the doses required to enhance the bone repair process. The result will be a more innate therapeutic strategy than currently available, which will result in significant cost savings, and most importantly, will eliminate many of the adverse side effects associated with high doses of BMPs. Furthermore, the results described herein can have a significant impact on the treatment of bone injuries in military personnel as well as the civilian population.

[0146] Example 6. Structural and biological characteristics of intracorporeal fracture hematomas. Bone defects were created in groups of male SAS Fischer rats (10-12 weeks old, n=38, n=5-8 / group) and stabilized with an external fixator (RISystem AG; Glatt V, Evans CH, Matthys R. Eur Cell Mater. 2012;23:289-98;discussion 299; and Glatt V, Miller M, Ivkovic A, Liu F, Parry N, Griffin D, et al. J Bone Joint Surg Am. 2012 Nov 21;94(22):2063-73) to characterize and compare the structural and biological properties of hematomas (0.5 mm) formed during normal bone healing with large segmental bone defects (5 mm). To assess the progress of fracture healing, a 0.5 mm osteotomy was performed, which was possible using the same external fixator. The reason for using osteotomy is that it is reproducible and allows for the formation of hematomas of more consistent size, which is important for the characterization of the structural properties of the hematoma. To evaluate the structural properties of the fracture hematoma after the clot has matured, the animals were killed on the third day after surgery. The structural properties of the hematoma, such as the thickness, density, and porosity of the fibrin fibers, were evaluated using scanning electron microscopy (SEM, n=8 / group) and ImageJ software. In addition, a different set of samples was used to analyze the differentially expressed genes involved in the initiation of the bone repair process using RNA sequencing (n=6 / group). Histology and immunohistochemistry (IHC) (n=5 / group) are also performed to characterize the tissue and confirm the presence of key proteins involved in the initiation of the repair process, such as macrophages (CD68, CD40, CD206) Osterix, PECAM1, vWF, VEGF, type I collagen, and H&E for the overall tissue morphology. The results were used to determine whether there was a correlation between structural characteristics and the expression of specific genes and proteins in 3-day old hematomas.

[0147] Methods: Rat, critical size defect and osteotomy model. Male SAS Fischer rats weighing approximately 200-250 g (10-12 weeks old) were anesthetized by administering isoflurane (2%, 2 liters / min) using a small animal vaporizer. The animals were then injected intramuscularly in the left femur with 20 mg / kg cefazolin (antibiotic) and 0.08 mg / kg buprenorphine (analgesic). A detailed surgical procedure can be found (Glatt V, Matthys R. Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models. J Vis Exp. 2014). Briefly, the right hind leg of each animal was shaved, disinfected with chlorhexidine, placed on a sterile field and covered with a sterile surgical drape exposing only the right leg. An incision of approximately 3.5–4 cm was made through the skin running anterolaterally over the surface of the right femur from the greater trochanter to the supracondylar region of the knee. The femoral shaft was exposed by gentle dissection between the quadriceps and hamstring muscles. The external fixator bar was first clipped to the Gigli wire saw guide and then placed on the anterior-posterior aspect of the femur to guide the drill and allow reproducible positioning of the four drill holes using a pen drill (RISystem AG, Davos Platz, CH). Mounting pins were inserted one at a time into the pre-drilled holes, starting from the proximal side. After the fixator was in place, the defect was created using the saw guide. For this, a Gigli wire saw was passed through two grooves under the femur to create a 5 mm segmental defect by a back-and-forth reciprocating motion, and a 0.5 mm defect was created using one wire saw. When the defect was created, the saw guide was removed and the wound was closed in several layers. Rats received analgesics every 12 hours and antibiotics every 24 hours for 3 days after surgery. Hematomas were collected on day 3 for structural and biological analysis.

[0148] Scanning electron microscopy. Intracellular fracture hematomas and extracellular thrombi were treated similarly. Samples were fixed overnight in 4% paraformaldehyde. Gross morphology was captured at 100-1,000 magnification using a Hitachi SU1510 VP-SEM. To analyze fiber diameter and density, samples were post-fixed in 4% osmium tetroxide and dehydrated in a gradient of ethanol solutions (25-100%). Slices of hematomas and thrombi were then dried using a Leica EM Critical Point Dryer, mounted on silicon chip specimen supports, sputter-coated with gold-palladium, and imaged at 10,000x (Hitachi S5500 SEM / STEM) to reveal structural features at high resolution. Images were analyzed using ImageJ.

