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WO2025166131A1 - Compositions et méthodes de traitement de lésions osseuses et musculaires - Google Patents

Compositions et méthodes de traitement de lésions osseuses et musculaires

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Publication number
WO2025166131A1
WO2025166131A1 PCT/US2025/013987 US2025013987W WO2025166131A1 WO 2025166131 A1 WO2025166131 A1 WO 2025166131A1 US 2025013987 W US2025013987 W US 2025013987W WO 2025166131 A1 WO2025166131 A1 WO 2025166131A1
Authority
WO
WIPO (PCT)
Prior art keywords
bmp
mrna
vivo
composition
hematoma
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/013987
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English (en)
Inventor
Vaida Glatt
Masatoshi Suzuki
Joshua A. CHOE
William L. Murphy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
Wisconsin Alumni Research Foundation
University of Texas at Austin
Original Assignee
University of Texas System
Wisconsin Alumni Research Foundation
University of Texas at Austin
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Filing date
Publication date
Application filed by University of Texas System, Wisconsin Alumni Research Foundation, University of Texas at Austin filed Critical University of Texas System
Publication of WO2025166131A1 publication Critical patent/WO2025166131A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3616—Blood, e.g. platelet-rich plasma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/30—Materials or treatment for tissue regeneration for muscle reconstruction

Definitions

  • the present application contains a sequence listing that is submitted concurrent with the filing of this application, containing the file name “21105_0097Pl_SL” which is 24,576 bytes in size, created on January 29, 2025. and is herein incorporated by reference in its entirety 7 pursuant to 37 C.F.R. ⁇ 1.52(e)(5).
  • VML Volumetric muscle loss
  • Therapeutic modalities include surgical debridement, bracing, extensive rehabilitation, and free muscle transfer, which is the most effective modality.
  • these interventions are severely limited by constraints like donor site availability, morbidity, motor dysfunction, and frequent rehabilitation failures leading to limb loss through amputation.
  • the limitations of current therapies underscore the need for the development of new treatments for VML.
  • compositions comprising: an ex vivo hematoma, wherein the ex vivo hematoma comprises: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • ex vivo hematoma comprises: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • multi-compartment devices comprising a first chamber comprising isolated whole blood; a second chamber comprising calcium chloride; thrombin; or thrombin and calcium chloride; and a third chamber comprising a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • biomimetic scaffolds comprising a scaffold and an ex vivo hematoma, wherein the ex vivo hematoma comprises: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger ribonucleic acid
  • a biomimetic scaffold comprising: a) dimensioning a scaffold in at least one of a shape and a size that facilitates implantation of the scaffold into a bone defect; and b) combining the scaffold in a) with an ex vivo hematoma comprising: (i) isolated whole blood and sodium citrate; (ii) calcium chloride; thrombin; or thrombin and calcium chloride; to create the biomimetic scaffold; c) combining the biomimetic scaffold in b) with a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • FIGS. 1 A-D show an overview of the healing muscle (FIGS. 1 A. 1C) indicating the location of magnified areas (FIGS. IB, ID). Masson’s Trichrome stained sections (FIGS. 1A, IB) ex vivo hematoma alone; (FIGS. 1C, ID) RSPO2 mRNA LNP + FC MCM + ex vivo hematoma.
  • FIG. 2 shows scanning electron microscopy images illustrating the architectural changes of the ex vivo hematoma (e.g., biomimetic hematoma (BH)) with introduced mRNA LNP (BH+LNP) or MCMs (BH+MCM) compared to the ex vivo hematoma alone (BH).
  • BH biomimetic hematoma
  • MCMs MCMs
  • FIG. 3 provides and ovendew of WNT and R-Spondin signaling.
  • R-Spondins bind LGRs and prevent the ubiquitin ligases ZNRF3/RNF43 from promoting degradation of the WNT receptor (LRPs).
  • R- Spondin-2/3, but not RSPO-1/4 contain a domain that inhibits BMP receptors.
  • FIG. 4 shows an ovendew of the in vitro methods used in Example 2.
  • Mineral coated microparticles were created with a ratio of 0.5X (Ca 2+: PC>4 3 ' ) and 5mM citric acid, 1 mM NaF mRNA for Gaussia Luciferase (G.Luc), Bone morphogenetic protein-2 (BMP-2), RSPO-2 and RSPO-1 were created and encapsulated in lipoplexes prior to delivery.
  • RSPOs mRNA +/- BMP-2 mRNA was delivered to human mesenchymal stromal cells (hMSC) for 14 days and assayed by alizarin red staining.
  • C2C12 myoblasts were treated with RSPO-2 mRNA, GLuc mRNA or rWNT3 A and differentiated for two weeks.
  • FIG. 5 shows the scanning electron microscopy of PTCP core material (left) and F-Cit MCMs (right). 300 nm scale shown.
  • FIG. 6 A shows that Mineral coatings (MCM) improve transfection of G.Luc mRNA compared to uncoated core material or free mRNA complexes alone.
  • FIG. 6B shows RSPO-2 ELISA: RSPO-2 mRNA delivery via MCMs over expresses RSPO-2 mRNA. EDTA was used to dissolve MCMs and release any bound RSPO-2. Significance by ANOVA. **** p ⁇ 0.0001.
  • FIGS. 7A-L show alizarin red staining of hMSCs on d!4 following mRNA treatment +/- MCMs. Top row: mRNA complexes alone, Bottom row: mRNA complexes + MCMs.
  • BMP-2 (FIGS. 7A-B).
  • RSPO-2 (FIGS. 7C-D).
  • RSPO-2 with a mutant TSP-1 domain from RSPO-1 lacking BMP-R binding domain (FIGS. 7E-F).
  • RSPO-2 (TSP-1) mRNA with equal dose of BMP-2 mRNA (FIGS. 7G-H).
  • RSPO-1 mRNA FIGS. 71- J). Osteogenic (Os) media (FIGS. 7K-L).
  • FIGS. 8A-H show myosin heavy chain staining (MYHC) on d!4 of myogenic differentiation of murine C2C12 cells. Top row: control treatment, bottom row: MCM treatment.
  • FIGS. 8A-B show negative control.
  • FIGS. 8C-D show RSPO-2 mRNA delivery.
  • FIGS. 8E-F show GLuc mRNA delivery'.
  • FIGS. 8G-H show rWNT3A delivery'.
  • FIGS. 9A-D gene expression data by RT-qPCR for pro-myogenic factors.
  • FIG. 9A shows myogenin (myog).
  • FIG.9B shows Myf5 (myf5).
  • FIG. 9C shows WNT marker Axin2.
  • FIG. 9D show myostatin.
  • FIGS. 10A-E show the in vivo methods and results.
  • FIG. 10A shows a schematic overview of an ex vivo hematoma comprising mRNA.
  • mRNA was encapsulated in SM-102 LNPs and bound to MCMs that were mixed with isolated whole blood and clotting factors.
  • FIG. 10B show rat tibialis anterior (TA) volumetric muscle loss (VML) model. 30% of the TA was excised and an ex vivo hematoma comprising mRNA was implanted for 2 weeks.
  • FIG. 10C shows the ex vivo hematoma implant 2 days after surgery'.
  • FIG. 10D shows 2 weeks post-surgery.
  • FIG. 10E shows Masson's tri chrome staining of TA muscles 2 weeks after surgery. Scale: 500 microns.
  • FIGS. 11A-D show Alizarin red staining of hMSCs on d21 of differentiation after treatment with mRNA WT-RSPO-2 (FIGS. 11 A-B) or Mut-RSPO-2 (FIGS. 11C-D) without (top row) or with (bottom row) MCM treatment.
  • FIGS. 11E-H show myosin heavy chain (MYHC) and nuclear (DAPI) staining of C2C 12 cells on d7 of differentiation after treatment with FIG. HE shows negative control.
