Compositions and methods for treating bone injury

Abstract

To provide compositions and methods of treating, improving, and accelerating the healing of large segmental bone defects in a subject.SOLUTION: The methods comprise administering to a patient in need of treatment an effective amount of whole blood, sodium citrate, ecarin and BMP-2.SELECTED DRAWING: None

Description

Background Art

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 732,534, filed on September 17, 2018, and U.S. Provisional Patent Application No. 62 / 845,500, filed on May 9, 2019. The entire contents of these previously filed applications are hereby incorporated by reference in their entirety.

[0002] On average, 7.9 million fractures occur each year in the United States alone, and about 5 - 10% result in delayed union, non - union, sub - critical size 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 the use of certain medications can greatly contribute to fracture healing problems (Zura, et al., JAMA Surg. 2016 Nov 16;151). The cost of treating non - union of the tibia, femur, or humerus ranges from $31,500 to $34,400 in each case, representing a significant burden on annual healthcare costs (Kanakaris NK, Giannoudis PV. Injury. 2007;38 Suppl2:S77 S84 and Wu, et al Orthopedic Research and Reviews. 2013:5 21 - 33).

[0003] Furthermore, more than 59,000 military personnel were wounded in combat-related operations while serving in Iraq and Afghanistan, 50% of whom had 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 the musculoskeletal injuries sustained in these conflicts were major limb injuries (Stansbury LG, Lalliss SJ, Branstetter JG, Bagg MR, Holcomb JB. Amputations in U.S. military personnel in the current conflicts in Afghanistan and Iraq. J Orthop Trauma. 2008;22:43-46). Such bone injuries, particularly those associated with high-energy blast injuries due to contaminated bone and soft tissue destruction combined with locally decreased tissue vascularity, are poorly healed. Even without such complications, bone defects exceeding critical size do not have the inherent ability to heal. The management of patients with major segmental bone defects remains one of the most difficult clinical problems faced by 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). These devastating injuries often carry a significant risk of failure and ultimate amputation, inevitably resulting in a long course of costly and ongoing medical care, so the importance of improving the capabilities of clinicians to manage these injuries cannot be overemphasized.As a result of military activities (OIF / OEF) in Iraq and Afghanistan, over 2,000 soldiers required at least one amputation (Fischer H. A Guide to U.S. Military 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 delays in returning to work or military duty, and accordingly, the quality of life of the individual is reduced. Thus, there is clearly an unmet clinical need, which serves as a strong motivation for the development of more effective treatment strategies to address these potentially devastating injuries.

Summary of the Invention

[0004] Disclosed herein is an ex vivo hematoma comprising (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, octacarin, and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, wherein the ex vivo hematoma comprises fibrin fibers having a thickness of at least 150 - 300 nm ± 10%.

[0005] Disclosed herein is an ex vivo hematoma comprising (a) platelet-rich plasma, plasma, or plasma having red blood cells, and (b) ecarin, octacarin, and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, wherein the ex vivo hematoma comprises fibrin fibers having a thickness of at least 150 - 300 nm ± 10%.

[0006] Disclosed herein is a method for constructing an implant, the method comprising: a) sizing a depot implant in at least one of a shape and 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 having red blood cells, and (ii) echalin, octacalin, and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, thereby creating a scaffold, the scaffold having a porosity of 55-75%.

[0007] Other features and advantages of the compositions and methods of the present invention are illustrated in the following description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0008]

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DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure can be more readily understood by reference to the following detailed description of the invention, drawings, and examples included herein.

[0010] Before the compositions and methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or to specific reagents unless otherwise specified, as such, of course, may 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. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but exemplary methods and materials are described hereinbelow.

[0011] Furthermore, it should be understood that, unless otherwise specified, none of the methods described in this specification are ever intended to be construed as requiring that their steps be performed in a particular order. Thus, where a method claim does not actually recite the order to follow for its steps, or where the steps are not otherwise particularly recited in the claims or specification as being limited to a particular order, in no way is an order to be inferred. This applies to any possible implicit basis for interpretation, including logical issues regarding the arrangement of steps or operation flows, the clear meaning derived from grammatical construction or punctuation, and the number or type of aspects described in this specification.

[0012] All publications mentioned in this specification are hereby incorporated by reference into this specification to disclose and explain the methods and / or materials related to the cited publications. The publications discussed in this specification are provided only for their disclosures prior to the filing date of this application. Nothing in this specification should be construed as an admission that the present invention has no right to antedate such publications by virtue of prior invention. Further, the dates of the publications provided in this specification may be different from the actual publication dates, and these may require independent verification.

[0013] Definitions 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.

[0014] The word "or" as used in this specification means any one member of a particular list and also includes any combination of members of that list.

[0015] Throughout the description and claims of this specification, the word "comprising", 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, in a method described as including one or more steps or operations, it is specifically contemplated that each step includes what is listed (except when that step includes a limiting term such as "consisting of"), which means that each step is not intended to exclude, for example, other additives, components, integers or steps not listed in the step.

[0016] 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, further aspects include from one particular value and / or to another particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about" or "approximately", it will be understood that the particular value forms further aspects. It will be further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. It is also to be understood that many values disclosed herein exist, and each value is disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also to be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0017] As used herein, the terms "any" or "optionally" mean that the event or circumstance recited thereafter may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0018] As used herein, the term "subject" refers to the target of administration, e.g., a human. Thus, the subject of the disclosed methods can be a vertebrate such as a mammal, fish, bird, reptile, or amphibian. The term "subject" also includes domesticated 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 indicate a particular age or gender. Thus, it is intended to cover adults, children, adolescent and neonatal subjects, as well as fetuses, regardless of male or female.

[0019] As used herein, the term "patient" refers to a subject suffering from a disease or disorder or condition. The term "patient" includes humans and veterinary subjects. In some aspects of the disclosed methods, the "patient" is diagnosed as in need of treatment for the healing of a bone injury, e.g., prior to an administration step.

[0020] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, relieving, delaying the onset of, inhibiting or delaying the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to subjects who do not exhibit signs of a disease, disorder, and / or condition and / or to subjects who exhibit only initial signs of a disease, disorder, and / or condition for the purpose of reducing the risk of developing the pathology associated with the disease, disorder, and / or condition. For example, the disease, disorder, and / or condition can be a bone injury or fracture.

[0021] Current methods for treating large segmental bone defects Many techniques have been employed for the treatment of skeletal defects, including autologous bone grafting, various bone graft substitutes, the Ilizarov method, arthroplasty using large external fixators, biological agents, and amputation as a last resort. However, these existing treatment options typically require long-term treatment involving multiple surgeries, are expensive, have a high complication rate, and are associated with a significant risk of treatment failure. For example, autograft remains the treatment of choice when curing 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). However, the supply of sufficient autograft material can be limited, especially in cases of severe traumatic injury or nonunion of fractures in civilian populations and severely injured soldiers, and is associated with significant morbidity at the donor site. In contrast, allografts are readily available in large quantities, but their use raises concerns about disease transmission and immune response (Khan SN, Cammisa FP, Sandhu HS, Diwan AD, Girardi FP, Lane JM. J Am Acad Orthop Surg. 2005;13:77~86), and more importantly, they consist of dead bone that does not resorb or reconstruct well. Also, microfractures gradually accumulate under load, and the failure rate has been shown to be over 30% at five years (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). Furthermore, various bone substitutes have been developed to assist in the treatment of large segmental bone defects, but these approaches are limited to only a slight improvement in outcomes. Inadequate bone formation, poor mechanical and handling properties, lack of biocompatibility, unpredictable resorption, and associated inflammatory reactions remain the major limitations of such materials (McKee MD. J Am Acad Orthop Surg. 2006;14:S163-7).Therefore, for bone defects exceeding 8 cm, distraction osteogenesis remains the treatment of choice by military surgeons. However, this technique is complex, painful, unreliable, complicated by pin-track infections, and may result in delayed healing (Pollak AN, Ficke JR, J Am Acad Orthop Surg. 2008;16:628-34). Above all, the main complicating factors of the available treatment options for large segmental bone defects are the inability to ensure rapid functional recovery and the ability to reduce refracture rates.

[0022] Biological research on bone formation has led to the discovery of bone morphogenetic proteins (BMPs), some of the most potent inducers of bone formation (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. BMPs have shown preclinical efficacy in animal models, but their clinical efficacy has been disappointing. Some of the clinical responses to these proteins are associated with delivery problems. Furthermore, most of the current practice of using very high supra-physiological doses of BMPs that rapidly leach from the site of application has the potential to increase the incidence and severity of metastatic / heterotopic ossification, as well as many other associated side effects such as antibody formation, graft detachment, 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 improve the efficacy of BMPs by significantly minimizing the required doses, and thus limit their potential side effects and associated treatment costs.

[0023] In recent years, platelet-rich plasma (PRP), which is plasma rich in high concentrations of platelets, has been tested in a wide range of applications, including the treatment of musculoskeletal injuries. PRP is thought to secrete various growth factors and cytokines at supra-physiological concentrations and generate a cell-supporting matrix in the form of a fibrin clot. Specifically, the effects of PRP to promote 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) have been suggested to result from increased concentrations of factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP), 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).However, the results of experimental (Simman R, Hoffmann A, Bohinc RJ, Peterson WC, Russ AJ. Ann Plast Surg. 2008;61:337-44) and clinical trials (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 craniofacial bone grafting surgeries are controversial, and there is currently little evidence suggesting that it actually improves or accelerates bone healing. In fact, in most cases, there was a decrease in bone formation (Choi B-H, Im C-J, Huh J-Y, Suh J-J, Lee S-H. 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 effectiveness extends to long bones and the spine. In a recent study in sheep, after using femoral shaft osteotomy and then distraction osteogenesis, the application of PRP did not show improvement in new bone formation (Hernandez-Fernandez A, Velez R, Soldado F, Saenz-Rios JC, Barber I, Aguirre-Canyadell M. Injury. 2013;44:901-7).Similarly, in rodent studies, PRP has not been shown to have a 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 Z. J Bone Joint Surg Am. 2007;89:139-147), or has shown only a low potential for regeneration (Sanchez AR, Sheridan PJ, Eckert SE, Weaver AL. J Periodontol. 2005;76:1637-1644). In studies to promote bone healing after spinal fusion, there has been no benefit in any animal (Li H, Zou X, Xue Q, Egund N, Lind M, Bunger C. Eur Spine J. 2004;13:354-8), or human subjects (Weiner BK, Walker M. Spine (Phila Pa 1976). 2003;28:1968-1970). The factors causing the variable outcomes in these studies are not known, but it has been suggested that proteases present in platelets can 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 effectiveness. Furthermore, PRP activation requires thrombin, which leads to a burst release of growth factors and a reduction in the total growth factor concentration. Bovine thrombin interferes with human coagulation proteins by stimulating antibodies against thrombin, which is thought to affect 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 appropriate structural properties.

[0024] Based on the low performance of PRP, a "second generation" of platelet concentrate called platelet-rich fibrin (PRF) was developed by French maxillofacial surgeon Joseph Choukroun. (Choukroun J, Diss A, Simonpieri A, Girard M - O, Schoeffler C, Dohan SL, et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:299 - 303). It has been described as a more natural fibrin matrix with specific advantages over PRP and containing blood components beneficial to healing and immunity. For example, the clotting process occurs by a slower natural polymerization compared to the rapid polymerization that occurs when thrombin is added to PRP. Most importantly, PRF does not require anticoagulant 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 have also shown that PRF has a more sustained release of growth factors such as PDGF, TGF - β, and BMP, lasting up to 28 days (He L, Lin Y, Hu X, Zhang Y, Wu H. Oral Surg 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.However, PRF has not shown any significant improvement when applied in oral and maxillofacial surgery (Choukroun J, Diss A, Simonpieri A, Girard M - O, 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). Again, this may be related to inappropriate structural properties and insufficient amounts of platelets delivered, similar to PRP.