[0149] RNA sequencing. Parallel sequencing of RNA (RNA-Seq) is a high-throughput method that can comprehensively measure gene transcript abundance (Wang Z, Gerstein M, Snyder M. RNA-Seq: A revolutionary tool for transcriptomics. 2009. pp. 57-63). To investigate differential expression of genes in fracture hematomas, samples were collected in microcentrifuge tubes, immediately flash frozen in liquid nitrogen, and stored at -80°C. RNA extraction was performed using QIAGEN RNeasy Plus Universal Tissue Mini (QIAGEN, Inc., Germantown, MD, USA) according to the manufacturer's protocol. RNA concentration and quality were measured using a Nanodrop spectrophotometer (ND-1000, Thermo Fisher Scientific, Inc.), and RNA integrity was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. Bone cylinders removed to create a 5 mm bone defect were used as controls to represent the gene expression state of healthy bone. Global transcriptome analysis was used to identify up- and / or down-regulated genes that significantly impact the initiation process of bone repair. This work was performed using an Ilumina HiSeq 3000.

[0150] Histology and immunohistochemistry. Samples are stained with hematoxylin and eosin to observe the overall tissue morphology. Standard immunohistochemistry protocols are applied to paraffin-embedded sections (5 μm). Sections are labeled with a panel of antibodies to determine the spatial expression of proteins such as macrophages (CD68 and CD206), osterix, PECAM1, collagen type II, and collagen type X (Maes C, Kobayashi T, Selig MK, Torrekens S, Roth SI, Mackem S, et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell. Elsevier Ltd; 2010; 19: 329-344).

[0151] To perform these experiments, 5 mm and 0.5 mm femoral defect models were used and stabilized with an external fixation device as described. This is a well-established research model. The primary outcome measures used were scanning electron microscopy, RNA sequencing, and histology / immunohistochemistry, which are routine procedures. As disclosed herein, data using SEM and RNA sequencing showed that there were discernible differences in structural and biological properties between the hematomas formed in the 0.5 mm defect compared to the 5 mm defect.

[0152] Scanning electron microscopy images show the structural characteristics of fracture hematomas. For example, clear structural differences are observed between normally healing fractures (0.5 mm) and large bone defects (5 mm). More specifically, normal fractures (0.5 mm) exhibited high porosity, low density, thin fibrin fibers, and a rougher surface, while large bone defects (5 mm) exhibited low porosity, high density, thick fibrin fibers, and a smooth surface.

[0153] Example 7. Structural characterization of thrombi / hematomas formed in vitro using snake venom clotting enzymes. SVCE, ecarin, was used to modify the structural properties of the clot to mimic the properties of natural hematomas formed in a 0.5 mm osteotomy model using various concentrations of ecarin. Whole blood was collected by cardiac puncture using a 21-gauge needle at the time of euthanasia from the same animals used in Example 4. The expected blood yield was approximately 5-10 mL per animal. To prevent clotting, blood was mixed with 1 part 4% sodium citrate solution to 9 parts blood. Ecarin, an enzyme purified from saw-tooth snake venom, was purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA). Variables such as pH, ionic strength, and calcium were kept constant, and final concentrations of the prothrombotic enzyme were applied in the picomolar to nanomolar range. Calcium chloride (CaCl2) and CaCl2 + recombinant human thrombin were used as controls. The ex vivo clot is designed to be cylindrical with a height of 5 mm spanning the defect size and a diameter of 4 mm matching the average diameter of the rat femur. Clots of 0.5 mm in height were used as a control to determine whether the addition of the same concentration of SVCE to a smaller volume of blood would result in the same structural characteristics in the clots formed. Structural changes, specifically the thickness and density of fibrin fibers, and overall clot structure, were assessed using SEM. Using the methods described herein, rat clots can be observed with different structural characteristics.

[0154] Preparation of ex vivo clots. To generate ex vivo clots, 5–10 mL of whole blood was collected from anesthetized rats by cardiac puncture at the time of euthanasia. Immediately after collection, blood was mixed 9:1 with 4% sodium citrate solution to prevent clotting. Various concentrations of ecarin were used to induce blood clotting. Samples were left at room temperature for 2 h to allow complete clotting. Ex vivo clots were subsequently fixed in 4% PFA overnight at 4 °C before being processed for scanning electron microscopy.