  • FIG. 1 IF shows MCM alone.
  • FIG. 11G shows RSPO2 mRNA alone.
  • FIG. 11H shows RSPO2 mRNA + MCM.
  • FIGS. 1 II- J shows gene expression on d7 of myogenic differentiation from C2C12 cells for myf5 (FIG. Ill) and myostatin (FIG. 11J).
  • the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps.
  • each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.
  • Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to “about” or “approximately” another particular value.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • values are expressed as approximations, by use of the antecedent “about,” or “approximately,” it will be understood that the particular value forms a further aspect.
  • the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint.
  • 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.
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • the term “subject” refers to the target of administration, e g., a human.
  • the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • a subject is a mammal.
  • the subject is a human.
  • the term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the term “patient” refers to a subject afflicted with a disease or disorder or condition.
  • the term “patient” includes human and veterinary subjects.
  • the “patient” has been diagnosed with a need for treatment for healing of bone injuries or the healing of muscle injuries, such as, for example, prior to the administering step.
  • treating refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of. reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
  • Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • the disease, disorder, and/or condition can be a bone injury, a bone fracture, muscle loss, or muscle injury.
  • an effective amount, a therapeutically effective amount, a prophylactically effective amount and a diagnostically effective amount can refer to an amount of RNA or mRNA adsorbed to or incorporated into the mineral layer of the mineral coated microparticle needed to elicit the desired biological response following administration.
  • mRNA complexes refer to complexes of mRNA with a complexing agent such as a lipopolyplex or a lipid nanoparticle complexing agent.
  • a “complexing agent”, as used herein, refers to a transfection agent that binds to mRNA and promotes cell transfection efficiency.
  • Complexing agents used herein include, but are not limited to, lipopolyplexes and lipid nanoparticles.
  • Lipopolyplex refers to a core-shell structure composed of nucleic acid, polycation, and lipid.
  • a “microparticle”, as used herein, refers to a particle that has a particle size in the micrometer range (or a particle size between about 0.2 um and about 1000 um).
  • a “mineral-coated substrate” is a substrate coated with a mineral coating layer containing at least calcium, phosphate, and carbonate.
  • Complex formation refers to the complex formation between mRNA and the complexing agent.
  • hematoma formation blood/fibrin clot
  • hematoma formation blood/fibrin clot
  • hematoma formation blood/fibrin clot
  • hematoma formation blood/fibrin clot
  • hematoma formation blood/fibrin clot
  • BH Biomimetic Hematoma
  • ex vivo hematoma is the only known carrier capable of efficiently delivering much lower doses of rhBMP-2.
  • the ex vivo hematoma can consistently and robustly initiate the natural bone repair cascade, successfully- reconstructing large bone defects without side effects.
  • tissue- engineered and regenerative medicine therapeutic strategies are being developed. These approaches aim to repair and replace damaged muscle using instructive biomaterial scaffolds, biologically active molecules, and cells (Langer R, Vacanti JP. 1993. Science: 260:920-6; and Nakayama KH, et al. 2019. Adv Healthc Mater:8:el801168). The goal of these approaches is to prevent scar tissue formation and promote enhanced functional muscle regeneration.
  • interleukins [IL-8, IL-6, IL-1 ] and tumor necrosis factor-a [TNF-a]) influences local blood flow, vascular permeability, and accelerates the inflammatory response (Carnes ME, Pins GD. 2020. Bioengineering (Basel):7:85). Additionally, the release of growth factors such as insulin-like growth factor- 1 (IGF-1), hepatocy te growth factor (HGF), transforming growth factors (TGF-a and TGF-[3), and platelet-derived growth factors (PDGF-AA and PDGF-BB) at the injured site regulates myoblast proliferation and differentiation, thereby promoting muscle regeneration and repair.
  • IGF-1 insulin-like growth factor- 1
  • HGF hepatocy te growth factor
  • TGF-a and TGF-[3 transforming growth factors
  • PDGF-AA and PDGF-BB platelet-derived growth factors
  • Biomaterial scaffolds employed to deliver biologically active molecules or cells lack these components that are present in a naturally formed hematoma. This deficiency may explain why these therapeutic approaches face challenges in achieving successful regeneration of functional muscle and seamless integration with the surrounding native muscle tissue.
  • the ex vivo hematoma disclosed herein can mimic the properties of a naturally healing muscle hematoma, and enhance the effectiveness of delivered biologically active molecules for the functional regeneration of VML.
  • the ex vivo hematoma closely resembles the structural and biological characteristics of normally healing fracture hematoma and can be used to efficiently deliver growth factors to stimulate bone repair.
  • Preclinical studies in small (Woloszyk A, et al. 2023. Biomater Adv: 148:213366) and large animals, along with a clinical study (Glatt V, Tetsworth K. 2023.
  • an ex vivo hematoma can comprise mRNA that can be delivered to the injury site using, for example, lipid nanoparticles with mineral coated microparticles (MCM) to promote regeneration.
  • MCM mineral coated microparticles
  • mRNA for R-spondin-2, Bone Morphogenetic Protein-2, or Bone Morphogenetic protein-2/-7 can be used.
  • R-spondin-2 is a WNT agonist involved in muscle differentiation, neuromuscular junction formation, and bone mineralization.
  • Bone morphogenetic proteins (BMP) are intrinsically involved in bone formation, mineralization and repair with BMP-2 and BMP-2/-7 heterodimers among the most potent.
  • mRNA and biomimetic and biocompatible mineral coated microparticles were used and combined within a biomimetic hematoma to deliver to a target site (site of injury) to repair large muscle, bone, and/or composite defects.
  • the results demonstrate beneficial improvements in muscle and bone differentiation in vitro.
  • the results described herein also show robust healing of a large rat tibialias anterior muscle defect in vivo and ectopic bone formation in vivo.
  • the disclosed compositions and methods have advantages over existing therapies.
  • the disclosed compositions and methods can be used for treating bone defects, as a recombinant protein therapy, and to deliver bone morphogenetic protein-2 (BMP-2), which can be used to initiate native repair of bone defects.
  • BMP-2 bone morphogenetic protein-2
  • extensive side effects related to supraphysiologic dosages and a poor delivery vehicle, an absorbable collagen sponge have limited adoption clinically.
  • Gene delivery via mRNA administered using the disclosed ex vivo hematoma can improve the potency of BMP-2 since this allows for local translation, native folding and post-translational modifications.
  • co-delivery or substitutions of different genes is simple since the packing and delivery mechanisms of mRNA with lipid nanoparticles (LNPs) are universally translatable between mRNA gene sequences.
  • LNPs lipid nanoparticles
  • mRNA therapeutics are stable and storage since mRNA LNPs are prone to hydrolysis and degradation.
  • Current mRNA vaccines have to be shipped and stored frozen and used within days of thaw ing to maximize efficacy that limit translation and increase expenses.
  • MCMs mineral coated microparticles
  • MCMs can improve the storage and delivery of therapeutic mRNA.
  • MCMs improve the storage of mRNA complexes with freeze-drying and promote long-term room temperature storage of the mRNA complexes up to 6 months. Additionally MCMs improve the transfection of mRNA lipid nanoparticles.
  • Intrinsic bone healing involves the formation of a hematoma that uses blood cells, migrating cells, and a fibrin scaffold to remodel into native bone. Described herein is the use of mRNA lipid nanoparticles with biocompatible mineral coated microparticles which are combined with or within an ex vivo hematoma to deliver to an injury site to promote and enhance muscle and/or bone healing.
  • These compositions and methods compared to other research grade technologies has the advantage of being quick to synthesize, simple and flexible without need for ex vivo expansion of cells or other materials.
  • This technology can also address reconstructive challenges associated with composite musculoskeletal defects. Bone fractures with overlying muscle loss is a challenging clinical scenario and is recalcitrant to healing by' BMP-2.