[0025] Effect of Hematoma Formation on Fracture Healing Hematoma formation at the fracture site occurs within minutes of bone injury and involves a series of biological events related to not only activated coagulation factors from the blood system but also several molecular factors derived from bone marrow and surrounding soft tissues, such as pro-inflammatory cytokines and growth factors including bone morphogenetic proteins and angiogenic factors (Lai BFL, Zou Y, Brooks DE, Kizhakkedathu JN. Biomaterials. Elsevier Ltd; 2010; 31: 5749-5758). At the fracture site, blood vessels constrict to prevent continuous blood loss, followed by a coagulation cascade in which a hematoma or thrombus forms between the fractured fragments (Schindeler A, McDonald MM, Bokko P, Little DG. Semin Cell Dev Biol. 2008; 19: 459-66). The coagulation process has two main pathways, the intrinsic pathway and the extrinsic pathway, and the common pathway is the conversion of coagulation factor X to Xa. Thrombin, the final 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 B.V.; 2012; 1824: 246-252). During normal blood coagulation, the coagulation cascade activates prothrombin by converting it to the serine protease thrombin. Thrombin then converts soluble fibrinogen to insoluble fibrin fibers. Finally, these fibrin fibers contribute to the formation of a mature web-like fibrin clot with the help of coagulation factor XIII (Chernysh IN, Nagaswami C, Purohit PK, Weisel JW. Sci Rep. 2012; 2: 879). Therefore, the concentration gradients of fibrinogen, thrombin, and coagulation factor XIII play important roles in regulating the three-dimensional structure of the fibrin clot (Wolberg AS, Campbell RA. Transfus Apher Sci. 2008; 38: 15-23).The structural parameters in fibrin clots can be characterized by the diameter, density, number of branch points, distance between branch points, and pore size of the fibers (Weisel JW, Litvinov RI. Blood. 2013;121:1712-1719). An 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. J R Soc Interface. 2005;2:309-318, and Kaur S, Sundarrajan S, Rana D, Matsuura T, Ramakrishna S. J Memb Sci. 2012;392-393:101-111). In addition, the fiber diameter and density affect the porosity and surface area of fibrin clots (Pham QP, Sharma U, Antonios G. Mikos. Biomacromolecules. 2006;7:2796-2805), and are responsible for biological functions of stem cells 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 concentration (<1 nM) generates a porous network of thick fibrin fibers that is highly susceptible to fibrinolysis, while high concentrations of thrombin result in 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, individual thick fibers have higher mechanical strength (rigidity), but fibrin clots composed of thick fibers often have low mechanical strength because the number of fibers is reduced (Carlisle CR, Coulai C, Guthold M. Acta Biomater. 2010;6:2997-3003, and Liu W, Thomopoulos S, Xia Y. Adv Healthc Mater. 2012;1:10-25).

[0026] Although there have been numerous studies on the importance of hematoma formation in fracture healing, there are no reports on the differences in the structural and biological properties of the hematomas formed between fractures that usually heal spontaneously and segmental bone defects that do not, and whether the hematoma properties can be modulated to enhance the repair of large segmental bone defects.

[0027] Instead, most studies appear to focus on refining the properties of PRP, as demonstrated by the "second-generation" product PRF. Some studies have suggested that PRF is a somewhat more effective product compared to its corresponding PRP, but the studies to date have been inconclusive and further investigation, particularly related to the repair and regeneration of long bones, is needed.

[0028] Disclosed herein is a method for improving the structural and biological properties of an induced thrombus to enhance the healing of large segmental bone defects. For example, to significantly improve and accelerate the healing of large segmental bone defects in soldiers and civilians, described herein is an ex vivo hematoma created by constructing a fibrin clot that mimics the structural properties of a congenital fracture hematoma. To this end, a rat model was used to evaluate whether the quality of the hematoma formed at the fracture site determines the potential for healing of large bone defects, and thrombin was used to form a thrombus that mimics the inherent structural properties of a normal fracture hematoma and was then used to enhance the healing of large bone defects.

[0029] Many snake venom toxins of the prothrombinotic snake contain proteolytic enzymes that affect hemostasis by the action of clotting thrombin-like enzymes and prothrombin-activating toxins (Figure 1). As shown in Figure 1, the conversion from factor X to (activated) Xa is the common junction between the extrinsic and intrinsic clotting pathways. Snake venom enzymes have evolved to utilize various stages of the clotting cascade. These venoms have evolved to exploit the fragile interactions that exist between platelets, endothelial cells, and plasma proteins and venom proteins that affect the stages of vertebrate hemostasis (Meier J, Stocker K. Crit Rev Toxicol. 1991;21:171-182). Depending on the type of snake, each has specific coagulation factors. For example, the procoagulant factor oscarine found in the venom of the coastal taipan (Oxyuranus scutellatus) is structurally and functionally similar to mammalian coagulation factor X. Oscarine is a serine protease belonging to the venom of group C prothrombin-activating factors and, unlike mammalian factor X, does not require non-enzymatic factor V because they contain their own factor Va-like molecules. (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, echistatin found in the venom of the saw-scaled viper (Echis carinatus) is a metalloprotease that acts without the involvement of cofactors such as calcium chloride (CaCl2) and phospholipids. Due to the lack of cofactor requirements, echistatin 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 has been 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+A metalloproteinase that requires but does not require 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 S. J Biol Chem. 1988;263:17471-17481). In comparison, at normal fracture sites, factor X binds to factor V on the platelet membrane, which accelerates thrombin generation by thousands of times. This is the mechanism leading to the formation of mature coagulation that stabilizes the primary hemostatic plug (Probst A, Spiegel H-U. J Investig Surg. 1997;10:77-86). From the above, it is suggested that the coagulation-promoting factors isolated from snake venom should be suitable as more natural clotting agents for modifying the structural properties of thrombi. In fact, snake venom proteins were important in elucidating the complex physiological mechanisms that govern the coagulation cascade and determine platelet function. Furthermore, they were useful for elucidating the structural and functional relationships of human clotting factors and platelet glycoproteins due to their potency, selectivity, and high biological efficacy (Hong T-T, Huang J, Lucchesi BR. Am J Physiol Heart Circ Physiol. 2006;290:H959-67, Han SM, Weaver FA, Comerota AJ, Perler BA, Joing M. J 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 toxic compounds have been identified, isolated, characterized, and purified and are currently being used for both diagnostic and pharmaceutical purposes (King GF. Expert Opin Biol Ther. 2011;11:1469-84; and Butler MS. Nat Prod Rep. 2008;25:475-516). Described herein are compositions containing snake venom coagulase (SVCE) as an alternative clotting agent, in contrast 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 be catalytically more active than their mammalian counterparts, are heat stable due to the presence of additional disulfide bridges, and are known to be more resistant to proteolysis (Kang TS, Georgieva D, Genov N, Murakami MT, Sinha M, Kumar RP, et al. FEBS J. 2011;278:4544-76). Disclosed herein are compositions and methods using a rat model to demonstrate the ability of snake venom enzymes to alter the properties of hematomas and their ability to heal large segmental defects. Disclosed herein are compositions and methods for modifying the properties of hematomas, including administering one or more of the compositions disclosed herein.

[0030] Coagulation factors derived from snake venom were preferred for several reasons, such as not affecting other clotting factors, having a small molecular size that makes it less likely to be recognized by the body's immune system during use and thus less likely to be attacked. Furthermore, the molecule is selective when binding to its targets in the body, minimizing the potential for unwanted side effects. Ecarin was specifically selected because it does not require a cofactor for activation.

[0031] This disclosure is important for the development of new and improved treatment strategies to enhance bone healing, improve quality of life, reduce high treatment costs, and lower the rate of amputations in both the general public and within the military as a result of severe trauma and battlefield injuries. The results disclosed herein may provide the requirements for ex vivo - generated hematomas having properties that increase the effectiveness of bone healing by mimicking the inherent structural and biological properties of natural healing fracture hematomas. This disclosure also functions as a reservoir for growth factors, serves as a biocompatible scaffold that is not only osteoinductive and osteoconductive but also enhances the healing of large - segmental bone defects and critically sized defects, and is important for the development of ex vivo hematomas for the treatment of non - healing fractures (delayed union or non - union).

[0032] In the operating room, without using highly specialized equipment in a civilian or military clinical setting, simply mixing the required amount of whole blood with a specific concentration of reptilase results in rapid product conversion and on - demand application. The results described herein may also benefit biomaterial researchers by incorporating the conforming structural properties of the hematoma into the design of biomaterial scaffolds, improving the capabilities of poorly performing scaffolds currently used in regenerative medicine to assist in bone repair. Furthermore, the clot - promoting factor isolated from snake venom can be used as a suitable clotting agent to modify the structural properties of thrombi and can also be used to stop uncontrollable bleeding in a hospital environment and on the battlefield. Also disclosed herein are products that can stop bleeding within seconds, are easy to carry, have a long shelf life, are absorbable or easily removable, and are inexpensive. These snakes evolved to kill their prey by instantaneously causing a massive coagulation disorder, thus developing a very specific biological agent that turns blood into gelatin. However, when properly isolated and carefully prepared under controlled conditions, the same clotting factors such as echistatin can be used instead to save lives. By immediately controlling blood loss, this remarkable property can limit further blood loss in civilian patients or battlefield - wounded soldiers.

[0033] Disclosed herein is a biocompatible snake venom-induced ex vivo hematoma for healing large bone defects. Also disclosed herein are methods of treating and manipulating an ex vivo hematoma (thrombus) in a specific way that modifies its ultrastructural properties and thereby changes its behavior in various clinical situations. Disclosed herein is a method of treating whole blood or blood products with an agent that changes its structural form and biological activity so as to be usable for the treatment of various different medical conditions.

[0034] Disclosed herein are compositions and methods that can be used to improve the regeneration and repair of large bone defects, size defects comparable to severe ones, and for the treatment of non-union fractures (delayed union or non-union). Disclosed herein is an organized thrombus (ex vivo hematoma / bioactive scaffold) that can be used to enhance bone healing by functioning as a temporary reservoir for the continuous release of important growth factors and providing an appropriate space to assist cell infiltration, proliferation, and differentiation simultaneously. Disclosed herein are compositions and methods for enhancing a repair process involving the production of a thrombus (ex vivo hematoma) that mimics the natural healing fracture hematoma. The concept is that the structural properties of the hematoma, an ex vivo product, are altered using the coagulation factor, echistatin, derived from snake venom, and when transplanted into a bone defect, it can mimic the native fracture hematoma that enhances and accelerates bone healing. The coagulation factor derived from snake venom, echistatin, can also be used to stop bleeding. In some embodiments, the composition can be formulated as a powder, liquid, or spray.

[0035] Compositions comprising whole blood, echistatin, and BMP-2 are disclosed herein. In some embodiments, echistatin can be present at a specific concentration. In some embodiments, low-dose or significantly reduced doses of BMP-2 can be used. In some embodiments, bone defect healing can be enhanced using the composition. In some embodiments, the composition can be formulated as a liquid or a gel.

[0036] The types of healing described herein are similar to those provided by commercially available products sold by Medtronic that use rhBMP-2 delivered with absorbable collagen sponge (Infuse (trademark)). The compositions or products described herein require significantly lower doses of BMP-2 (e.g., rhBMP-2) 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 times or more lower than any commercially available product or composition.

[0037] 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 endochondral bone neogenesis. 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.