[0155] Scanning electron microscopy. Intracorporeal fracture hematomas and excorporeal thrombi were treated in essentially the same way. Samples were fixed overnight in 4% paraformaldehyde. Gross morphology was captured at 100-1,000 magnification using a Hitachi SU1510 VP-SEM. To analyze fiber diameter and density, samples were postfixed in 4% osmium tetroxide and dehydrated in a gradient of ethanol solutions (25-100%). Slices of hematoma and thrombi were then dried using a Leica EM Critical Point Dryer, mounted on silicon chip specimen supports, sputter-coated with gold-palladium, and imaged at 10,000x (Hitachi S5500 SEM / STEM) to reveal structural characteristics at high resolution. Images were analyzed using ImageJ. In the SVCE disclosed herein, varying concentrations of ecarin were used to modify the structural properties of the thrombus (ex vivo hematoma) using either whole blood or platelet-rich plasma (PRP) to mimic the properties of the hematoma formed in a 0.5 mm osteotomy model. Ecarin (0.1 and 0.5 U / mL) or CaCl2 (10 mM) were used as coagulants. The results disclosed herein demonstrated that it is possible to generate ex vivo hematomas with the required structural properties by varying the concentration of ecarin using either whole blood or PRP.

[0156] Scanning electron microscopy images show the structural characteristics of ex vivo snake venom-induced hematomas. These results demonstrate that clot morphology can be manipulated using snake venom enzymes or calcium chloride to affect the thickness and density of fibrin fibers, and that the structural characteristics of ex vivo hematomas differ from those created with whole blood compared to those created with PRP.

[0157] Example 8, Determine whether an extracorporeal hematoma provides a viable environment for mesenchymal stem cells (MSCs). These experiments are performed to determine the ability of MSCs to survive in the presence of ecarin and when seeded within ecarin-induced clots, and to evaluate the biocompatibility of these scaffolds. Cell viability and potential cytotoxicity of ecarin at various concentrations are measured in 2D and 3D assays. Rat bone MSCs are cultured in the presence of different concentrations of ecarin to determine cell viability. Cell proliferation (e.g., CyQUANT™ Cell Proliferation Assay Kit) and cytotoxicity (e.g., Vybrant™ Cytotoxicity Assay Kit) are measured on days 1, 7, 14, and 21. Ecarin is then added to the citrated blood mixture at a concentration established in the previous experiment, taking into account the cell survival along with the specific structural properties determined. After clotting, the clots are transferred to a 24-well plate containing growth medium.

[0158] To test the biocompatibility of ecarin-induced clots, samples are removed from the cultures on days 1, 7, 14, and 21 (n=3 / group) to study the viability and differentiation capacity of the seeded cells, as well as their ability to form extracellular matrix. Furthermore, the number of cells in the clot required to maximize the regenerative potential of bone tissue is determined. Cell viability within the clot is assessed using the LIVE / DEAD® cell viability assay and confocal imaging.

[0159] To test the differentiation ability of the cells, the cells are cultured in adipogenic, chondrogenic, and osteogenic differentiation media, and qRT-PCR is used to determine the differential expression of genes at selected time points. Once cell viability is established, the hematoma, which most closely resembles the structural and biological characteristics of spontaneous hematomas during bone healing with a 0.5 mm defect / osteotomy, is implanted into an in vivo rat 5 mm femoral defect model to study their ability to heal large segmental bone defects. Based on the results in Figure 5A, Figure 5B, it is expected that the use of a lower concentration of the clotting enzyme, ecarin (<0.5 U / mL), will be less toxic to the cells.

[0160] Cell culture. Rat bone marrow stem cells are cultured according to standard protocols. The medium is changed every 3-4 days.

[0161] 2D cell viability assay. PrestoBlue™ Cell Viability Reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) is added directly to the culture medium of rat bone marrow stem cells grown in well plates on days 1, 3, and 7. After 20 min of incubation at 37 °C, fluorescence is read on a multiplate reader according to the manufacturer's instructions.