  • a dual mRNA ex vivo hematoma can be used to promote muscle and bone formation with the inhibition of spillover bone formation in the muscle (heterotopic ossification) through native actions of R-spondin-2 mRNA.
  • mRNA for R-Spondin-2 (RSPO-2), or bone morphogenetic proteins can be used to promote bone and/or muscle regeneration at an injury site.
  • R-spondin- 2 is a WNT agonist involved in muscle and bone formation.
  • Another advantage of RSPO-2 is that it is involved in neuromuscular junction formation which is required for proper muscle integration and function in tandem with the nervous system.
  • RSPO-2 promotes muscle differentiation
  • WT wild type
  • RSPO-2 inhibits signaling, and subsequent mineralization induced by BMP -2.
  • a mutant RSPO-2 with the TSP-1 domain from RSPO-1 has been shown to promote BMP -2 signaling and improve osteogenesis. Therefore, for composite musculoskeletal defect healing wild type RSPO-2 can be used to regenerate native muscle and prevent heterotopic ossification, while mutant RSPO-2 mRNA can be used in tandem with BMP mRNA to promote bone defect healing, and these effectshave been demonstrated in vitro (see Examples).
  • RSPO-2 mRNA can be used to heal a large muscle defect in a rodent within two weeks.
  • compositions and methods disclosed herein can be used for civilian or battlefield treatment of bone and muscle defects.
  • the compositions and methods disclosed herein are simple and can be applied within a single surgical window; wherein blood from the patient, or blood bank, can be harvested. This blood can then be mixed with mRNA lipid nanoparticles with or without mineral coated microparticles, and clotting factors to form a composition comprising mRNA combined with ex vivo hematoma which can be achieved within 20 minutes, followed by directly implanting the ex vivo hematoma into the defect site.
  • mineral coated microparticles can allow for a freeze-dried mRNA product to be generated permitting an "off-the-shelf mRNA therapy to be manufactured and stored for months prior to operation.
  • compositions disclosed herein can be produced, packaged in a kit and used for muscle and bone regeneration.
  • the compositions disclosed herein takes advantage of the patient's own cells, in the blood, without need for external expansion or complicated manipulation.
  • Other approaches require synthesis of exogenous materials and combination with cells that need to be harvested and expanded for weeks to months prior to therapeutic application.
  • Total combination of the materials for this therapeutic can be created in 20 minutes, within a single surgical window;
  • Blood can be harvest from the patient directly or from a blood bank and combined with the other materials on demand.
  • the blood is combined with simple, FDA approved materials to form a blood clot (e.g., ex vivo hematoma) that can be made to fit the musculoskeletal defect architecture.
  • mRNA lipid nanoparticles and MCMs can be stored frozen over months. Furthermore, mRNA LNPs with MCMs can be stored at room temperature with MCMs allowing for an ‘off-the-shelf product immediately available for use in the operating room.
  • the compositions and methods disclosed herein can be adapted for other regenerative medicine applications by changing the mRNA sequence without impacting deliver ⁇ ' characteristics within the blood clot.
  • compositions comprising: an ex vivo hematoma, wherein the ex vivo hematoma comprises: (a) isolated whole blood; (b) sodium citrate; and (c) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride; and a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the compositions can further comprise a bone substitute.
  • compositions comprising: an ex vivo hematoma, wherein the ex vivo hematoma comprises: (a) platelet rich plasma, plasma, or plasma with red blood cells; and (b) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride; and a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the composition can further comprise a bone substitute.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride; thrombin; or thrombin and calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) thrombin.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) thrombin; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) thrombin and calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) platelet rich plasma, plasma, or plasma with red blood cells; and (b) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride.
  • the ex vivo hematoma can further comprise sodium citrate.
  • the ex vivo hematomas can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%. In some aspects, the ex vivo hematomas can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • the phrase “plasma with red blood cells” means plasma without platelets.
  • the ecarin; oscutarin 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%.
  • the phrase “plasma with red blood cells” means plasma without platelets.
  • the ecarin; oscutarin 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 100-400 nm ⁇ 10%.
  • ex vivo refers to a hematoma that can be formed outside of an organism, for example, in an external environment.
  • the ex vivo hematoma can comprise (a) isolated whole blood and sodium citrate; platelet rich plasma, plasma alone, plasma with red blood cells (without platelets) or other blood products; and (b) one or more coagulating factors.
  • the ex vivo hematomas can comprise whole blood and one or more coagulating factors.
  • the terms “whole blood” and “blood” are used here to mean blood that can be draw n directly from the body from which none of the components, including plasma or platelets, have been removed.
  • the whole blood or blood can be from the subject or patient that will be the receipt of any of the compositions described herein or any of the ex vivo hematomas described herein.
  • the whole blood or blood can be from a donor subject or patient.
  • Whole blood is made up of red blood cells, white blood cells, platelets and plasma.
  • a fibrin gel can be used in place of whole blood.
  • blood is a specialized body fluid that delivers important substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells.
  • blood is composed of blood cells suspended in blood plasma.
  • Blood can comprise different components, for example, plasma, red blood cells (ery throcytes), platelets (thrombocytes) and white blood cells (leukocytes).
  • Plasma is the main component, making up about 55%, of blood, consisting of mostly water with ions, proteins, nutrients and wastes. Plasma can contain some of every protein produced by the body.
  • plasma can comprise about 90% water and 10% of a mix of the following: ions (Na + , K + , Mg +2 , Ca +2 , Cl’, HCOv.
  • SOT 2 Neuronal et al., 2012
  • proteins e.g., mainly albumin-55%, globulin, growth factors, enzymes, hormones, antibodies
  • clotting factors Factors I-XI1I
  • sugars glucose
  • lipids cholesterols
  • minerals sodium, calcium, magnesium, potassium, iron, zinc, copper, and selenium
  • Red blood cells erythrocytes
  • erythrocytes are responsible for carrying oxygen and carbon dioxide. They are about 7-8 pm in size, contain no mitochondria or nucleus when mature, and have an average life span of 120 days.
  • WBCs white blood cells
  • a normal white blood cell count ranges from about 5, 000-10, 000/mm 3 .
  • White blood cells can be divided into 5 major types that are further divided into two different groups: Granulocytes: Neutrophils: 60-70% of WBCs or 3, 000-7, 000/mm 3 , Eosinophils: 1-3% of WBCs or 50-400/mm 3 . and Basophils: 0.3-0.5% of WBCs or 25-200/mm 3 ; and Agranulocytes: Lymphocytes: 20-
  • platelet rich plasma refers to a concentrated form of platelet rich plasma protein that is derived from whole blood.
  • whole blood can be centrifuged to remove red blood cells.
  • blood plasma alone”, “plasma alone” or “plasma” can refer to a yellowish liquid component derived from whole blood that normally holds blood cells in whole blood in suspension.
  • blood plasma can be separated from whole blood by centrifuging 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.
  • the term “plasma with red blood cells” can refer to “plasma alone” with added red blood cells.
  • red blood cells can be derived by centrifuging whole blood until they fall to the bottom of the tube, and are retrieved after removing plasma, white blood cell and platelets from the top of the tube.
  • the mRNA-loaded lipid nanoparticles can be combined with a mineral coated microparticle.
  • the mineral coated microparticle comprises a mineral coating layer containing at least calcium, phosphate, and carbonate.
  • the mRNA-loaded lipid nanoparticles bind to the mineral coated microparticle.
  • “microparticle” can refer to a particle that has a particle size in the micrometer range (or a particle size between 0.2 pm and about 100 pm).
  • the mRNA-loaded lipid nanoparticles can be adsorbed to the mineral coating layer of the mineral coated microparticle.