[0038] Composition Disclosed herein is an ex vivo hematoma. In some embodiments, the ex vivo hematoma can comprise (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, octacarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the ex vivo hematoma can comprise (a) platelet-rich plasma, plasma, or plasma having red blood cells, and (b) ecarin, octacarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the ex vivo hematoma can further comprise sodium citrate. In some embodiments, the phrase "plasma having red blood cells" means plasma without platelets. In some embodiments, the ex vivo hematoma can comprise fibrin fibers having a thickness of at least 150 - 300 nm ± 10%. In some embodiments, ecarin, octacarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride can result in the formation of one or more fibrin fibers having a thickness of at least 150 - 300 nm ± 10%. As used herein, the term "ex vivo" refers to a hematoma that can be formed outside of an organism, for example, in an external environment. In some embodiments, the ex vivo hematoma can comprise (a) isolated whole blood and sodium citrate platelet-rich plasma, plasma alone, plasma having red blood cells (without platelets) or other blood products and (b) one or more clotting factors. In some embodiments, the ex vivo hematoma can comprise whole blood and one or more clotting factors.

[0039] As used herein, the terms "whole blood" and "blood" are used herein to mean blood that can be directly obtained from the body without any of the components including plasma or platelets being removed. In some embodiments, the whole blood or blood can be from a subject or patient who is a recipient of any of the compositions described herein or any of the ex vivo hematomas described herein. In some embodiments, 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 embodiments, a fibrin gel can be used in place of whole blood.

[0040] One of ordinary skill in the art will understand that blood is a specialized body fluid that delivers important substances such as nutrients and oxygen to cells and transports metabolic waste products away from those same cells. In vertebrates, blood consists of blood cells suspended in plasma. Blood can contain different components, such as plasma, red blood cells (erythrocytes), platelets (thrombocytes), and white blood cells (leukocytes). Plasma is the main component that accounts for about 55% of blood and is mainly composed of water containing mainly ions, proteins, nutrients, and waste products. Plasma can contain some of all the proteins produced in the body. For example, plasma can contain about 90% water and 10% of the following mixture: 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), coagulation factors (factors I - XIII) (labtestsonline.org.au), sugars (glucose), lipids (cholesterol), minerals (sodium, calcium, magnesium, potassium, iron, zinc, copper, and selenium) (Harrington et al., 2014), waste products, and soluble gases. Red blood cells (erythrocytes) play the role of carrying oxygen and carbon dioxide. They are about 7 - 8 μm in size, do not contain mitochondria or a nucleus when mature, and have an average lifespan of 120 days. Women have about 3.6 - 5 million / mm 3 red blood cells, and men have about 4.2 - 5.4 million / mm 3 red blood cells. Platelets (thrombocytes) are the cause of blood clots. The normal platelet count is about 150,000 - 450,000 / mm 3is in the range. White blood cells (WBC; leukocytes) are part of and function in the immune system in an immune response. Approximately 1% of those cells are found in the blood. They are larger than red blood cells and contain a normal nucleus and mitochondria. The normal white blood cell count is approximately 5,000 - 10,000 / mm 3 is in the range. White blood cells can be divided into five major types and further into two different groups. Granulocytes: Neutrophils: 60 - 70% of WBC or 3,000 - 7,000 / mm 3 , Eosinophils: 1 - 3% of WBC or 50 - 400 / mm 3 and Basophils: 0.3 - 0.5% of WBC or 25 - 200 / mm 3 and Agranulocytes: Lymphocytes: 20 - 30% of WBC or 1,000 - 4,000 / mm 3 and Monocytes: 3 - 8% of WBC or 100 - 600 / mm 3 .

[0041] As used herein, the term "platelet-rich plasma" (also known as autologous conditioned plasma) refers to a concentrated form of platelet-rich plasma proteins derived from whole blood. For example, whole blood can be centrifuged to remove red blood cells. In some embodiments, the terms "blood plasma alone", "plasma alone", or "plasma" can generally refer to the yellowish liquid component derived from whole blood that holds blood cells 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 then the plasma can be drawn off from the top of the tube. In some embodiments, the term "plasma with red blood cells" can refer to "plasma alone" with additional red blood cells. For example, red blood cells are obtained by centrifuging whole blood until it falls to the bottom of the tube and are recovered after removing plasma, white blood cells, and platelets from the top of the tube.

[0042] In some embodiments, the ex vivo hematoma may contain one or more growth factors. In some embodiments, the one or more growth factors may be one or more of the 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 to 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 ex vivo hematoma may further contain BMP-2. In some embodiments, the one or more growth factors may be BMP-2.

[0043] In some embodiments, whole blood may contain viable cells. In some embodiments, about 50% to 70% of the viable cells in whole blood remain viable after hematoma formation. In some embodiments, at least 50% of the viable cells in whole blood remain viable after hematoma formation. In some embodiments, at least 60% of the viable cells in whole blood remain viable after hematoma formation. In some embodiments, at least 70% of the viable cells in whole blood remain viable after hematoma formation. In some embodiments, at least 80% of the viable cells in whole blood remain viable after hematoma formation. In some embodiments, at least 90% of the viable cells in whole blood remain viable after hematoma formation. In some embodiments, more than 90% of the viable cells in whole blood remain viable after hematoma formation.

[0044] In some embodiments, whole blood can 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 excluding water. Examples of other biological factors include, but are not limited to, ions, proteins, clotting factors, sugars, lipids, and minerals.

[0045] In some embodiments, one or more biological factors present in whole blood can be endogenous biological factors. Platelets are present in whole blood. Many growth factors can be found in platelets. Growth factors in platelet-rich plasma alpha granules include, but are not limited to, platelet-derived growth factor (PDGF), transforming growth factor beta1, beta2, beta3 (TGF-β1, TGF-β2, TGF-β3), platelet-derived angiogenesis factor (PDAF), insulin-like growth factor 1 (IGF-1), platelet factor 4 (PF-4), epidermal growth factor (EGF), epithelial cell growth factor (ECGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and other cytokines, and have been shown to contain mitogenic and chemotactic growth factors along with related healing molecules in an inactive form important in wound healing. In addition, plasma fluid also contains many biologically active proteins such as growth factor IGF-I and hepatocyte growth factor (HGF). During normal wound healing, trapped platelets are activated and degranulated, and alpha granule components are released. Examples of growth factors present in platelets include, but are not limited to, platelet-derived growth factor, transforming growth factor beta1, beta2, beta3, platelet-derived angiogenesis factor, insulin-like growth factor 1, platelet factor 4, epithelial growth factor, epithelial cell 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 include, but are not limited to, insulin-like growth factor 1, and hepatocyte growth factor.

[0046] In some embodiments, the ex vivo hematoma can include whole blood, ecarin, and sodium citrate. In some embodiments, the ex vivo hematoma can include whole blood, calcium chloride, and sodium citrate. In some embodiments, the ex vivo hematoma can include platelet-rich plasma and ecarin. In some embodiments, the ex vivo hematoma can include platelet-rich plasma and calcium chloride. In some embodiments, the ex vivo hematoma can include whole blood, calcium chloride, or octarine and calcium chloride, and sodium citrate. In some embodiments, (a) one combination of isolated whole blood and sodium citrate, platelet-rich plasma, or plasma having red blood cells can be combined with (b) one of ecarin, octarine and calcium chloride, or calcium chloride. In some embodiments, (a) one combination of isolated whole blood and sodium citrate, platelet-rich plasma, or plasma having red blood cells can be combined with (b) one of thrombin or thrombin and calcium chloride. In some embodiments, any of the ex vivo hematoma combinations described herein can further include one or more antibiotics.

[0047] In some embodiments, the concentration of calcium chloride present in the ex vivo hematoma can be in the range of 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.

[0048] In some embodiments, the concentration of thrombin can be in the range of 0.1 to 1 U / mL. In some embodiments, the concentration of thrombin present in the ex vivo hematoma can 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 thereabove between these. In some embodiments, the concentration of thrombin present in the ex vivo hematoma can be 0.5 U / mL.

[0049] In some embodiments, the concentration of ecarin present in the ex vivo hematoma can be at least 0.05 U / mL. In some embodiments, the concentration of ecarin present in the ex vivo hematoma can 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 greater. In some embodiments, the concentration of ecarin present in the ex vivo hematoma can be 0.3 U / mL. In some embodiments, the concentration of ecarin present in the ex vivo hematoma can be 0.6 U / mL. In some embodiments, the concentration of ecarin present in the ex vivo hematoma can be 0.75 U / mL.

[0050] In some embodiments, the ex vivo hematoma 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 ex vivo hematoma may be at least 0.01 mg. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma may be from 0.01 to 5 mg. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma 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 mg, or any number therebetween. In some embodiments, the recombinant BMP-2 may be used at a dose of about 0.01 mg to about 12 mg. In some embodiments, the 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 number therebetween. In some embodiments, the recombinant BMP-2 may be used at a dose greater than 12.0 mg. In some embodiments, the dose of BMP-2 may be from about 1 mg to 5 mg. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma may be at least 0.01 μg. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma may be from 0.01 to 5 μg. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma 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, the recombinant BMP-2 may be used at a dose of about 0.01 μg to about 12 μg.In some embodiments, recombinant BMP-2 can 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 number in between. In some embodiments, recombinant BMP-2 can be used at a dose greater than 12.0 μg. In some embodiments, the dose of BMP-2 can be about 1 μg to 5 μg. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma can be 0.3 to 0.4 μg. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma can be lower than the standard dose. In some embodiments, the dose of BMP-2 present in the ex vivo hematoma can be 10 to 50 times lower than the standard dose or the minimum effective dose of BMP-2 / ACS.

[0051] In some embodiments, the amount of ecarin present in the ex vivo hematoma can be at least 0.05 U / mL, and the amount of BMP-2 present in the ex vivo hematoma can be at least 0.01 mg.

[0052] In some embodiments, the amount of ecarin present in the ex vivo hematoma can be at least 0.05 U / mL, and the amount of BMP-2 present in the ex vivo hematoma can be at least 0.01 μg.

[0053] In some embodiments, the concentration of sodium citrate can be about 3.2 to 4 mg / ml. In some embodiments, the solution is about 3.2 to 4% (weight / volume) sodium citrate, and then one part of this solution can be mixed with nine parts of whole blood.

[0054] In some embodiments, the ex vivo hematoma 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 a 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.

[0055] In some embodiments, the ex vivo hematoma can be formulated as a liquid or a gel. In some embodiments, the ex vivo hematoma can be formulated in a lyophilized form or a powder form. The lyophilized form or the powder form can make the ex vivo hematoma more stable for storage. In some embodiments, growth factors (BMP and others), coagulation factors (echalin, calcium chloride, etc.) and sodium citrate can be available in a lyophilized form or a powder form. In some embodiments, the compounds used to make the ex vivo hematoma disclosed herein can be dissolved in sterile distilled water before being mixed 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 collected from a patient before surgery (e.g., immediately before surgery) and can be citrated to prevent coagulation. In some embodiments, donor blood can be used for patients with blood disorders or diseases including, but not limited to, anemia, hemophilia, leukemia, HIV, etc. The remaining components of the ex vivo hematoma do not require any additional stabilizers for storage. For example, BMP-2 is commercially available in ready-to-use vials, CaCl2 is available in powder form and may in some cases already be dissolved in sterile distilled water (very stable after dissolution). Both BMP-2 and CaCl2 can be stored at room temperature. Echalin is available in a lyophilized form (lyophilized), stored at -20°C and can be dissolved in sterile distilled water before use. The ex vivo hematoma can be prepared relatively simply using the components described herein in an amount based on the volume of the defect to be filled. Thus, after the components are prepared, they can be mixed together in a tube / mold. Generally, the ex vivo hematoma is formed in about 30 - 45 minutes and can then be inserted (or transplanted) into the bone defect. In some embodiments, the ex vivo hematoma described herein can be stored using, for example, a "smart storage system" that uses a radio frequency identification-based system (e.g., Smartstorage (trademark)), which is an almost real-time tissue tracking system that can rationalize inventory management including maintaining an accurate usage history and temperature log.