[0162] 2D cytotoxicity assay. Rat bone marrow stem cells are cultured in 96-well plates. Supernatants are collected on days 1, 3, and 7 and used to measure lactate dehydrogenase (LDH) cytotoxicity according to the manufacturer's instructions (Pierce LDH Cytotoxicity Assay Kit, Thermo Fisher Scientific AG, Reinach BL, Switzerland). Using a microplate reader, measure the absorbance at 490 nm and subtract background at 680 nm.

[0163] 3D in vitro clots and live / dead staining in confocal microscopy. Rat bone marrow stem cells are cultured in 3D clots. On days 1, 3, and 7, clots are removed from the medium, cut in half sagittally, and then immersed in serum-free medium containing 10 μM calcein AM stock and 1 μM ethidium homodimer-1 (Thermo Fisher Scientific, Inc.) in a 24-well plate. After 3 h of incubation at 4 °C and 1 h at 37 °C, 5% CO2, and 100% humidity, samples are imaged to a depth of 200 μm using a confocal microscope (Gantenbein-Ritter B, Sprecher CM, Chan S, Illien-Junger S, Grad S. Confocal imaging protocols for live / dead staining in three-dimensional carriers. Methods Mol Biol. 2011;740:127-40).

[0164] 3D cell differentiation assay. Cell-seeded clots are cultured in either adipogenic, chondrogenic, or osteogenic media (StemPro® Differentiation Kits, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cell differentiation is assessed using qRT-PCR and customized TaqMan® PCR array plates (Thermo Fisher Scientific, Inc.).

[0165] Real-time quantitative PCR (qRT-PCR). Ex vivo clots are collected in microcentrifuge tubes, immediately flash frozen in liquid nitrogen, and stored at -80 °C. RNA extraction is performed using QIAGEN RNeasy Plus Universal Tissue Mini (QIAGEN, Inc., Germantown, MD, USA) according to the manufacturer's protocol. RNA concentration and quality are measured with a Nanodrop spectrophotometer (ND-1000, Thermo Fisher Scientific, Inc.), and RNA integrity is assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. One microgram of extracted RNA was reverse transcribed into cDNA using the TaqMan™ High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Inc.), and then the expression of inflammation-, angiogenesis-, and osteogenesis-related genes was analyzed using customized TaqMan® PCR array plates (Thermo Fisher Scientific, Inc.).

[0166] As disclosed herein, the ability of bone marrow MSCs to survive in the presence of ecarin and bone marrow mesenchymal stem cells when seeded within ecarin-induced thrombi will be further studied to evaluate the biocompatibility of these scaffolds. The use of various concentrations of clotting enzymes from snake venom was not toxic to MSCs added to the ex vivo thrombi. The enzyme ecarin is highly purified. Therefore, it is expected that ecarin will not be toxic to migrating cells when the ex vivo hematoma is implanted in the body. Furthermore, in vivo experiments in a rat model were performed, demonstrating that two different concentrations of ecarin (0.3 and 0.6 U / ml) do not cause toxicity. However, since higher concentrations are required to generate thrombi with specific structural and biological properties for human subjects, if ecarin proves to be toxic to cells, calcium chloride or other clotting factors described herein will be considered as clotting agents. As mentioned herein, thrombin has been used previously to activate the coagulation cascade in platelet-rich plasma, but this product performed poorly in bone repair (Diesen DL, Lawson JH. Vascular. 2008; and Sands JJ, Nudo SA, Ashford RG, Moore KD, Ortel TL. Am J Kidney Dis. 2000; 35: 796-801). This may be related to the fact that the thrombin added to the PRP was used purely as an activator, without consideration of the structural properties of the clot. In some embodiments, growth factors such as VEGF, PDGF, hFGF-2, BMP-2 (and other BMPs) can also be added in small amounts.

[0167] Scanning electron microscopy images show the structural characteristics of an in vitro hematoma induced by snake venom in vitro. These results indicate that cell viability was not significantly affected by the presence of snake venom enzymes, suggesting biocompatibility.