  • the messenger ribonucleic acid (mRNA)-based therapeutic composition comprises: a mineral-coated substrate; a mRNA complex(es) bound to the mineral-coated substrate, wherein the mRNA complexes include mRNA complexed with a DOTAP-substituted lipid nanoparticle; and a lyoprotectant, wherein the mRNA-based therapeutic composition is lyophilized to a dry powder.
  • the ribonucleic acid (RNA)-based therapeutic composition comprises: a mineral-coated substrate; an RNA complex(es) bound to the mineral-coated substrate, wherein the RNA complexes include RNA complexed with a DOTAP-substituted lipid nanoparticle; and a lyoprotectant, wherein the RNA-based therapeutic composition is lyophilized to a dry powder.
  • the mRNA complex can comprise mRNA.
  • the mRNA-loaded lipid nanoparticles can comprise mRNA.
  • the mRNA-based therapeutic composition comprises a mineral- coated substrate; a mRNA complex(es) bound to the mineral-coated substrate, wherein the mRNA complexes include mRNA complexed with lipid nanoparticle.
  • the mRNA-based therapeutic composition can further comprise a ly coprotectant. In some aspects, the mRNA-based therapeutic composition can be lyophilized to a dr ' powder.
  • the mRNA-based therapeutic compositions can comprise mRNA.
  • the mRNA can be a therapeutic mRNA.
  • the mRNA or the therapeutic mRNA can include an mRNA complex formed of mRNA complexed with a complexing agent (e.g., lipid nanoparticle (LNP)), and a mineral coated microparticle (MCM) having one or more mineral coating layers that stabilize the mRNA complexes and improve transfection efficiency of the composition after freeze drying/lyophilization.
  • a complexing agent e.g., lipid nanoparticle (LNP)
  • MCM mineral coated microparticle
  • Further included in the composition can be one or more lyoprotectants that protect the mRNA complexes from damage that may occur during freeze drying/lyophilization.
  • the resulting composition can be provided as a dry medication or powder that is reconstituted in solution prior to administration to a subj ect.
  • the complexing agent can be a transfection reagent that forms a complex with the mRNA and improves transfection efficiency.
  • Suitable complexing agents may include positively charged lipid-based complexes such as lipopolyplexes (LPP) and lipid nanoparticles (LNP).
  • LNP lipopolyplexes
  • LNP lipid nanoparticles
  • Such an LNP may comprise DLin-MC3-DMA, DMG-PEG(2000), and 1,2-DSPC (LNP-MC3 Exploration Kit, Cayman Chemical).
  • SM-102 DMG-PEG(2000). 1,2- DSPC (LNP- 102 Exploration Kit, Cayman Chemical), or any other suitable lipids.
  • the lyoprotectant can be a disaccharide that interacts with the polar head groups of the complexing agent to prevent damage during lyophilization and help with long term storage of the composition.
  • Preferred lyoprotectants include trehalose or sucrose.
  • the therapeutic composition may contain from about 5 millimolar (mM) to about 1 molar (M), or about 30 mM to about 1 M of the disaccharide (prior to lyophilization).
  • the composition may contain about 150 mM trehalose.
  • the composition may contain about 250 mM or about 254 mM sucrose.
  • lyoprotectants may also be used including, but not limited to, glucose, maltose, lactose, inositol, dextran, hydroxypropyl-P-cyclodextrin, polyethylene glycol, and combinations thereof.
  • the MCMs can include at least one mineral coating layer.
  • the mineral coating layer may have different ratios of calcium, phosphate, and carbonate.
  • the calcium: phosphate ratio in the mineral coating layer can vary from about 0. 1 to about 10, or from about 2 to about 5.
  • the carbonate concentration of the mineral coating layer can vary from about 1 mM to about 150 mM, or from about 3 mM to about 100 mM. Other ratios/concentrations of calcium, phosphate, and carbonate may be used in alternative embodiments.
  • Different morphologies of the mineral coating layer e.g.. plate-like, spherulite-like, etc.
  • the MCM can include a core material in the form of a microparticle coated with the mineral coating layer.
  • suitable core materials on which the mineral coating layer is formed may include polymers, ceramics, metals, glass, and combinations thereof in the form of microparticles.
  • suitable microparticles include ceramics (e.g.. hydroxyapatite, beta-tricalcium phosphate (0-TCP). magnetite, neodymium), plastics (e g., polystyrene, poly-caprolactone), hydrogels (e.g., polyethylene glycol, poly(lactic-co-glycolic acid) and the like, and combinations thereof.
  • Particularly suitable core materials include those that are dissolved in vivo such as P-TCP and hydroxyapatite.
  • the mRNA complexes can be adsorbed to the mineral coating layer of the MCM. Upon cell transfection or administration, the mRNA complexes adsorbed to the mineral coating layer may be released as the mineral coating layer degrades. Upon introduction to the cytoplasm (via endocytosis, micropinocytosis or other mechanism), the mRNAs may proceed to translation for protein production. After translation and processing, the MCMs used for initial delivery of the mRNA complexes may bind and sequester the secreted protein. The protein may be released from the MCM over time back to the cell, prolonging the biological response.
  • the mRNA can include any therapeutically active mRNA.
  • the mRNA can encode a protein of interest.
  • the mRNAs can encode any protein of interest.
  • the mRNAs can encode proteins including growth factors and reporters. Suitable reporters can be, for example, green fluorescent protein, chloramphenicol acetyl -transferase, P-galactosidase, P-glucuronidase, and luciferase.
  • U.S. Patent No. 11,779,542 is incorporated by reference herein for its disclosure of compositions and making of compositions that include mineral coated microparticles having a mineral layer and a ribonucleic acid.
  • the mRNA used in the disclosed compositions can encode roof platespecific sondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2), transforming growth factors (e.g., TGF-a and TGF-P), and platelet-derived growth factors (e.g., PDGF-AA and PDGF-BB), insulin-like growth factor-1 (IGF-1), tumor necrosis factor-a (TNF-a), vascular endothelial growth factor (e.g... VEGF A and B). or basic fibroblast growth factor (bFGF).
  • RSPO-2 roof platespecific sondin-2
  • NRF2 nuclear factor erythroid 2-related factor 2
  • TGF-a and TGF-P transforming growth factors
  • platelet-derived growth factors e.g., PDGF-AA and PDGF-BB
  • IGF-1 insulin-like growth factor-1
  • TNF-a tumor necrosis factor-a
  • VEGF A and B vascular endothelial growth factor
  • the mRNA can encode bone morphogenetic protein-2 (BMP- 2), BMP-2/BMP-4, BMP-2/BMP-7, BMP-4/BMP-7, BMP-6, or BMP-9.
  • BMP-2 bone morphogenetic protein-2
  • BMP-2/BMP-4 bone morphogenetic protein-2
  • BMP-2/BMP-7 BMP-4/BMP-7
  • BMP-6 BMP-9
  • the mRNA can encode roof plate-specific spondin-2 (RSPO-2).
  • RSPO-2 roof plate-specific spondin-2
  • the mRNA can encode a growth factor.
  • the growth factor is roof plate-specific spondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2), a transforming growth factors (TGF-a and TGF-P), and a platelet-derived growth factor (PDGF-AA and PDGF-BB), insulin-like growth factor- 1 (IGF-1), tumor necrosis factor-a [TNF-a]), a vascular endothelial growth factor (VEGF A and B), basic fibroblast growth factor (bFGF; known as FGF-2) bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4. BMP-6, BMP-9. BMP-14, or BMP-2/-7.
  • the growth factor can be BMP-2 or BMP-2/-7.
  • the mRNA sequences can comprise or consist of a 5’ cap 5’ untranslated region (UTR), coding sequence, 3’ UTR and polyA tail.
  • the 5’ Cap can be commercially available, such as Clean Cap M6 or Clean Cap AG (Trilink Biotechnologies) or ARCA Cap (New England Biosciences).
  • the beta-globin 5’ UTRA sequence can be: acatttgcttctgacacaactgtgttcactagcaacctcaaacagacacc (SEQ ID NO: 1).