[0056] In some embodiments, the ex vivo hematoma described herein may further include a carrier. For example, the carrier can be a biodegradable biomaterial scaffold (e.g., a silk fibroin scaffold, poly(lactide-co-glycolide) (PLGA), or other similar absorbable products or materials). Such carriers can be used to provide additional mechanical support for the ex vivo hematoma.

[0057] In some embodiments, the ex vivo hematoma can be formulated for local administration. In some embodiments, the compositions disclosed herein (e.g., in liquid form) or ex vivo hematomas (e.g., in gel form) can be administered locally, surgically implanted, or injected percutaneously. In some embodiments, the liquid formulation can be delivered through a syringe. In some embodiments, the gel formulation can be implanted at the bone defect site. The gel formulation can be prepared using an external mold corresponding to the bone size and shape for implantation at the bone defect site. In some embodiments, the formulation can be in an intermediate form between a liquid and a gel. In some embodiments, the intermediate formulation can be applied to a solid biologic scaffold and can crosslink gaps (e.g., large gaps) that may exist within the solid biologic scaffold itself while independently providing mechanical support. Examples of solid biologic scaffolds include, but are not limited to, titanium cages or other porous metal implants. Such biologic scaffolds can be used to reconstruct skeletal defects or achieve spinal fusion. The formulations disclosed herein can be used to enhance healing when a PEEK spinal cage is used in interbody spinal fixation, considering that PEEK itself is biologically inert and does not have inherent bone healing capabilities. Alternatively, any of the formulations disclosed herein can be injected or topically applied to an absorbable biologic scaffold so as to be used to reconstruct a segmental or sub-segmental skeletal defect. When used in combination with either a metal porous implant or an absorbable biologic scaffold, this also includes bone defects associated with opening wedge osteotomy (of the femur, tibia, or other long bones), temporary distraction arthroplasty sites, and arthroplasty. Further, any of the formulations disclosed herein can be applied in the same manner to other joint fixation sites with bone defects, such as the ankle, knee, wrist, shoulder, hip, or other smaller joints including, but not limited to, the Lisfranc joint, hand, wrist, or foot, and is extended to include applications for filling bone defects that occur when harvesting bone grafts for transplantation to a secondary anatomic location.

[0058] In some embodiments, ecarin can be used alone or in combination with one or more of the components described herein to stop bleeding. In some embodiments, the bleeding can be severe. In some embodiments, the bleeding can be arterial, venous, or capillary bleeding. In some embodiments, ecarin can be formulated as a powder, liquid, or spray. In some embodiments, ecarin can be formulated as beads (e.g., collagen beads). In some embodiments, ecarin can be formulated as nanoparticles.

[0059] Method Disclosed herein is a method for promoting bone healing. Also disclosed herein is a method for producing a bone replacement material. Further disclosed herein is a method for producing an implant. In some embodiments, the methods disclosed herein can be combined. Disclosed herein is a method for promoting bone healing, a method for producing a bone replacement material, an implant, or a combination thereof. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising an ex vivo hematoma disclosed herein. Disclosed herein is a method for promoting bone healing in a subject, a method for producing a bone replacement material, an implant, or a combination thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an ex vivo hematoma disclosed herein.

[0060] In some embodiments, the ex vivo hematoma may comprise (a) isolated whole blood, (b) sodium citrate, and (c) ecarin, octacarin, and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the ex vivo hematoma may comprise (a) platelet-rich plasma, plasma, or plasma having red blood cells, and (b) ecarin, octacarin, and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the ex vivo hematoma may further comprise sodium citrate. In some embodiments, the ex vivo hematoma may comprise fibrin fibers having a thickness of at least 150-300 nm ± 10%. In some embodiments, ecarin, octacarin, and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride may result in the formation of one or more fibrin fibers having a thickness of at least 150-300 nm ± 10%. In some embodiments, the ex vivo hematoma may comprise (a) isolated whole blood and sodium citrate platelet-rich plasma, plasma alone, plasma having red blood cells (platelet-free) or other blood products and (b) one or more coagulation factors. In some embodiments, the ex vivo hematoma may comprise whole blood and one or more coagulation factors. In some embodiments, the whole blood may comprise one or more viable cells. In some embodiments, the whole blood may comprise one or more biological factors. In some embodiments, the ex vivo hematoma may comprise whole blood, ecarin, and sodium citrate. In some embodiments, the ex vivo hematoma may comprise whole blood, calcium chloride, and sodium citrate. In some embodiments, the ex vivo hematoma may comprise platelet-rich plasma and ecarin. In some embodiments, the ex vivo hematoma may comprise platelet-rich plasma and calcium chloride. In some embodiments, the ex vivo hematoma may comprise plasma and ecarin. In some embodiments, the ex vivo hematoma may comprise plasma and calcium chloride. In some embodiments, the ex vivo hematoma may comprise plasma having red blood cells and ecarin. In some embodiments, the ex vivo hematoma may comprise plasma having red blood cells and calcium chloride.In some embodiments, the ex vivo hematoma may contain plasma having octreotide and calcium chloride. In some embodiments, the ex vivo hematoma may contain plasma having thrombin and calcium chloride. In some embodiments, the ex vivo hematoma may further contain bone morphogenetic protein 2 (BMP-2). In some embodiments, BMP-2 may be recombinant BMP-2. In some embodiments, the recombinant BMP-2 may contain human BMP-2. In some embodiments, the composition may further contain one or more growth factors, one or more platelets, and one or more cells. In some embodiments, the composition may be formulated as a thrombus or a scaffold. In some embodiments, the scaffold may be chemotactic. In some embodiments, the scaffold may attract endogenous growth factors beneficial for bone healing.

[0061] In some embodiments, the subject can be human. In some embodiments, the subject has a skeletal defect. In some embodiments, the skeletal defect can be a large segmental bone defect. In some embodiments, the subject has one or more fractures. In some embodiments, the subject has one or more bone injuries.

[0062] In some embodiments, the composition can be formulated as a clot or a scaffold. In some embodiments, the composition can be formulated as an ex vivo hematoma. In some embodiments, the composition can be formulated for local administration. In some embodiments, the composition can be administered locally. In some embodiments, the composition can be transplanted. In some embodiments, the composition can be injected percutaneously. In some embodiments, the composition can be injected with a syringe. In some embodiments, the amount of ecarin present in the composition can be at least 0.05 U / mL, and the amount of BMP-2 present in the composition can be at least 0.01 - 5 mg or any amount therebetween. In some embodiments, the amount of ecarin present in the composition can be at least 0.05 U / mL, and the amount of BMP-2 present in the composition can be at least 0.01 - 1 μg or any amount therebetween.

[0063] In some embodiments, the treatment regimen can be a standard treatment regimen for treating any bone defect. Briefly stated, the defective wound can be crushed and fixed with an internal plate, an external fixator, or an intramedullary nail. The compositions and ex vivo hematomas described herein can then be inserted into the skeletal defect prior to closing the wound. The treatment regimen can be consistent and unchanged if there is no infection and otherwise the defect is ready for final treatment. The implants disclosed herein can be inserted into the bone region by entering the body through the skin or through a body cavity or anatomical opening, minimizing additional damage to nearby structures. The selection of the type, including the size and shape of the implant, can be based on many factors including, but not limited to, the shape and / or size of the bone into which the implant is implanted, the rate of bone density (i.e., the porosity of the remaining bone), and / or the desired rate and distribution of scaffold or implant diffusion into the bone, or a combination of such factors. In some embodiments, the shape of the implant can be constructed to conform to the shape of the bone or vertebral body, thus allowing for a more uniform distribution of the scaffold, implant or ex vivo hematoma, or components present in the scaffold, implant or ex vivo hematoma. Application of the implant or ex vivo hematoma can occur during surgery or by any other suitable method.

[0064] Disclosed herein is a method of constructing an implant. In some embodiments, the method of constructing an implant comprises: a) determining the dimensions of a depot implant in at least one of a shape and a size that can facilitate transplantation of the depot implant into a bone defect; and b) structuring the depot implant to have a scaffold and creating a scaffold by introducing (i) isolated whole blood and sodium citrate, or platelet-rich plasma, plasma alone, plasma having erythrocytes (platelet-free), and (ii) ecarin, calcium chloride, osteocalcin and calcium chloride, thrombin, or thrombin and calcium chloride. In some embodiments, the scaffold can have a porosity of 55-75%. In some embodiments, the scaffold can comprise fibrin fibers having a thickness of at least 150-300 nm ± 10%. 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 matrix can be at least 0.01 mg. 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 μg. In some embodiments, the scaffold can contain at least one viable blood cell. In some embodiments, the scaffold can contain appropriate biological factors.

[0065] In some embodiments, the shape of the depot implant can be spherical or cylindrical. In some embodiments, the shape of the depot implant can be spherical or any other patient-specific geometries, forms, or shapes dictated by clinical exigency. 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 have a diameter of at least 1 mm to about 60 mm (or more). In some embodiments, the cylindrical shape can be straight and / or can be curved. In some embodiments, the cylindrical shape can be a straight rod or a curved rod. The cylindrical or rod shape can be any shape having a longitudinal axis that is longer along one direction than in other directions. The cross-sectional shape of the depot across the longitudinal axis can be any shape. In some embodiments, the cross-sectional shape can be oval, circular, trilobed, or any other shape. In some embodiments, the implant can be either straight or curved in such a longitudinal direction. The end face of the implant can be shaped such that it is either flat, round, or folded.

[0066] The dimensions of the implant can depend on the size of the bone defect and the anatomical site being treated. In some embodiments, the scaffold can be about 20% longer than the actual size of the defect, fit snugly, and completely fill the volume of the missing bone. For example, if the size of the bone defect is 3 cm and it is in the midshaft femur of an adult, the implant will likely need to be constructed, for example, with dimensions of about 3 - 4 cm in diameter and about 3.6 cm in length. In some embodiments, the scaffold can be similar in size and shape to a given bone defect. In some embodiments, the scaffold can be chemotactic.

[0067] Disclosed herein is a method of constructing an implant. The method of constructing an implant includes: a) sizing with at least one of a shape and size that can facilitate the insertion or implantation of a 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 having red blood cells, and (ii) ecarin, octacarin, and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride, and manufacturing the scaffold. In some embodiments, the scaffold can have a porosity of 55-75%. In some embodiments, the scaffold can be constructed in the form of a thrombus. In some embodiments, the ex vivo hematoma can further include one or more growth factors. 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, vascular endothelial growth factor, fibroblast growth factor 2, or a combination thereof. In some embodiments, BMP-2 can be introduced into the scaffold or ex vivo hematoma. Additionally, the scaffold or clot or ex vivo hematoma can be biodegradable so as to degrade without the need for surgical removal.

[0068] Also disclosed herein is a method of using any of the compositions described herein to initiate or enhance bone healing. Also disclosed herein is a method of using any of the compositions described herein to reconstruct a segmental bone defect. Also disclosed herein is a method of using any of the compositions described herein to reconstruct a segmental bone defect resulting from a tumor, trauma, or infection, creating a biomimetic hematoma 12q that initiates the normal fracture healing cascade by delivering a catalytic amount of BMP using ecarin, and locally multi-activating endogenous growth factors.