[0168] Example 9, to investigate whether an ex vivo generated extracorporeal hematoma inserted into a large bone defect can promote the bone regeneration process in a 5 mm large critical size defect model in the rat femur. Groups of male SAS Fischer rats (10–12 weeks old, n=8–16; pilot study n=4; external hematoma, BMP-2, PRP) had 5 mm femoral defects created and stabilized with the external fixator described herein. External hematoma was implanted into the fracture gap to determine its ability to promote healing of large segmental bone defects. Two control groups were used to compare the bone healing process to the experimental groups. Healing in the first control group was enhanced using recombinant human BMP-2 delivered on an absorbable collagen sponge, the same product currently used in clinical practice (Infuse®, Medtronic plc., Minneapolis, MN, USA).This is a well-established research model using BMP-2, with healing reported within 8 weeks (Glatt V, Bartnikowski N, Quirk N, Schuetz M, Evans C. Reverse Dynamization: Influence of Fixator Stiffness on the Mode and Efficiency of Large-Bone-Defect Healing at Different Doses of rhBMP-2. J Bone Joint Surg Am. 2016;98:677-87; Yasko AW, Lane JM, Fellinger EJ, Rosen V, Wozney JM, Wang EA. The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. J Bone Joint Surg Am. The American Orthopedic Association;1992;74:659-70; and Gantenbein-Ritter B, Sprecher CM, Chan S, Illien-Junger S, Grad S.Confocal imaging protocols for live / dead staining in three-dimensional carriers.Methods Mol Biol.2011;740:127-40), therefore reducing the number of animals used (n=4). In the second control group, PRP was used to determine whether an extracorporeal hematoma would provide superior healing results compared to a fibrin clot enriched with high platelets. In the experimental group, an extracorporeal hematoma was used to determine whether the created scaffold had the ability to regenerate the bone defect. The advantages of an extracorporeal hematoma are:(1) Osteoinductive - key growth factors persist for a long time to stimulate new bone formation, and (2) osteoconductive - the well-organized fibrin structure creates a favorable microenvironment for mesenchymal stem cell migration and early mineralization. The success of this series of proof-of-concept experiments is expected to lead to the development of extracorporeal hematomas that act as natural growth factor reservoirs, as well as biocompatible autologous scaffolds that improve the healing of large bone defects without the addition of growth factors such as rhBMP-2. To evaluate this, animals were monitored weekly by X-ray and euthanized at 8 weeks. After euthanasia, healed defects were harvested for evaluation by micro-computed tomography (μCT, all samples), histology / IHC (n=4 / group, BMP-2 group n=2), and biomechanical testing (n=12 / group, BMP-2 group n=6).

[0169] Surgery. Rat surgeries were performed as described herein.

[0170] Preparation of ex vivo hematomas for implantation. To generate clots ex vivo, 5–10 mL of whole blood was collected from anesthetized rats by cardiac puncture at the time of euthanasia. Immediately after collection, blood was mixed 9:1 with 4% sodium citrate solution to prevent clotting. Ecarin was used at pre-determined concentrations of 0.3 and 0.6 U / ml in combination with 0.55 μg of rhBMP-2 to induce blood clotting. Samples were allowed to clot for 45 min to 1 h at 22°C (room temperature) before implantation into a 5 mm rat bone defect.

[0171] Preparation of platelet-rich plasma (PRP) and BMP-2. To prepare PRP from rat blood, 5–10 mL of whole blood was collected from anesthetized rats by cardiac puncture at the time of euthanasia. Immediately after collection, blood was mixed 9:1 with 4% sodium citrate solution to prevent coagulation. Whole blood was centrifuged at 150 x g for 10 min at room temperature with a soft brake to separate the platelet layer from the plasma and red blood cells. The lower red blood cell layer was discarded, and the middle platelet layer and the upper plasma layer were collected. Platelet counts in whole blood and PRP were measured using a cell counting chamber to check the quality of PRP. Calcium chloride was added to create a PRP gel, which was then embedded into the defect gap. Recombinant human BMP-2 (5.5 μg) was applied to an absorbable collagen sponge carrier (Infuse™ Bone Graft, Medtronic, Minneapolis, MN, USA) in the shape of a bone defect and used as an implant.

[0172] Evaluation of bone healing. Femurs from each group of 16 animals are radiographed weekly and evaluated in vivo by μCT after euthanasia. Twelve samples undergo biomechanical testing and four are used for histology. In the pilot study, four animals per group were used.