  • the beta-globin 3’ UTR sequence can be: gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcat ctggattctgcctaataaaaaacatttattttcattgcaa (SEQ ID NO: 2).
  • the polyA-tail sequence can be: (120 As): (AAA)40.
  • the mRNA coding sequences can be any of the following listed below for human: RSPO-2, RSPO-1, RSPO2(dTSP-l) a mutant version of RSPO-2 with the TSP-1 domain from RSPO-1, hBMP-2, hBMP-7, hBMP- 2-P2A-hBMP-7, hBMP-2-(GSG)4-hBMP-7. hBMP-6, hBMP-2-(GSG)4-hBMP-6.
  • P2A and (GSG)4 sequences are bicistronic sequences with a single mRNA encoding for 2 growth factors with stoichiometric expression (P2A) or linked together using 4 glycine-serine-glycine amino acid spacers. Therefore, functional mRNA sequences for the coding sequences below would have following structure:
  • the mRNA coding sequence for hRSPO-2 is:
  • the mRNA coding sequence for hRSPO-1 is: atgcggcttgggctgtgtgtggtggccctggttctgagctggacgcacctcaccatcagcagcccgggggatcaaggggaaaggca gaggcggatcagtgccgaggggagccaggcctgtgccaaaggctgtgagctctgctgaagtcaacggctgctcaagtgctcac ccaagctgttcatcctgctggagaggaacgacatccgccaggtgggcgtctgcttgcctgcccacctggatacttcgacgcccg caaccccgacatgaacaagtgcatcaaatgcaagatcgaggcctgcgcacctggatacttcgacg
  • dTSP-1 The mRNA coding sequence for hRSPO-2 (dTSP-1) is:
  • the mRNA coding sequence for hBMP-2 is:
  • the mRNA coding sequence for hBMP-7 is: atgcacgtgcgctcactgcgagctgcggcgccgcacagcttcgtggcgctctgggcacccctgttcctgctgcgctccgccctggcc gacttcagcctggacaacgaggtgcactcgagctcatccaccggcgcctccgcagccaggagcggcgggagatgcagcgcgag atcclctccalttlgggcttgccccaccgcccgcgcacctccagggcaagcacaaclcggcacccatgtlcatgclcatgclcatgclggacctgta caacgccatggcggtggaggagggcggcgggccggcggccagggctct
  • hBMP-2-P2A-hBMP-7 The mRNA coding sequence for hBMP-2-P2A-hBMP-7 is:
  • hBMP-2-(GSG)4-hBMP7 The mRNA coding sequence for hBMP-2-(GSG)4-hBMP7 is:
  • the mRNA coding sequence for hBMP-6 is:
  • hBMP-2-(GSG)4-hBMP-6 The mRNA coding sequence for hBMP-2-(GSG)4-hBMP-6 is:
  • the mRNA can comprise the any of nucleic acid sequences of SEQ
  • the composition can comprise one or more bone substitutes.
  • the one or more bone substitutes can be derived from biological products, can be a synthetic bone substitute or a combination thereof.
  • bone substitutes derived from biological products include but are not limited to demineralized bone matrix (DBM), bone morphogenetic proteins (BMPs), hydroxyapatite (HA) and corals, allogeneic cancellous bone chips, or bone marrow aspirate concentrate (BMAC), including bone graft from long bones harvested using the reamer irrigation aspirator (RIA).
  • the one or more bone substitutes can be derived from a biological product, wherein the biological product can be bone morphogenetic proteins (BMPs), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF). demineralized bone matrix (DBM), hydroxyapatite (HA), corals, allogeneic cancellous bone chips, or bone marrow aspirate concentrate (BMAC), including bone graft from long bones harvested using the reamer irrigation aspirator (RIA).
  • BMPs bone morphogenetic proteins
  • PDGF platelet derived growth factor
  • VEGF vascular endothelial growth factor
  • demineralized bone matrix (DBM) demineralized bone matrix
  • HA hydroxyapatite
  • corals hydroxyapatite
  • allogeneic cancellous bone chips or bone marrow aspirate concentrate (BMAC), including bone graft from long bones harvested using the reamer irrigation aspirator (RIA).
  • RIA bone marrow aspirate
  • synthetic bone substitutes can include but not are limited to calcium sulfate, calcium phosphate cements, beta-tri-calcium phosphate (TCP) ceramics, biphasic calcium phosphates (Hydroxyapatite (HA) and Beta-TCP ceramics), bioactive glasses, and polymer-based bone substitutes.
  • Further examples of synthetic bone substitutes include but are not limited to Cal cigen® S Calcium Sulfate Bone Void Filler, STIMULAN® Beads, HydroSet Injectable Bone Substitute (calcium phosphate), Ossilix calcium phosphate cement.
  • Syntoss Synthetic Beta-Tricalcium Phosphate Bone Graft Material CERASORB® Tri-Calcium Phosphate Bone Graft, GL1894P/-20 58S BIOACTIVE GLASS, UniGraft Bioactive Glass 200-600um, BonAlive (BonAlive Biomaterials Ltd, Finland), Cerament (bone void filler) and Cerament G (Bonesupport Holding AB, Lund Sweden).
  • polymers include but are not limited to collagen, gelatin, chitosan, and synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly caprolactone (PCL) - GalaFlex P4HB biopolymer, and the like.
  • the bone substitutes can be available in a variety 7 of forms including but not limited to dry, moldable or injectable forms, and pastes, powders, putty, granules, gels, sponges, or strips.
  • the bone substitute can be a commercially available product.
  • the bone substitute can be demineralized bone matrix (DBX; MTF Biologies, Edison, NJ), RegenaVate DBM, Puros DBM, StaGraft DBM, or FiberStack DBM (Zimmer Biomet; Warsaw, IN).
  • the DBM can be an allograft cancellous or cortical bone that has been decalcified to produce a product of collagen and non-collagenous protein.
  • DBMs include but are not limited to Grafton DBM (Osteotech, Inc, Eatontown. New Jersey), Allosource (Denver, Colorado), Dynagraft II (Integra LifeSciences, Plainsboro, New Jersey), DBX (Musculoskeletal Transplant Foundation and Synthes, Paoli, Pennsylvania), Osteofil (Medtronic Sofamor Danek, Minneapolis, Minnesota).
  • corals include but are not limited to animalia, coelenterate, scleractenia, poratidae, porites species, and gonioporas species; each of which can be used for developing coralline hydroxyapatite (CHA) bone substitute.
  • the bone substitute is not BMP, rhBMP-2 or BMP-2.
  • the ratio of the ex vivo hematoma to bone substitute can be from 1000: 1 to 1: 1000 or any ratio in between. In some aspects, the ratio of the ex vivo hematoma to bone substitute can be 1 : 1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10. In some aspects, the ratio ofthe bone substitute to ex vivo hematoma can be 1:1, 1:2, 1 :3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1 : 10.
  • the composition, ex vivo hematoma, bone substitute or biomimetic scaffold can comprise one or more growth factors.
  • the one or more growth factors can be one or more of the bone morphogenetic proteins.
  • 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 BMPs are contemplated, including BMP-1 through BMP-18.
  • the one or more growth factors can be platelet-derived growth factor.
  • the one or more growth factors can be vascular endothelial growth factor.
  • the one or more grow th factors can be fibroblast growth factor 2.
  • the one or more growth factors can be one or more of the bone morphogenetic proteins, platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor 2, or a combination thereof.
  • the composition, ex vivo hematoma, bone substitute or biomimetic scaffold can further comprise BMP -2.
  • the one or more grow th factors can be BMP-2.
  • the one or more growth factors is not BMP, rhBMP- 2, BMP-2, BMP-7. BMP-4, BMP-6, BMP-9, or BMP-14.