[0069] Also disclosed herein is a method of using any of the compositions described herein to treat at-risk fractures (e.g., in osteoporosis, diabetes, the elderly, or smokers). Also disclosed herein is a method of using any of the compositions described herein to treat at-risk fractures, by using ecarin to create a biomimetic hematoma that initiates the normal fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0070] Also disclosed herein is a method of using any of the compositions described herein to treat atypical femoral fractures. Also disclosed herein is a method of using any of the compositions described herein to treat atypical femoral fractures percutaneously, by using ecarin to create a biomimetic hematoma that initiates the normal fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0071] Also disclosed herein is a method of using any of the compositions described herein to treat minimally displaced femoral neck fractures. Also disclosed herein is a method of using any of the compositions described herein to treat percutaneously minimally displaced femoral neck fractures, by using ecarin to create a biomimetic hematoma that initiates the normal fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0072] Also disclosed herein is a method of using any of the compositions described herein to treat osteoporotic insufficiency fractures (pelvis, spine). Also disclosed herein is a method of using any of the compositions described herein to treat percutaneously osteoporotic insufficiency fractures (e.g., pelvis, spine), by using ecarin to create a biomimetic hematoma that initiates the normal fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0073] Also disclosed herein is a method of using any of the compositions described herein to enhance a spinal fusion procedure in conjunction with a spinal cage (either ceramic, PEEK, or a metal alloy). Also disclosed herein is a method of using any of the compositions described herein to enhance a spinal fusion procedure in conjunction with a spinal cage (either ceramic, PEEK, or a metal alloy) that allows for full immediate weight bearing, provides more stable fixation, and enhances postoperative recovery, using ecarin to induce local formation of a biomimetic hematoma embedded in a substrate containing the cage, initiating a fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0074] Also disclosed herein is a method of using any of the compositions described herein to treat delayed union of a long bone fracture (either percutaneous or open). Also disclosed herein is a method of using any of the compositions described herein to treat delayed union of a long bone fracture (either percutaneous or open), using ecarin to create a biomimetic hematoma, initiating a fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0075] Also disclosed herein is a method of using any of the compositions described herein to treat established non-union of a long bone fracture (either percutaneous or open). Also disclosed herein is a method of using any of the compositions described herein to treat established non-union of a long bone fracture (either percutaneous or open), using ecarin to create a biomimetic hematoma, initiating a fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0076] Also disclosed herein is a method of using any of the compositions described herein to improve (e.g., accelerate) the healing of long bone fractures. Also disclosed herein is a method of using any of the compositions described herein to improve (e.g., accelerate) the recovery from long bone fractures in selected candidates (such as high-performance athletes) by creating a biomimetic hematoma using ecarin, to promote more rapid recovery, initiating the fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0077] Also disclosed herein is a method of using any of the compositions described herein to accelerate the healing of long bone fractures in the selected veterinary candidates (such as thoroughbred racehorses). Also disclosed herein is a method of using any of the compositions described herein to accelerate the healing of long bone fractures in selected veterinary candidates (such as thoroughbred racehorses) by creating a biomimetic hematoma using ecarin, to promote more rapid recovery, initiating the fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0078] Also disclosed herein is a method of using any of the compositions described herein to facilitate more rapid and predictable dental and craniofacial reconstruction. Also disclosed herein is a method of using any of the compositions described herein to facilitate more rapid and predictable dental and maxillofacial reconstruction by creating a biomimetic hematoma using ecarin, initiating the fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0079] Also disclosed herein is a method of using any of the compositions described herein to reverse a condition that results in spontaneous jawbone resorption. Also disclosed herein is a method of using any of the compositions described herein to reverse a condition that results in spontaneous jawbone resorption, and using ecarin to create a biomimetic hematoma and locally regenerate bone.

[0080] Also disclosed herein is a method of using any of the compositions described herein to treat and / or reverse a condition that results in spontaneous osteonecrosis. Also disclosed herein is a method of using any of the compositions described herein to reverse a condition that results in spontaneous osteonecrosis, and using ecarin to create a biomimetic hematoma that is delivered percutaneously or openly (such as in Kienböck's disease, avascular necrosis of the femoral head, and osteonecrosis at various other anatomical locations including but not limited to the femoral condyles).

[0081] Also disclosed herein is a method of using any of the compositions described herein to treat and / or reverse a condition that results in spontaneous avascular necrosis of the femoral head with a collapsed femoral head. Also disclosed herein is a method of using any of the compositions described herein to treat and / or reverse a condition that results in spontaneous avascular necrosis of the femoral head with a collapsed femoral head, and using ecarin to create a biomimetic hematoma that is delivered by an open procedure after surgical dislocation of the hip joint.

[0082] Also disclosed herein is a method of using any of the compositions described herein to treat osteonecrosis resulting from chemotherapy, alcohol dependence, smoking, or other exogenous agents. Also disclosed herein is a method of using any of the compositions described herein to treat osteonecrosis resulting from chemotherapy, alcohol dependence, smoking, or other exogenous agents, and using ecarin to create a biomimetic hematoma that is delivered percutaneously or openly.

[0083] Also disclosed herein is a method of using any of the compositions described herein to enhance any standard fusion procedure. Also disclosed herein is a method of using any of the compositions described herein to enhance any standard fusion procedure (e.g., any limited fusion in the hip, knee, ankle, wrist, elbow, shoulder, subtalar joint, calcaneus or midfoot, or any fusion in any small joint such as a toe or finger, such as the great toe, thumb, or smaller fingers), by using ecarin to create a biomimetic hematoma, initiating the fracture formation cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous growth factors.

[0084] Also disclosed herein is a method of using any of the compositions described herein to accelerate the healing of a scaphoid waist fracture. Also disclosed herein is a method of using any of the compositions described herein to accelerate the healing of a scaphoid waist fracture and promote a more rapid recovery, by using ecarin to create a biomimetic hematoma that initiates the fracture healing cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous proliferation factors.

[0085] Also disclosed herein is a method of using any of the compositions described herein to reconstruct complex skeletal defects. Also disclosed herein is a method of using any of the compositions described herein to reconstruct complex skeletal defects in the skull, whether due to trauma, tumor, or infection, by using ecarin to create a biomimetic hematoma, initiating the bone formation cascade by delivering a catalytic amount of BMP, and locally multi-activating endogenous proliferation factors.

[0086] Also disclosed herein is a method of using any of the compositions described herein to accelerate the healing of a sternotomy. Also disclosed herein is a method of creating a biomimetic hematoma using ecarin, initiating a bone formation cascade by delivering a catalytic amount of BMP, and accelerating the healing of a sternotomy associated with open heart surgery and promoting more rapid recovery by locally multi-activating endogenous growth factors, using any of the compositions described herein.

[0087] Also disclosed herein is a method of using any of the compositions described herein in an arthroplasty component having an endosteal growth surface of bone specially adapted and enhanced with ecarin to induce local formation of a biomimetic hematoma embedded on a structural matrix and initiate the bone healing cascade more rapidly.

[0088] Also disclosed herein is a method of using any of the compositions described herein in an osseointegration stem and an arthroplasty component having an endosteal growth surface of bone specially adapted and enhanced with ecarin to induce local formation of a biomimetic hematoma embedded on a structural matrix and initiate the bone healing cascade more rapidly.

[0089] As used herein, the term "biomimetic hematoma" can be used to refer to an "ex vivo hematoma".

[0090] Also disclosed herein is a method of using any of the compositions described herein to reduce or manage bleeding.

[0091] Also disclosed herein is a method of using any of the compositions described herein to manage extensive venous bleeding / oozing during a surgical procedure. In some embodiments, in the methods disclosed herein, any of the compositions described herein can be formulated to be locally sprayed as an aqueous aerosol (using an atomizer for ecarin distribution to the affected area).

[0092] Also disclosed herein is a method of using any of the compositions described herein to manage or stop bleeding from individual damaged blood vessels (e.g., large bleeding-prone areas) during surgery or in the context of an emergency victim. In some embodiments, in the methods disclosed herein, any of the compositions described herein can be administered with beads (e.g., magnetic beads). In some embodiments, in the methods disclosed herein, any of the compositions described herein can be applied as a clamp / clamshell at the end of a blood vessel, simultaneously clamped off, deliver ecarin locally, and limit application to a specific damaged blood vessel end. The clamp or clamp element can contract adjacent damaged blood vessels adjacent to each other and can eliminate or minimize the risk of systemic administration of the composition.

[0093] Also disclosed herein is a method of using any of the compositions described herein as selective embolization. In some embodiments, in the methods disclosed herein, any of the compositions described herein can be delivered to one or more target blood vessels using a long fluoroscopy catheter via an interventional radiologist, manage or stop pelvic / intra-abdominal / esophageal / intracranial bleeding, and enable selective and highly specific administration of ecarin limited to a distinct pathology as directed (e.g., similar to the method performed using angiographic coils).

[0094] Also disclosed herein is a method of using any of the compositions described herein to treat meniscal bleeding. In some embodiments, the methods disclosed herein can be used to direct placement or disposition of any of the compositions described herein in the uterus of an affected female. In some embodiments, ecarin can be formulated to be delivered as part of biodegradable collagen beads (s).

[0095] Also disclosed herein is a method of using any of the compositions described herein for treating hemophilia-related spontaneous arthropathy. In some embodiments, ecarin can be formulated to be delivered as part of biodegradable collagen beads (s).

[0096] Also disclosed herein is a method of using any of the compositions described herein for treating spontaneous rheumatoid arthritis associated with anticoagulant overdose (e.g., warfarin, coumadin, etc.). In some embodiments, ecarin can be formulated to be delivered as part of biodegradable collagen beads (s).

[0097] Also disclosed herein is a method of using any of the compositions described herein for treating spontaneous intramuscular hemorrhage associated with anticoagulant overdose (e.g., warfarin, coumadin, etc.). In some embodiments, in the methods disclosed herein, any of the compositions described herein can be used as selective embolization.

[0098] Also disclosed herein is a method of using any of the compositions described herein for treating spontaneous intramuscular hemorrhage associated with hemophilia. In some embodiments, in the methods disclosed herein, any of the compositions described herein can be used as a selective plug.

[0099] Also disclosed herein is a method of using any of the compositions described herein for treating postoperative hemarthrosis in any elective total knee arthroplasty. In some embodiments, ecarin can be formulated to be delivered as part of biodegradable collagen beads (s) or nanoparticles (s). In some embodiments, the biodegradable collagen beads (s) or nanoparticles (s) can be freely delivered or dispersed into the joint immediately before wound closure.

[0100] Also disclosed herein is a method of using any of the compositions described herein for treating epistaxis. In some embodiments, echalin can be formulated to be delivered as part of biodegradable collagen beads. In some embodiments, the biodegradable collagen beads can be embedded in a fabric packing material or encapsulated within a fabric sheath to limit their distribution and contain them locally. In some embodiments, echalin is formulated to be part of biodegradable collagen beads and delivered in the form of a nasal pack so as to be embedded in a fabric packing material or encapsulated within a fabric sheath.

[0101] Also disclosed herein is a method of using any of the compositions described herein for treating retinal hemorrhage. In some embodiments, in the methods disclosed herein, any of the compositions described herein can be used as a selective embolization. In some embodiments, echalin can be formulated to be delivered as part of biodegradable collagen beads or nanoparticles. In some embodiments, biodegradable collagen bead or nanoparticle formulations are used to create a Velcro-type effect by creating a self-adhering shape, minimizing the risk of recurrence, and actively addressing retinal detachment

[0102] In some embodiments, "bleeding" can be hemorrhage. In some embodiments, blood can escape the circulatory system from one or more damaged blood vessels. In some embodiments, the hemorrhage can be internal or external.