[0173] X-rays. Bone healing was monitored weekly by radiography. Under general anesthesia as described in the surgical procedure, rats were placed in a ventral position with the hindlimb rotated laterally to obtain reproducible standardized images perpendicular to the defect.

[0174] Micro-computed tomography (μCT). Femurs are scanned using a tabletop micro-tomography imaging system (Bruker Skyscan 1172, Belgium) equipped with a microfocus X-ray tube with a 10 mm focal spot. Femurs are scanned with approximately 600 μCT slices per specimen using an isotropic voxel size of 16 μm at an energy of 75 keV and an integration time of 250 ms. The evaluation is applied to a central defect area of ​​4 mm, ensuring that the existing cortical bone is not included in the analysis. To evaluate the region of interest, the total cross-sectional volume of the defect (TV, mm 3 ), bone volume (BV, mm3 ), bone volume fraction (BV / TV, %), bone density (BMD, mg HA / ccm), polar moment of inertia (pMOI, mm 4 ) variables are assessed. Images are thresholded using an adaptive iterative algorithm and morphometric variables are calculated from the binarized images using a direct 3D technique that does not rely on a priori assumptions about the underlying structure.

[0175] Mechanical testing. After non-invasive imaging, 12 specimens from each group undergo torsional fracture testing. The ends of each specimen are embedded in polymethylmethacrylate to provide a suitable and reproducible gripping interface with the testing module. Specimens are tested to failure under normal deformation control at a constant deformation rate of 5 rad / min. Angular deformation and applied load data are acquired at 10 Hz. Torque and rotation data are used to calculate the torsional stiffness and strength of the repaired defects.

[0176] Histology of bone samples. Femurs (n=4) are fixed in ice-cold 4% paraformaldehyde for 48 hours and then decalcified in 14% EDTA for up to 4 weeks. Pins are removed from the bones before embedding and sectioning. Fixed and decalcified tissues are dehydrated in graded ethanol up to 100%, transferred to xylene and embedded in paraffin. Five micron paraffin sections are placed on poly-L-lysine-coated slides and either dried overnight and stained immediately or stored at 4°C. Separate sections are stained with hematoxylin and eosin or Safranin O and fast green before examination under a light microscope. Safranin O is included to stain cartilage as part of monitoring the endochondral ossification process.

[0177] Power analysis and statistics. Sample sizes for all individual groups were based on a coefficient of variation of 15% for the type of data collected, using an alpha level of 0.05 and a power of 80% (beta = 0.20). Power analysis revealed that n = 8–16 animals per group would allow for detection of significant differences between groups for each outcome parameter, based on an effect size of 1.3 using Student's t-test. Previous experience with these rat models confirms that an n of 10 would provide sufficient statistical power. In vivo experiments are performed in triplicate and compared for statistical significance using ANOVA tests. Sample size and power calculations were determined using the nQuery Advice software program, version 4.0 (Statistical Solutions, Boston, MA). Statistical analyses are performed using SAS version 6.12 software (SAS Institute, Cary, NC). A two-tailed p < 0.05 is considered statistically significant.

[0178] Experiments to determine the dose response in vivo are important for creating extracorporeal hematomas with specific structural properties that mimic those of naturally healing fracture hematomas.If healing cannot be achieved using extracorporeal clots alone, the extracorporeal hematomas will be combined with either rat bone marrow mesenchymal stem cells, rhBMP-2 in significantly reduced amounts compared to the supraphysiological doses currently used in clinical practice, or other growth factors.

[0179] As disclosed herein, the in vivo results of the in vivo study at the end of the 8-week and 4-week bone defect healing periods (groups with 0.6 U / ml + 0.55 μg BMP-2) clearly show that whole blood + ecarin (0.1 U / mL) and platelet-rich plasma (PRP) + CalCl2 (10 mM) do not promote bone healing / regeneration at the concentrations of clotting factors tested. In contrast, 5 mm rat femoral defects were healed when 0.3 U / mL ecarin and 1.1 μg or 0.55 μg BMP-2 were added to the defect. It is important to note that BMP-2 delivered on a collagen sponge sold by Medtronic (Infuse™) using the same amount of BMP-2 did not initiate healing of the bone defect. Interestingly, when 0.6 U / mL ecarin + 0.55 μg BMP-2 was used, healing was much better compared to that observed with the lower concentration of ecarin. These results seem to indicate that the ultrastructural characteristics of blood clots (external hematomas) have a significant impact on promoting healing of bone defects.