  • the whole blood can comprise viable cells. In some aspects, about 50% to 70% of the viable cells of the whole blood remain viable after formation of the ex vivo hematoma. In some aspects, at least 50% of the viable cells of the whole blood remain viable after formation of the hematoma. In some aspects, at least 60% of the viable cells of the whole blood remain viable after formation of the hematoma. In some aspects, at least 70% of the viable cells of the whole blood remain viable after formation of the ex vivo hematoma. In some aspects, at least 80% of the viable cells of the whole blood remain viable after formation of the ex vivo hematoma. In some aspects, at least 90% of the viable cells of the whole blood remain viable after formation of the ex vivo hematoma. In some aspects, more than 90% of the viable cells of the whole blood remain viable after formation of the ex vivo hematoma.
  • the whole blood can comprise one or more biological factors.
  • biological factors biological factors
  • other biological factors refers to the plasma component of whole blood excluding water. Examples of other biological factors include but are not limited to ions, proteins, clotting factors, sugars, lipids, and minerals.
  • the one or more biological factors present in the whole blood can be endogenous biological factors.
  • Platelets are present in whole blood. Many grow th factors can be found in platelets. Growth factors in platelet-rich plasma platelet a-granules have been shown to contain mitogenic and chemotactic growth factors along with associated healing molecules in an inactive form, which are important in wound healing, including but not limited to platelet-derived growth factor (PDGF), transforming growth factors 01, 02, 03 (TGF-01, TGF-02, TGF-03, platelet-derived angiogenesis factor (PDAF), insulin-like growth factor 1 (IGF-1), platelet factor 4 (PF-4), epidermal growth factor (EGF), epithelial cell grow factor (ECGF).
  • PDGF platelet-derived growth factor
  • transforming growth factors 01, 02, 03 TGF-01, TGF-02, TGF-03
  • PDAF platelet-derived angiogenesis factor
  • IGF-1 insulin-like growth factor 1
  • PF-4 platelet factor 4
  • EGF epi
  • VEGF vascular endothelial cell growth factor
  • bFGF basic fibroblast growth factor
  • HGF hepatocyte growth factor
  • growth factors present in platelets include but are not limited to platelet-derived growth factors, transforming growth factors 01, 02, 03, platelet-derived angiogenesis factor, insulin-like growth factor 1, platelet factor 4, epidermal growth factor, epithelial cell growth factor, vascular endothelial cell growth factor, basic fibroblast growth factor, and others cytokines; as well as platelet-derived endothelial growth factor (PDEGF).
  • interleukin 1 osteocalcin and osteonectin.
  • Growth factors present in plasma fluid include but are not limited to insulin-like growth factor 1, and hepatocyte growth factor.
  • the ex vivo hematoma can comprise whole blood, ecarin and sodium citrate. In some aspects, the ex vivo hematoma can comprise whole blood, calcium chloride and sodium citrate. In some aspects, the ex vivo hematoma can comprise platelet rich plasma and ecarin. In some aspects, the ex vivo hematoma can comprise platelet rich plasma and calcium chloride. In some aspects, the ex vivo hematoma can comprise whole blood; calcium chloride; or oscutarin and calcium chloride; and sodium citrate.
  • a combination of one of (a) isolated whole blood and sodium citrate; platelet rich plasma, or plasma with red blood cells can be combined with one of (b) ecarin, oscutarin and calcium chloride, or calcium chloride.
  • a combination of one of (a) isolated whole blood and sodium citrate; platelet rich plasma, or plasma with red blood cells can be combined with one of (b) thrombin or thrombin and calcium chloride.
  • any of the ex vivo hematoma combinations described herein can further comprise one or more antibiotics.
  • the concentration of calcium chloride present in the ex vivo hematoma can be in the range of 1 mM to 20 rnM. In some aspects, 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 in between. In some aspects, the concentration of calcium chloride can be about 10 mM.
  • the concentration of thrombin can be in the range of 0.1 to 1 U/mL. In some aspects, 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 in between or higher. In some aspects, the concentration of thrombin present in the ex vivo hematoma can be 0.5 U/mL
  • the concentration of ecarin present in the ex vivo hematoma can be at least 0.05 U/mL. In some aspects, 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, 1.1. 1.2, 1.3, 1.4, 1.5. 1.6, 1.7, 1.8, 1.9, 2 U/mL or any number in between or higher. In some aspects, the concentration of ecarin present in the ex vivo hematoma can be 0.3 U/mL. In some aspects, the concentration of ecarin present in the ex vivo hematoma can be 0.6 U/mL.
  • the concentration of ecarin present in the ex vivo hematoma can be 0.75 U/mL. In some aspects, the concentration of ecarin present in the ex vivo hematoma can be any value (rational or irrational) between 0 and 2.
  • the ex vivo hematomas, bone substitutes, biomimetic scaffolds or compositions described herein can further comprise BMP-2.
  • the BMP -2 can be a recombinant BMP -2.
  • the recombinant BMP-2 can comprise human BMP-2.
  • the dose of BMP-2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be at least 0.01 mg. In some aspects, the dose of BMP-2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be 0.01 to 5 mg.
  • the dose of BMP-2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mg or any number in between.
  • recombinant BMP -2 can be used at a dose of about 0.01 mg to about 12 mg. In some aspects, recombinant BMP -2 can be used at a dose of 0.
  • recombinant BMP-2 can be used at a dose higher than 12.0 mg.
  • dose of BMP-2 can be about 1 mg to 5 mg.
  • the dose of BMP-2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be at least 0.01 pg.
  • the dose of BMP-2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be 0.01 to 5 pg. In some aspects, the dose of BMP-2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 pg or any number in between. In some aspects, recombinant BMP -2 can be used at a dose of about 0.01 pg to about 12 pg. In some aspects, recombinant BMP-2 can be used at a dose of 0.
  • recombinant BMP-2 can be used at a dose higher than 12.0 pg. In some aspects, dose of BMP -2 can be about 1 pg to 5 pg.
  • the dose of BMP -2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be between 0.3 and 0.4 pg. In some aspects, the dose of BMP-2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be lower than the standard dose. In some aspects, the dose of BMP - 2 present in the ex vivo hematoma, bone substitute, composition, or biomimetic scaffold can be 10-50 times lower than the standard dose or the lowest effective dose of BMP-2/ ACS.
  • the amount of the 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.
  • the amount of the 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 pg.
  • the concentration of sodium citrate can be about 3.2 to 4 mg/ml.
  • the solution is about 3.2 to 4% (weight/volume) sodium citrate, in which one part of this solution can then be mixed with nine parts whole blood.
  • the ex vivo hematomas or compositions described herein can further comprise one or more therapeutic agents.
  • the therapeutic agent can be a grow th factor.
  • the therapeutic agent can be BMP -2.
  • the therapeutic agent can be recombinant BMP -2.
  • the therapeutic agent can be stem cells or pre-differentiated stem cells, including but not limited to mesenchymal stem cells, adipose stem cells, and induced pluripotent stem cells.
  • the therapeutic agent can be ecarin.
  • the ex vivo hematoma, biomimetic scaffolds or compositions disclosed herein can be formulated as a liquid or a gel. In some aspects, the ex vivo hematoma, biomimetic scaffolds or compositions disclosed herein can be formulated as a paste or a putty. In some aspects, the ex vivo hematomas can be formulated as a lyophilized or powder form. Said lyophilized or powder forms can make the ex vivo hematoma more stable for storage.
  • the ex vivo hematoma, biomimetic scaffolds or compositions disclosed herein can be formulated as a liquid, a gel, a powder, granules, a paste or a putty.
  • the composition can be formulated as a lyophilized or powder form. Said lyophilized or powder forms can make the ex vivo hematoma, biomimetic scaffolds or compositions disclosed herein more stable for storage.
  • the growth factors (BMPs and others), coagulating factors (ecarin, calcium chloride, etc.) and sodium citrate can be available as a lyophilized or powder forms.