[0103] Manufactured product The compositions and ex vivo hematomas described herein can be packaged in suitable labeled containers, for example, as a therapy for treating bone defects or for use in any of the methods disclosed herein. Accordingly, at least isolated whole blood and sodium citrate, or plasma, plasma with platelets, or erythrocytes, and a packaged product comprising ecarin, octacarin and calcium chloride, calcium chloride, thrombin, or thrombin and calcium chloride (e.g., a sterile container containing the compositions or ex vivo hematomas described herein and packaged for storage, shipping, or sale at a concentrated or usable concentration) and kits, as well as instructions for use are also within the scope of the present disclosure. The product can include a container (e.g., a vial, jar, bottle, bag, etc.) containing the compositions or ex vivo hematomas described herein. Further, the manufactured product can further include, for example, packaging materials, instructions for use, syringes, buffers, or other control reagents for treating or monitoring a condition that requires prophylaxis or treatment. The product can also include legends (e.g., printed labels or inserts, or other media (e.g., audio or video tapes) that explain the use of the product). The legend can be associated with the container (e.g., affixed to the container) and can explain the method by which the composition or ex vivo hematoma therein should be administered (e.g., the frequency and route of administration), and thus, the instructions, as well as other uses. The composition or ex vivo hematoma can be ready for administration (e.g., present in units suitable for dosing) and can include pharmaceutically acceptable adjuvants, carriers, or other diluents. Alternatively, the compound can be provided in concentrated form together with a diluent and an accompanying instruction for dilution.

Examples

[0104] Example 1: Structure and biological properties of the hematoma. Approaches for healing large segmental bone defects It is well established that the hematoma formed at the fracture site significantly affects the method of fracture healing. For example, studies have shown that removal of the hematoma delays 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, there are reports suggesting that the structural properties of the formed fibrin clot, such as the porosity and thickness of the fibrin fibers, affect 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 Y. J 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 (not healing without intervention; 320±64 nm) three days after surgery (Figures 2, 7, 8, and 9). Furthermore, a less porous network was also observed in the 5 mm defect compared to the 0.5 mm defect (42.56% vs. 50.03%), resulting in a 16% difference between the groups. To investigate whether there are differences in biological properties between hematomas formed in large bone defects (5 mm) and hematomas formed in normally healing fractures (0.5 mm), in vivo studies were conducted using RNA sequencing analysis (Table 1).

[0105] There were significant differences in genes mediating the inflammatory response (e.g., those produced by Il1b-activated macrophages, expressed in the area of inflammatory bone destruction, and mediating their inhibitory effects on osteoclast formation), which were mainly upregulated in 5 mm defects compared to 0.5 mm defects. Genes important for extracellular matrix (ECM) structural components (e.g., Col1a1, Col2a1, Col3a1), as well as 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 showed that angiogenic factor (Ang), a potent stimulator of new blood vessel formation, was downregulated, while endothelin (Edn1), an angiogenic cytokine that also stimulates cells in the osteoblast lineage, a potent vasoconstrictor, was upregulated. 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 - playing an important role in the transformation of mesenchymal cells into bone and cartilage). Table 1. Gene regulation TIFF2025096273000001.tif117161

[0106] These results first demonstrated important differences in gene expression between normally healing fractures and large bone defects at the early stage of bone healing. The most prominent differences were found in the set of genes involved in the inflammatory response, which is a key event after fracture. The upregulation of inflammatory genes in 5-mm defects compared to 0.5-mm defects suggests that large bone defects induce a stronger inflammatory response than normal fractures, which leads to an increase in the recruitment of macrophages, fibroblasts, MSCs, and bone progenitor cells. The invading inflammatory cells also produce angiogenesis-promoting cytokines, which explains the upregulation observed when comparing 5-mm defects to 0.5-mm defects. At the same time, the downregulation of many genes important for skeletal development, bone mineral metabolism, and ECM formation suggests a decrease in the bone-forming response in large bone defects.

[0107] In vitro tests were also performed to determine whether ex vivo thrombi could be produced with specific structural properties using SVCE and ecarin, and to determine whether ecarin is toxic to stem cells. The results innovatively showed that the structural properties of the thrombi changed according to the concentration of ecarin (Figure 11). For example, higher concentrations of ecarin resulted in thinner fibrin fibers with an average thickness of 93 ± 3 nm, while at the lowest concentration, the thickness of the fibrin fibers was 173 ± 9 nm. The cell growth rate from 1 to 7 days was low with increasing ecarin concentration (Figure 12). For example, there was a 4.3 ± 0.7-fold increase at the highest concentration compared to a 14.8 ± 2.6-fold increase without ecarin, and a 13.7 ± 3.1-fold increase at the lowest concentration. Cells cultured in ex vivo hematomas had a stable cell number over 7 days. Therefore, these results suggest that ecarin does not cause toxicity, but the higher the concentration, the more likely the cell growth rate is to decrease (Figure 13).

[0108] Based on these observations, additional experiments are conducted to determine the structural and biological properties of the formed hematoma in normal fractures that heal and compare them to large bone defects that do not heal without intervention (see the following examples). For example, well-organized fibrin clots are studied for their ability to enhance bone healing by serving as a temporary reservoir for the continuous release of growth factors and by providing sufficient space to assist cell infiltration, proliferation, and differentiation. Thus, the main opportunity to enhance the repair process lies in the production of thrombi that mimic the inherent properties of fracture hematoma healing. The structural properties of ex vivo-produced hematomas can be modified using SVCE, thereby mimicking endogenous fracture hematomas that enhance and accelerate bone healing when transplanted into segmental bone defects. Various snake venoms have been successfully used for diagnostic purposes in patients with heart disease, cancer, and stroke, as well as in diseases such as lupus. Thus, these experiments are also the first to examine the use of echistatin, a snake venom enzyme, 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 is explored as an improved treatment strategy because it can be used as a bio-scaffold that can more reliably enhance bone healing either by completely eliminating the need for growth factors such as BMP or by significantly minimizing the doses required to enhance the bone repair process. As a result, an inherent treatment strategy is born that is better than what is currently available. This results in significant cost savings and, most importantly, eliminates many of the harmful side effects associated with high doses of BMP. Furthermore, the results described herein can have a significant impact on the treatment of bone injuries in the general public and military personnel.

[0109] Example 2: Structure and biological properties of in vivo fracture hematomas. Bone defects were created in groups of male Sprague-Dawley 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). The structural and biological properties of the hematomas formed during normal bone healing (0.5 mm, Figure 3A) and those formed in large segmental bone defects (5 mm, Figure 3B) were characterized and compared. To evaluate the course of fracture healing, an osteotomy of 0.5 mm, which was possible with the same external fixation device, was performed. The reason for using osteotomy was that they are reproducible and allow for the formation of more consistent-sized hematomas, which are important for the characterization of hematoma structural properties. After the thrombus had matured, the animals were sacrificed 3 days after surgery to evaluate the structural properties of the fracture hematomas (Figure 3A, B). The structural properties of the hematomas, such as the thickness, density, and porosity of fibrin fibers, were evaluated using scanning electron microscopy (SEM; n = 8 / group) and ImageJ software. Additionally, different sample sets were used and RNA sequencing was employed to analyze genes with differential expression involved in the initiation of the bone repair process (n = 6 / group). Histology and immunohistochemistry (IHC) were also performed (n = 5 / group) to characterize the tissue and to confirm the presence of major proteins involved in the initiation of the repair process, such as macrophages (CD68, CD40, and CD206), osteocalcin, PECAM1, vWF, VEGF, type I collagen, and the overall tissue morphology with H&E. The results were used to determine whether there was a correlation between the structural properties in 3-day-old hematomas and the expression of specific genes and proteins.

[0110] Method Rat, large clinical size defect and osteotomy model. Male SAS Fischer rats (Charles River Laboratories, Inc., Wilmington, MA, USA) weighing approximately 200 - 250 g (10 - 12 weeks old) were anesthetized by administration of isoflurane (2%, 2 l / min) with a small animal vaporizer. Subsequently, the animals received an intramuscular injection of 20 mg / kg cefazolin (antibiotic) and 0.08 mg / kg buprenorphine (analgesic) in the left thigh. Detailed information on the surgical procedure can be verified (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 hindlimb of each animal was shaved, disinfected with chlorhexidine, placed in a sterile area, and covered with a sterile surgical drape that exposed only the right foot. A skin incision of approximately 3.5 - 4 cm was made through the skin running in the anterior direction on the surface of the right femur from the greater trochanter to the supracondylar region of the knee. The shaft of the femur was exposed by gentle dissection between the quadriceps femoris and hamstring muscles. The external fixator bar was first clipped to a Gigli wire saw guide and placed on the anterior surface of the femur to guide the drill and enable a reproducible placement of four drill holes using a pendrill (RISystem AG, Davos Platz, CH). The attachment pins were inserted into the pre-drilled holes one by one starting from the proximal side. After the fixator was placed in position, a defect was created using the saw guide. For this, the Gigli wire saw was passed through two grooves under the femur and a 5 mm segmental defect was created by reciprocating motion, and a 0.5 mm defect was created using a single wire saw. Once the defect was created, the saw guide was removed and the wound was closed in layers. For three days postoperatively, the rats were given analgesics every 12 hours and antibiotics every 24 hours. Hematomas were collected on the third day for structural and biological analysis.

[0111] In vivo fracture hematomas and ex vivo thrombi were treated similarly by scanning electron microscopy. Samples were fixed overnight in 4% paraformaldehyde. Using a Hitachi SU1510VP-SEM, the overall morphology was captured at magnifications of 100 - 1,000 times. For the analysis of fiber diameter and density, samples were post-fixed in 4% osmium tetroxide and dehydrated through a gradient of ethanol solutions (25 - 100%). Subsequently, slices of hematomas and thrombi were dried using a Leica EM Critical Point Dryer, mounted on silicon chip specimen supports, sputter-coated with gold palladium before imaging at 10,000 times (Hitachi S5500SEM / STEM) to reveal the structural characteristics at high resolution. Images were analyzed using ImageJ.

[0112] Parallel sequencing of RNA sequencing (RNA-Seq) is a high-throughput method that can globally measure the abundance of gene transcripts (Wang Z, Gerstein M, Snyder M. RNA-Seq: A revolutionary tool for transcriptomics. 2009. pp. 57-63). To determine the gene expression differences in fracture hematomas, samples were collected in microcentrifuge tubes, immediately snap-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. The concentration and quality of RNA were determined using a nanodrop spectrophotometer (ND-1000, Thermo Fisher Scientific, Inc.), and the integrity of RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. Bone cylinders removed to create 5-mm bone defects were used as controls representing the gene expression status of healthy bone. Global transcriptome analysis was used to identify upregulated and / or downregulated genes that have a major impact on the initial process of bone repair. This work was performed using an Illumina HiSeq 3000.

[0113] Histology and immunohistochemistry. Samples were stained with hematoxylin and eosin, and the overall tissue morphology was observed. A standard immunohistochemistry protocol was applied to paraffin-embedded sections (5 μm). The sections were labeled with an antibody panel to determine the spatial expression of proteins such as macrophages (CD68 and CD206), osteocalcin, PECAM1, type II collagen, and type X collagen (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).

[0114] To perform these experiments, 5 mm and 0.5 mm femoral defect models were used and stabilized with the described external fixation device. This is an established research model. The main outcome measures used were scanning electron microscopy, RNA sequencing, and histology / immunohistochemistry, which are routine procedures. As disclosed herein, data using SEM (Figures 2, 7, 8, and 9) and RNA sequencing analysis showed significant differences in the structural and biological properties between the hematomas formed in the 0.5 mm defect compared to the 5 mm defect (Table 1).

[0115] Figures 7-9 show scanning electron microscope images demonstrating the structural properties of fracture hematomas. These data show distinct structural differences between fractures that heal normally (0.5 mm) and large bone defects (5 mm). Normal fractures (0.5 mm) showed more porous, less dense, thinner fibrin fibers, and a rougher surface. Large bone defects (5 mm) showed less porosity, higher density, thicker fibrin fibers, and a smooth surface.