[0180] Example 10. Biomimetic hematoma: Effect of coagulant and rhBMP-2 concentration on rhBMP-2 / ACS on bone healing. Various concentrations of the coagulants, ecarin, calcium / thrombin, and BMP-2, were tested and demonstrated the ability to initiate healing of large segmental bone defects. Concentrations of ecarin tested included 0.3, 0.6, and 0.75 U / mL, which successfully initiated healing, however, an ecarin concentration of 0.6 U / mL showed the best results among the doses tested. Similarly, a combination of the coagulants, 10 mM CaCl2 and 0.5 U / mL thrombin, also effectively healed large bone defects in a similar manner as 0.6 U / mL ecarin and 0.33 μg BMP-2. The concentration of BMP-2 that consistently initiated healing of 5 mm bone defects in this rat model was 0.33 μg. This dose is 33-fold lower than the standard dose of 11 μg, and this dose is 17-fold lower than the lowest effective dose of BMP-2 / ACS (ACS = absorbable collagen sponge) that efficiently heals large segmental bone in a 5 mm femoral defect rat model (5.5 μg). The other two doses tested to initiate bone defect healing were 0.165 μg and 0.0825 μg, however the response at these doses was less consistent, 75% and 50%, respectively, compared to 0.33 μg.

Claims

1. 1. A composition comprising: 1) an extracorporeal hematoma, the extracorporeal hematoma comprising (a) isolated whole blood, (b) sodium citrate, and (c) calcium chloride, thrombin, or thrombin and calcium chloride; 2) a bone substitute, wherein the bone substitute is a demineralized bone matrix or is derived from a biological product, a synthetic bone substitute, or a combination thereof; The composition comprising:

2. The composition of claim 1, wherein the extracorporeal hematoma comprises fibrin fibers having a thickness of at least 150-300 nm ± 10%.

3. 10. The composition of claim 1, wherein the biological product is demineralized bone matrix, hydroxyapatite, or coral.

4. 10. The composition of claim 1, wherein the synthetic bone substitute is calcium sulfate, calcium phosphate cement, β-tricalcium phosphate ceramic, bioactive glass, or a polymer.

5. The composition of claim 1 , wherein the extracorporeal hematoma further comprises an antibiotic.

6. 10. The composition of claim 1, wherein the extracorporeal hematoma further comprises one or more growth factors, wherein the one or more growth factors are bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4, BMP-6, BMP-9, BMP-14, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), or a combination thereof.

7. The composition of claim 1 , wherein the whole blood comprises viable cells and one or more biological factors.

8. 8. The composition of claim 7, wherein after the formation of the hematoma, about 50% to 70% of the viable cells in the whole blood remain viable.

9. The composition of claim 1 , wherein the extracorporeal hematoma further comprises a therapeutic agent.

10. 10. The composition of claim 1, wherein the extracorporeal hematoma comprises isolated whole blood, calcium chloride, thrombin, and sodium citrate.

11. 10. The composition of claim 9, wherein the therapeutic agent is bone morphogenetic protein 2 (BMP-2).

12. The composition of claim 11, wherein the BMP-2 present in the extracorporeal hematoma is at a dose of at least 0.01 mg.

13. The composition of claim 1 , wherein the composition is formulated as a gel, liquid, powder, paste, granules, or putty.

14. The composition of claim 1, wherein the ratio of the extracorporeal hematoma to the bone substitute is 1000:1 to 1:1000.

15. 10. Use of the composition of claim 1 in a method for promoting bone healing or for producing a bone substitute material or implant in a subject in need thereof.

16. 16. The use of the composition of claim 15, wherein the composition is formulated as a clot or scaffold.

17. 16. The use of the composition of claim 15, wherein the composition is administered topically, implanted, or delivered transdermally.

18. 16. The use of the composition of claim 15, wherein the subject has a skeletal abnormality, a bone defect, one or more fractures, a non-union of a fracture, or one or more bone injuries.

19. The composition of claim 1, further comprising a scaffold.

20. The composition of claim 19, wherein the scaffold is PEEK, titanium, or a metal alloy.

Images