  • compounds used for making the ex vivo hematomas disclosed herein can be dissolved in sterile distilled water prior to mixing with whole blood or other blood products (e.g., PRP, plasma, etc.).
  • the diluent is sterile distilled water. No additional components are needed for preparation or storage.
  • Whole blood (or other blood products) can be drawn from a patient before (e g., immediately before) the surgery, and citrated to prevent clotting.
  • donor blood can be used, for example, for patients with a blood disorder or diseases including but not limited to anemia, hemophilia, leukemia, HIV, etc. The remainder of the components of the ex vivo hematoma do not require any additional stabilizers for storage.
  • BMP-2 is commercially available in a bottle, ready to use
  • CaCL is available in a powder form, and in some cases may already be dissolved in sterile distilled water (it is very stable after it is dissolved).
  • Both BMP -2 and CaCh can be stored at room temperature.
  • Ecarin is available in a lyophilized form (freeze-dried), stored at -20°C, and can be dissolved in sterile distilled water prior to use.
  • the ex vivo hematoma can be prepared relatively simply, using the components described herein in amounts based on the volume of the defect to fill. For this, after the components are prepared, they can be mixed together in a tube/mold.
  • the ex vivo hematoma will form in about 30 to 45 minutes and then can be inserted (or implanted) into the bone defect.
  • the ex vivo hematomas described herein can be stored, for example, using a ‘"smart storage system’” that uses radiofrequency identification-based system (e.g., SmartstorageTM) which involves a near real-time tissue tracking system that can streamline inventory management including keeping accurate usage history and temperature logs.
  • radiofrequency identification-based system e.g., SmartstorageTM
  • biomimetic scaffolds can comprise any of the scaffolds described herein and any of the ex vivo hematomas described herein.
  • biomimetic scaffolds comprising a scaffold and an ex vivo hematoma, wherein the ex vivo hematoma comprises: (a) isolated whole blood; (b) sodium citrate; and (c) ecarin; oscutarin and calcium chloride: calcium chloride; thrombin; or thrombin and calcium chloride.
  • biomimetic scaffolds comprising a scaffold and an ex vivo hematoma, wherein the ex vivo hematoma comprises: (a) platelet rich plasma, plasma, or plasma with red blood cells; and (b) ecarin; oscutarin and calcium chloride; calcium chloride: thrombin; or thrombin and calcium chloride.
  • the ex vivo hematoma can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%. In some aspects, the ex vivo hematoma can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • the biomimetic scaffold further comprises one or more bone substitutes. In some aspects, the ex vivo hematoma of the biomimetic scaffold comprises one or more bone substitutes.
  • biomimetic scaffolds comprising a scaffold and an ex vivo hematoma
  • the ex vivo hematoma comprises: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematoma can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%.
  • the ex vivo hematoma can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • the biomimetic scaffold further comprises one or more bone substitutes.
  • the ex vivo hematoma of the biomimetic scaffold comprises one or more bone substitutes.
  • the mRNA-loaded lipid nanoparticles can be combined with a mineral coated microparticle.
  • the mRNA-loaded lipid nanoparticles can comprise mRNA.
  • the messenger ribonucleic acid (mRNA)-based therapeutic composition can comprise mRNA.
  • the mRNA can encode a protein of interest. The mRNAs can encode any protein of interest.
  • the mRNA can encode roof plate-specific sondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2), transforming grow th factors (e.g., TGF-a and TGF-0), and platelet-derived grow th factors (e.g., PDGF-AA and PDGF-BB), insulin-like growth factor-1 (IGF-1), tumor necrosis factor-a (TNF-a), vascular endothelial growth factor (e.g., VEGF A and B), or basic fibroblast growth factor (bFGF).
  • RSPO-2 roof plate-specific sondin-2
  • NRF2 nuclear factor erythroid 2-related factor 2
  • transforming grow th factors e.g., TGF-a and TGF-0
  • platelet-derived grow th factors e.g., PDGF-AA and PDGF-BB
  • IGF-1 insulin-like growth factor-1
  • TNF-a tumor necrosis factor-a
  • VEGF A and B vascular endo
  • the mRNA can encode bone morphogenetic protein-2 (BMP-2), BMP-2/BMP-4, BMP-2/BMP-7, BMP-4/BMP-7, BMP-6, or BMP-9.
  • BMP-2 bone morphogenetic protein-2
  • BMP-2/BMP-4 BMP-2/BMP-7
  • BMP-4/BMP-7 BMP-6
  • BMP-9 BMP-9
  • the mRNA can encode roof plate-specific spondin-2 (RSPO-2).
  • the mRNA can encode a grow th factor.
  • the growth factor is roof plate-specific spondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2).
  • TGF-a and TGF-0 transforming growth factors
  • PDGF-AA and PDGF-BB platelet-derived growth factor
  • IGF-1 insulin-like growth factor- 1
  • TGF-a tumor necrosis factor-a
  • VEGF A and B vascular endothelial growth factor
  • BMP-2 basic fibroblast growth factor
  • BMP-7 basic fibroblast growth factor
  • BMP-4 basic fibroblast growth factor
  • BMP-6 BMP-9
  • BMP- 14, or BMP-2/-7 BMP-2
  • BMP-2 basic fibroblast growth factor
  • BMP-7 BMP-4, BMP-6, BMP-9, BMP- 14, or BMP-2/-7
  • the growth factor can be BMP-2 or BMP-2/-7.
  • the mRNA can comprise the any of nucleic acid sequences of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • biomimetic scaffolds comprising a scaffold and an ex vivo hematoma, wherein the ex vivo hematoma comprises: (a) isolated whole blood; (b) sodium citrate; (c) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition .
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • biomimetic scaffolds comprising a scaffold and an ex vivo hematoma
  • the ex vivo hematoma comprises: (a) platelet rich plasma, plasma, or plasma with red blood cells; (b) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride; (c) and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematoma can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%.
  • the ex vivo hematoma can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • the biomimetic scaffold further comprises one or more bone substitutes.
  • the ex vivo hematoma of the biomimetic scaffold comprises one or more bone substitutes.
  • the mRNA-loaded lipid nanoparticles can be combined with a mineral coated microparticle.
  • the mRNA-loaded lipid nanoparticles can comprise mRNA.
  • the messenger ribonucleic acid (mRNA)-based therapeutic composition can comprise mRNA.
  • the mRNA can encode a protein of interest.
  • the mRNAs can encode any protein of interest.
  • the mRNA can encode roof plate-specific sondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2), transforming grow th factors (e.g., TGF-a and TGF-P), and platelet-derived growth factors (e.g., PDGF-AA and PDGF-BB), insulinlike growth factor-1 (IGF-1), tumor necrosis factor-a (TNF-a), vascular endothelial grow th factor (e.g., VEGF A and B), or basic fibroblast growth factor (bFGF).
  • the mRNA can encode bone morphogenetic protein-2 (BMP-2), BMP-2/BMP-4.
  • the mRNA can encode roof platespecific spondin-2 (RSPO-2). In some aspects, the mRNA can encode a grow th factor.
  • RSPO-2 roof platespecific spondin-2
  • the growth factor is roof plate-specific spondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2), a transforming growth factors (TGF-a and TGF-P), and a platelet-derived growth factor (PDGF-AA and PDGF-BB), insulin-like growth factor- 1 (IGF-1), tumor necrosis factor-a [TNF-a]), a vascular endothelial growth factor (VEGF A and B), basic fibroblast growth factor (bFGF; known as FGF-2) bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4. BMP-6, BMP-9, BMP- 14. or BMP-2/-7.
  • the growth factor can be BMP -2 or BMP-2/-7.
  • the mRNA can comprise the any of nucleic acid sequences of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the ex vivo hematomas described herein can further be combined with a carrier such as a scaffold.