[0116] Example 3: Structural properties of ex vivo thrombi / hematomas formed using snake venom coagulase. To mimic the properties of a natural hematoma formed in a 0.5 mm bone cut model by altering the structural properties of thrombi within the study, ecarin of various concentrations was used. Whole blood was collected from the same animals used in Example 2 by cardiac puncture using a 21-gauge needle at euthanasia. The expected blood yield was approximately 5 - 10 mL per animal. To prevent clotting, the blood was mixed with 1 part of 4% sodium citrate solution to 9 parts of blood. The enzyme, ecarin, purified from cobra venom, was purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA). Variables such as pH, ionic strength, and calcium were kept constant while the thrombus-promoting enzyme was applied over a range from picomolar to nanomolar for the final concentration. Calcium chloride (CaCl2) and CaCl2 + recombinant human thrombin were used as controls. Ex vivo thrombi were intended to have a cylinder with a height of 5 mm spanning the defect size and a diameter of 4 mm matching the average diameter of the rat femur. Thrombi with a height of 0.5 mm were used as controls to determine if the same concentration of SVCE added to a smaller volume of blood would produce the same structural properties in the thrombi formed. Scanning electron microscopy (SEM) was used to evaluate the structural changes, particularly the thickness and density of fibrin fibers, and the overall clot structure. Figures 4A - D show thrombi in rats with different structural properties using the methods described herein.

[0117] Preparation of ex vivo thrombi. To create ex vivo thrombi, 5 - 10 mL of whole blood was collected by cardiac puncture from anesthetized rats at euthanasia. Immediately after collection, the blood was mixed 9:1 with 4% sodium citrate solution to prevent clotting. Thrombosis was induced using various concentrations of ecarin. The samples were left at room temperature for 2 hours to fully clot. Subsequently, the ex vivo thrombi were fixed with 4% PFA overnight at 4°C before being processed for scanning electron microscopy.

[0118] Scanning electron microscopy. In vivo fracture hematomas and ex vivo thrombi were treated essentially in the same way. Samples were fixed overnight in 4% paraformaldehyde. Using a Hitachi SU1510VP-SEM, the overall morphology was captured at magnifications of 100 - 1,000 times. For the analysis of fiber diameter and density, the samples were post-fixed in 4% osmium tetroxide and dehydrated through a gradient of ethanol solutions (25 - 100%). Next, slices of the hematomas and thrombi were dried using a Leica EM Critical Point Dryer, attached to silicon chip specimen supports, sputter-coated with gold palladium before imaging at 10,000 times (Hitachi S5500SEM / STEM) to reveal the structural characteristics at high resolution. The images were analyzed using ImageJ. As disclosed herein, various concentrations of ecarin were used with SVCE, and in an attempt to mimic the characteristics of hematomas formed in a 0.5 mm bone cut model, either whole blood or platelet-rich plasma (PRP) was used to alter the structural characteristics of the thrombi (ex vivo hematomas). Ecarin (0.1 and 0.5 U / mL) or CaCl2 (10 mM) was used as a coagulant. The results disclosed herein demonstrate that it is possible to create ex vivo hematomas with the required structural characteristics by various concentrations of ecarin using either whole blood or PRP.

[0119] Figures 10 - 11 show scanning electron microscope images demonstrating the structural characteristics of venom-induced ex vivo hematomas. These results show that the morphology of the thrombi can be manipulated using venom enzymes or calcium chloride, which affect the thickness and density of the fibrin fibers (Figure 11), and that the structural characteristics of ex vivo hematomas differ between those made with whole blood and those made with PRP (Figure 10).

[0120] Example 4: Determine whether this ex vivo hematoma provides a viable environment for mesenchymal stem cells (MSCs). These experiments are conducted to determine the ability of MSCs to survive when seeded within ecarin-induced thrombi as well as in the presence of ecarin alone, and to evaluate the biocompatibility of these scaffolds. The 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. Thereafter, ecarin is added to the citrated blood mixture at a concentration established from previous experiments, taking into account cell viability along with the determined specific structural characteristics. After clotting, the thrombi are transferred to 24-well plates containing growth medium.

[0121] To test the biocompatibility of ecarin-induced thrombi, samples are removed from the cultures on days 1, 7, 14, and 21 (n = 3 / group) to study the viability and differentiation potential of the seeded cells and their ability to form extracellular matrix. Further, the number of cells required in the thrombus to maximize the regenerative capacity of bone tissue is determined. Cell viability within the thrombi is evaluated using the LIVE / DEAD® Cell Viability Assay and confocal imaging.

[0122] To test the differentiation ability of the cells, the cells are cultured in adipogenic, chondrogenic, and osteogenic differentiation media. qRT-PCR is used to determine the gene expression differences at selected time points. Once cell viability is established, a hematoma most similar in its structural and biological properties to a natural hematoma during 0.5 mm defect / osteotomy bone healing is transplanted into an in vivo rat, 5 mm femoral defect model to study their ability to heal their large segmental bone defects. Based on the results in FIGS. 5A, B, it is expected that even lower concentrations of thrombin (<0.5 U / mL) ecarin will be non-toxic to the cells.

[0123] Cell culture. Rat bone marrow stem cells are cultured according to a standard protocol. The medium is changed every 3 - 4 days.

[0124] 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 a well plate on days 1, 3, and 7. After incubation at 37 °C for 20 minutes, fluorescence is read on a multiplate reader according to the manufacturer's instructions.

[0125] 2D cytotoxicity assay. Rat bone marrow stem cells are cultured in a 96 - well plate. On days 1, 3, and 7, the supernatant is collected and used to determine 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, absorbance is measured at 490 nm with background subtraction at 680 nm.

[0126] Live / Dead Staining and Confocal Microscopy in 3D Ex Vivo Thrombi. Rat bone marrow stem cells are cultured within 3D thrombi. On days 1, 3, and 7, the thrombi are removed from the culture medium, cut sagittally in half, and then immersed in serum-free medium containing 10 μM Calcein AM stock and 1 μM ethidium homodimer-1 (Thermo Fisher Scientific, Inc.) in 24-well plates. After incubation at 4 °C for 3 h and then at 37 °C, 5% CO2, and 100% humidity for 1 h, the 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).

[0127] 3D Cell Differentiation Assay. Thrombi seeded with cells are cultured in either adipogenic medium, chondrogenic medium, or osteogenic medium (StemPro® Differentiation Kits, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cell differentiation is evaluated using qRT-PCR and custom TaqMan® PCR array plates (Thermo Fisher Scientific, Inc.).

[0128] Real-time quantitative polymerase chain reaction (qRT-PCR). Ex vivo thrombi are collected in microcentrifuge tubes, immediately snap-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. The concentration and quality of RNA are determined using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher Scientific, Inc.), and the integrity of RNA is evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. One microgram of extracted RNA is reverse-transcribed into cDNA using the TaqMan (trademark) High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Inc.), and the expression of genes related to inflammation, angiogenesis, and bone neogenesis is analyzed using a customized TaqMan (registered trademark) PCR array plate (Thermo Fisher Scientific, Inc.).

[0129] As disclosed herein, the ability of bone marrow MSCs to survive in the presence of ecarin is further investigated not only when seeded into ecarin-induced thrombi but also to evaluate the biocompatibility of these scaffolds. Even when using various concentrations of coagulase from snake venom, there was no toxicity to MSCs added to ex vivo thrombi (Figs. 12-13). The enzyme, ecarin, is highly purified. Thus, it is expected that ecarin will be non-toxic to migrating cells when an ex vivo hematoma is transferred in vivo. Furthermore, in vivo experiments in a rat model were performed, which demonstrated that two different concentrations (0.3 and 0.6 U / ml) of ecarin did not cause toxicity (Figs. 14D-F). However, if it is demonstrated that ecarin is toxic to cells due to the need for higher concentrations to create thrombi with specific structural and biological properties for human subjects, calcium chloride or other coagulation factors described herein will be considered clotting agents. As mentioned herein, thrombin has been previously used to activate the coagulation cascade in platelet-rich plasma, but this product has shown insufficient performance 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 adding thrombin to PRP has been used purely as an activator and the structural properties of the clot have not been considered. In some embodiments, small doses of growth factors such as VEGF, PDGF, hFGF-2, or BMP-2 (other BMPs) may be added.

[0130] Figs. 12-13 show scanning electron microscope images showing the structural properties of ex vivo snake venom-induced ex vivo hematomas. These results indicate that cell viability was not significantly affected by the presence of snake venom enzymes, suggesting biocompatibility.

[0131] Example 5: Investigate whether an ex vivo-produced ex vivo hematoma inserted into a large bone defect can enhance the bone regeneration process in a rat femoral 5 mm large critical size defect model. A 5-mm femoral defect was created in a group of male SAS Fischer rats (10 - 12 weeks old, n = 8 - 16; pilot study n = 4; ex vivo hematoma, BMP-2, PRP) and stabilized with the external fixator described herein. Ex vivo hematoma was transplanted into the fracture gap to determine the ability to enhance the healing power of large segmental bone defects (Figure 6). Two control groups were used to compare the bone healing process with the experimental group. Healing in the first control group was enhanced using recombinant human BMP-2 delivered on an absorbable collagen sponge (Infuse®, Medtronic plc., Minneapolis, MN, USA), the same product currently used clinically.This is an established research model using BMP-2, which has been reported to heal 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), and thus, a reduced number of animals were used (n = 4). In the second control group, PRP was used to determine whether ex vivo hematoma has superior healing outcomes compared to fibrin clots rich in high concentrations of platelets. In the experimental group, ex vivo hematoma was used to determine whether the scaffolds created have the ability to regenerate bone defects. The advantages of ex vivo hematoma are as follows: (1) osteoinductivity - important growth factors persist for a long period and stimulate new bone formation, and (2) osteoconductivity - well-organized fibrin structures create a favorable microenvironment for MSC migration and early mineralization.By successfully conducting this proof-of-concept experiment, it is expected to result in the development of a biocompatible autograft that can improve the healing of large segmental bone defects without adding growth factors such as rhBMP-2, as well as ex vivo hematomas that can function as natural growth factor reservoirs. To evaluate this, the animals were monitored by weekly X-rays and euthanized at 8 weeks. After euthanasia, the recovered defects were harvested for evaluation by micro-computed tomography (μCT; all samples), and used for histology / IHC (n = 4 / group; n = 2 (BMP-2 group)) and biomechanical testing (n = 12 / group; n = 6 (BMP-2 group)).

[0132] Surgery. Rat surgery was performed as described herein.

[0133] Preparation of ex vivo hematoma for transplantation. To create an ex vivo thrombus, 5 - 10 mL of whole blood was collected from anesthetized rats by cardiac puncture at the time of euthanasia. Immediately after collection, the blood was mixed with 4% sodium citrate solution at a ratio of 9:1 to prevent coagulation. Ecarin was used at previously determined concentrations of 0.3 and 0.6 U / ml and combined with 0.55 μg of rhBMP-2 to induce blood clots. The samples were allowed to clot at 22°C (room temperature) for 45 minutes to 1 hour and then transplanted into 5 mm rat bone defects.

[0134] 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, the blood was mixed with 4% sodium citrate solution at a ratio of 9:1 to prevent coagulation. The whole blood was centrifuged at 150×g for 10 minutes 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 central platelet layer and upper plasma layer were collected. The platelet counts in the whole blood and PRP were determined using a cell counting chamber to confirm the quality of the PRP. Calcium chloride was added to prepare the PRP gel, which was transplanted 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 the bone defect, which was used as an implant.

[0135] Evaluation of bone healing. The femurs of each group of 16 animals were evaluated in vivo by X-ray weekly and after euthanasia by μCT. Twelve samples were subjected to biomechanical tests and four were used for histology. For the pilot study, four animals per group were used.

[0136] Radiography. Bone healing was monitored by weekly radiography. Under general anesthesia, as described in the surgical procedure, the rats were placed in the abdominal position and the hindlimbs were rotated laterally to obtain reproducible and standard images normal to the defect.

[0137] Micro-computed tomography (μCT). The femurs were scanned using a desktop microscopic imaging system (Bruker Skyscan 1172, Belgium) equipped with a 10 mm focal spot microfocus X-ray tube. The femurs were scanned at approximately 600 μCT slices per specimen with an isotropic voxel size of 16 μm using an energy of 75 keV and an integration time of 250 ms. The evaluation was applied to a 4 mm central defect region to confirm that the existing cortical bone was not included in the analysis. To evaluate the region of interest, the total cross-sectional area of the defect (TV, mm3 ) and bone volume (BV, mm 3 ), bone volume fraction (BV / TV, %), bone mineral density (BMD, mgHA / ccm), and polar moment of inertia (pMOI, mm 4 ) are evaluated. Images are thresholded using an adaptive iterative algorithm, and morphometric variables are calculated from the binary images using a direct 3D technique that does not depend on any prior assumptions about the underlying structure.

[0138] Mechanical testing. Following non-invasive imaging, 12 specimens from each group are tested from torsion to failure. The ends of each specimen are embedded in polymethyl methacrylate to provide a proper and reproducible grip interface with the test module. The specimens are tested to failure at a constant deformation rate of 5 rad / min under regular deformation control. Angular deformation and load force data are acquired at 10 Hz. Torque and rotation data are used to calculate the torsional stiffness and strength of the healed defects.

[0139] Histology of bone samples. Femurs (n = 4) are fixed in ice-cold 4% paraformaldehyde for 48 hours and subsequently decalcified in 14% EDTA for up to 4 weeks. Pins are removed from the bone before embedding and sectioning. Fixed and decalcified tissues are dehydrated in gradient ethanol up to 100%, transferred to xylene, and embedded in paraffin. Five-micron paraffin sections are placed on slides coated with poly-L-lysine, dried overnight, and either stained immediately or stored at 4°C. Alternate sections are stained with hematoxylin and eosin or safranin O and fast green before examination with an optical microscope. Safranin O is included to stain cartilage as part of monitoring the endochondral ossification process.

[0140] Power analysis and statistics. The sample size for each individual group was based on a coefficient of variation of 15% in the type of data collected, using an alpha level of 0.05 and 80% power (beta = 0.20). Power analysis revealed that animals with n = 8 - 16 per group, based on an effect size of 1.3 using the Student t-test, would enable detection of significant differences between groups for each outcome parameter. Previous experience with these rat models confirmed that n = 10 provides sufficient statistical power. In vitro experiments were performed in triplicate and compared for statistical significance using ANOVA tests. Sample size and power calculations were determined using version 4.0 of the nQuery Advisor software program (Statistical Solutions, Boston, MA). Statistical analyses were performed using SAS version 6.12 software (SAS Institute, Cary, NC). Two-sided p < 0.05 was considered statistically significant.

[0141] Experiments to determine the dose response in vitro are important for creating ex vivo hematomas with specific structural properties that mimic the structural properties of naturally healing fracture hematomas. If healing cannot be achieved using only ex vivo thrombi, the ex vivo hematomas are combined with either rat bone marrow mesenchymal stem cells, significantly reduced amounts of rhBMP-2 compared to currently clinically used supra-physiological doses, or any of several other growth factors.

[0142] As disclosed herein, the in vivo results of the in vivo tests at the end of the bone defect healing times (Figures 14A - F) for 8 weeks and 4 weeks (group having 0.6U / ml + 0.55μg of BMP - 2) clearly demonstrate that whole blood + ecarin (0.1U / mL) (Figure 14A) and platelet - rich plasma (PRP) + CalCl2 (10mM) (Figure 14B) do not enhance bone healing / regeneration at the concentrations of the coagulation factors tested. In contrast, when either 0.3U / mL of ecarin and 1.1μg (Figure 14D) or 0.55μg (Figure 14E) of BMP - 2 was added to the defect, the 5 - mm femoral rat defect healed. 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 (Figure 14C). Interestingly, when 0.6U / mL of ecarin + 0.55μg of BMP - 2 was used (Figure 14F), the healing was much better compared to that observed with the lower concentration of ecarin. This result would seem to indicate that the ultrastructural properties of the thrombus (ex vivo hematoma) have a major impact in enhancing the healing of bone defects.

[0143] Example 6: Biomimetic Hematoma: Effect of Coagulants and rhBMP - 2 Concentrations on Bone Healing with rhBMP - 2 / ACS. Not only coagulants, ecarin, calcium / thrombin, but also various concentrations of BMP-2 were tested and demonstrated the ability to initiate the healing of large segmental bone defects. The concentrations of ecarin tested included 0.3, 0.6, and 0.75 U / mL, and these concentrations initiated healing normally (Figure 15, columns 4 - 8), but the concentration of ecarin at 0.6 U / mL showed the best results among the doses tested. Similarly, the combination of 10 mM CaCl2 of the coagulant and 0.5 U / mL of thrombin (Figure 15, column 3) also effectively healed large bone defects in a manner similar to 0.6 U / mL of ecarin and 0.33 μg of BMP-2 (Figure 15, column 4). The concentration of BMP-2 that consistently initiated the 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 minimum 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, Figure 15, first column). The other two tested doses that initiated the healing of bone defects were 0.165 μg and 0.0825 μg (see Figure 15, columns 7 and 8), but the responses to these doses were less consistent compared to 0.33 μg, being 75% and 50% respectively (for example, see column 5).

Claims

1. An ex vivo hematoma comprising: (a) isolated whole blood; (b) sodium citrate; and (c) ecarin; oscutin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride, wherein the ex vivo hematoma comprises fibrin fibers having a thickness of at least 150-300 nm ± 10%.

2. 1. An ex vivo hematoma comprising: (a) platelet-rich plasma, plasma, or plasma with red blood cells; and (b) ecarin; oscutin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride, wherein the ex vivo hematoma comprises fibrin fibers having a thickness of at least 150-300 nm±10%.

3. 3. The ex vivo hematoma of claim 2 further comprising sodium citrate.

4. The ex vivo hematoma of claim 1 or 2, further comprising an antibiotic.

5. The ex vivo hematoma of claim 1 or 2, further comprising one or more growth factors.

6. 6. The ex vivo hematoma of claim 1 or 5, 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 ex vivo hematoma of claim 1 , wherein the whole blood comprises viable cells and one or more biological factors.

8. 8. The ex vivo hematoma of claim 7, wherein about 50%-70% of the viable cells of the whole blood remain viable after formation of the hematoma.

9. The ex vivo hematoma of claim 1 or 2, further comprising a therapeutic agent.

10. 2. The ex vivo hematoma of claim 1 comprising whole blood, ecarin, and sodium citrate.

11. 10. The ex vivo hematoma of claim 1 comprising whole blood, calcium chloride, and sodium citrate.

12. The ex vivo hematoma of claim 2 comprising platelet-rich plasma and ecarin.

13. The ex vivo hematoma of claim 2 comprising platelet-rich plasma and calcium chloride.

14. 2. The ex vivo hematoma of claim 1 comprising whole blood, sodium citrate, and thrombin.

15. 2. The ex vivo hematoma of claim 1 comprising whole blood, calcium chloride, or oscutaline and calcium chloride, and sodium citrate.

16. 3. The ex vivo hematoma of claim 1 or 2, wherein the concentration of ecarin present in the ex vivo hematoma is at least 0.05 U / mL.

17. 3. The ex vivo hematoma of claim 1 or 2, wherein the concentration of ecarin present in the ex vivo hematoma is 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 U / mL.

18. The ex vivo hematoma of claim 1 or 2, further comprising bone morphogenetic protein 2 (BMP-2).

19. The ex vivo hematoma of claim 9, wherein the therapeutic agent is bone morphogenetic protein 2 (BMP-2).

20. 20. The ex vivo hematoma of claim 18, wherein the dose of BMP-2 present in the ex vivo hematoma is at least 0.01 mg.

21. The ex vivo hematoma of claim 18, wherein the BMP-2 is recombinant BMP-2.

22. The ex vivo hematoma of claim 21 , wherein the recombinant BMP-2 comprises human BMP-2.

23. The ex vivo hematoma of claim 1 or 2, further comprising growth factors, platelets, and cells.

24. The ex vivo hematoma of claim 1 or 2, formulated as a gel or liquid.

25. The ex vivo hematoma of claim 1 or 2, formulated for local administration.

26. 19. The ex vivo hematoma of claim 18, wherein the amount of ecarin present in the ex vivo hematoma is at least 0.05 U / mL and the amount of BMP-2 present in the ex vivo hematoma is at least 0.01 mg.

27. A method for promoting bone healing or producing a bone replacement material or implant, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the ex vivo hematoma described in claim 1 or 2.

28. 30. The method of claim 27, wherein the whole blood comprises viable cells and one or more biological factors.

29. 28. The method of claim 27, wherein the ex vivo hematoma comprises whole blood, ecarin and sodium citrate; whole blood, calcium chloride and sodium citrate; platelet-rich plasma and ecarin; or platelet-rich plasma and calcium chloride.

30. 28. The method of claim 27, wherein the ex vivo hematoma further comprises bone morphogenetic protein 2 (BMP-2).

31. The method of claim 30, wherein the BMP-2 is recombinant BMP-2.

32. The method of claim 31 , wherein the recombinant BMP-2 comprises human BMP-2.

33. 28. The method of claim 27, further comprising growth factors, platelets, and cells.

34. 28. The method of claim 27, wherein the subject is a human.

35. 28. The method of claim 27, wherein the composition is formulated as a clot or scaffold.

36. 28. The method of claim 27, wherein the composition is formulated for topical administration.

37. 28. The method of claim 27, wherein the composition is administered topically, implanted, or delivered transdermally.

38. The method of claim 27 , wherein the composition is implanted.

39. 31. The method of claim 30, wherein the amount of ecarin present in the composition is at least 0.05 U / mL and the amount of BMP-2 present in the composition is at least 0.01-5 mg.

40. 28. The method of claim 27, wherein the subject has a skeletal defect.

41. 41. The method of claim 40, wherein the skeletal defect is a large segmental bone defect.

42. 30. The method of claim 27, wherein the subject has one or more fractures.

43. 28. The method of claim 27, wherein the subject has one or more bone injuries.

44. 1. A method of constructing an implant, comprising: a) dimensioning the depot implant in at least one of a shape and a size that facilitates implantation of the depot implant into a bone defect; b) constructing said depot implant for having a scaffold by introducing (i) isolated whole blood and sodium citrate; or platelet rich plasma, plasma, or plasma with red blood cells; and (ii) ecarin; oscutin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride to create said scaffold; wherein the scaffold has a porosity of 55-75%.

45. 45. The method of claim 44, wherein the scaffold comprises fibrin fibers having a thickness of at least 150-300 nm ± 10%.

46. 45. The method of claim 44, wherein the shape of the depot implant is that of a cylinder or a sphere.

47. The method of claim 44, wherein the scaffold is constructed as a clot.

48. 45. The method of claim 44, further comprising one or more growth factors.

49. 45. The method of claim 44, 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.

50. The method of claim 49, wherein the BMP-2 is introduced into the scaffold.

51. 50. The method of claim 49, wherein the amount of ecarin present in the scaffold is at least 0.05 U / mL and the amount of BMP-2 present in the scaffold is at least 0.01 mg.

52. 45. The method of claim 44, wherein the scaffold resembles the size and shape of a given bone defect.

53. 45. The method of claim 44, wherein the scaffold is chemotactic.

54. 45. The method of claim 44, wherein the scaffold comprises viable blood cells and appropriate biological factors.

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