  • a carrier such as a scaffold.
  • the carrier can be a biodegradable biomaterial scaffold (e.g., silk fibroin scaffolds, poly(lactide-co-glycolide (PLGA), or other similar resorbable products or materials).
  • PLGA poly(lactide-co-glycolide
  • Such carrier can be used to provide additional mechanical support of the ex vivo hematoma.
  • scaffolds for use in the disclosed biomimetic scaffolds include, but are not limited to a biocompatible scaffold, an osteoconductive scaffold, a three-dimensionally printed scaffold, collagen, absorbable collagen scaffold (collagen type 1 bovine or porcine), collagen bovine membrane, or collagens derived from other mammalian or non-mammalian (such as marine) sources, chitins, bioabsorbable polymers such PLA, or nonabsorbable polymers such as PEEK, or titanium or other biocompatible metallic alloys such as resorbable magnesium (such as magnesium calcium alloys) or a three-dimensionally printed scaffold.
  • the scaffolds that can be combined with the ex vivo hematomas described herein can be biocompatible and biodegradable.
  • the scaffold should elicit a negligible immune reaction in order to prevent it from causing such a severe inflammatory' response that it might reduce healing or cause rejection by the body.
  • the scaffold is amendable to cells adhering to it and so that the cells can function normally and migrate to the surface and eventually through the scaffold to proliferate.
  • the scaffold disclosed herein can also be biodegradable to that cells can produce their own extracellular matrix. The by-products of this degradation can also be non-toxic and able to exit the body without interference from other organs.
  • the scaffold can have mechanical properties that are consistent with the anatomical site into which it is to be implanted and strong enough to allow surgical handling during implantation. Further, the scaffold can be sufficient to allow cell infiltration and vascularization. In designing the appropriate scaffold, it is important to consider the architecture of the scaffold. For instance, the scaffolds can be have an interconnected pore structure and high porosity to ensure cellular penetration and adequate diffusion of nutrients to cells. A porous interconnected structure is important to allow- diffusion of waste products out of the scaffold, and the products of scaffold degradation should be able to exit the body without interference with other organs and surrounding tissues.
  • the scaffold can be a three-dimensionally printed scaffold. In some aspects, the three-dimensionally printed scaffold can be custom produced.
  • the three-dimensional scaffold can be produced using a 3-D printing technique referred to as robotic deposition or direct write (DW) technology.
  • This technique uses a computer controlled printing process and colloidal inks to form three-dimensional structures. These structures can form on the self-components or can be custom formed for filling individual bone defects from tomographic data (X-ray, sonographic or MRI).
  • Ink fabrication and the printing system itself uses water-based Theologically controlled inks that become solid as they leave the print nozzle. These inks consist of finely controlled ceramic particles in a water-based slurry containing organic chemicals that control the handling characteristics of the colloidal ink. This allows 3-D lattice-like structures to be printed, in layers, without or with minimal sag of unsupported structural elements.
  • the elements of the first layer can be printed by forcing the ink through a small ( ⁇ 50-400 pm diameter) nozzle onto a support plate, using the x and y coordinate control system of an x-y-z control gantry system. Then the z control system is used to move the nozzle up slightly less than 1 nozzle diameter. Then the next layer is printed over the first layer. This is continued layer-by-layer until the entire 3-D structure is finished.
  • the entire structure can be printed in an oil bath to prevent drying.
  • the system can have 3 nozzles and ink reservoirs so that up to three materials can be used to print a single structure.
  • Fugitive inks, inks consisting entirely of material that bum off during firing, can also be used as part of the printing process. These can be used to print support structures for complex parts requiring temporary’ supports.
  • the resulting structures are then removed from the oil bath, dried, and fired in a programmable furnace to produce the final ceramic structure. Firing is currently done at approximately 1100° C for about four hours, which substantially bums off the organic components, sintering the ceramic particles together into a solid structure. This may cause a small amount of predictable shrinkage that can be calculated into the printing process to produce precise and predictable structures.
  • the print nozzles can be routinely cylindrical producing cylindrical rod printed structures. However, nozzles can be made that are shaped to produce non-cylindrical structures or structures with surface striations of sizes designed to control cell migration, growth, and differentiation.
  • the biomaterials can be ceramics, synthetic polymers and/or natural polymers or combinations thereof.
  • ceramics include but are not limited hydroxyapatite (HA) and tri-calcium phosphate (TCP).
  • examples of synthetic polymers include but are not limited to polystyrene, poly-l-lactic acid (PLLA), polyglycolic acid (PGA) and poly-dl-lactic-co-glycolic acid (PLGA).
  • Examples of natural polymers include but are not limited to collagen, various proteoglycans, alginate-based substrates and chitosan.
  • the scaffold can be made of a combination of biomaterials.
  • collagen can be combined with a polysaccharide (e.g., glycosaminoglycan).
  • the scaffolds can be prepared by using chemical cross-linking methods.
  • compositions or biomimetic scaffolds comprising the scaffold and ex vivo hematoma can be formulated for local administration.
  • the compositions (e.g., liquid form) or ex vivo hematomas (e.g., gel form) disclosed herein can be combined with any of the carriers or scaffolds disclosed herein and administered locally or implanted surgically or injected percutaneously.
  • the liquid formulation can be delivered through a syringe to the scaffold.
  • the gel formulation can be implanted into the bone defect site.
  • the gel formulation can be prepared using a mold outside of the body that corresponds to the bone size and shape for implantation into the bone defect site.
  • the formulation can be in an intermediate form, between liquid and gel.
  • an intermediate formulation can be applied to a solid scaffold or carrier to bridge gaps that may be present in the solid scaffold itself (e g., large gaps) while also providing mechanical support independently.
  • a solid scaffold includes but is not limited to titanium cages or other porous metallic implants. Such scaffolds can be used to reconstruct a skeletal defect or to achieve spinal fusion.
  • the formulations disclosed herein can be used to augment healing when PEEK spinal cages are used for interbody spinal fusions, considering PEEK is itself biologically inert and has no intrinsic bone healing capacity 7 .
  • any of the formulations disclosed herein can be infused or applied topically to resorbable scaffolds as may be used to reconstruct skeletal defects, segmental or subsegmental.
  • this would also include opening wedge osteotomies (of the femur, tibia, or other long bones), distraction arthrodesis sites, and bone defects related to arthroplasty 7 .
  • any of the biomimetic scaffolds, compositions and ex vivo hematoma formulations disclosed herein can be applied in the same fashion to other arthrodesis sites with bone defects such as the ankle, knee, wrist, shoulder, hip, or other smaller joints, including but not limited to the Lisfranc joint, smaller joints in the hand, wrist, or foot, and extends to include applications to fill bone defects created when harvesting bone grafts for transposition to a secondary anatomic location.
  • the multicompartment device can comprise two or more chambers or two or more syringes can be used to deliver components of the ex vivo hematoma, a messenger ribonucleic acid (mRNA)- based therapeutic composition (or mRNA or mRNA-loaded lipid nanoparticles or mRNA- loaded lipid nanoparticles combined with mineral coated microparticles) or a ribonucleic acid (RNA)-based therapeutic composition, and/or the bone substitutes separately.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • a first chamber or syringe can comprise the coagulants (e.g., calcium chloride, calcium chloride and thrombin, or ecarin) at pre-determined concentrations.
  • a second chamber or syringe can comprise whole blood alone.
  • a second chamber or syringe can comprise whole blood in combination with exogenous growth factors (e.g., BMP2, PDGF, VEGF).
  • exogenous growth factors e.g., BMP2, PDGF, VEGF
  • a second chamber or syringe can comprise whole blood in combination with bone substitutes (e.g... DBM, allogeneic cancellous bone chips).
  • a second chamber or syringe can comprise whole blood in combination with exogenous growth factors (e.g., BMP2, PDGF, VEGF) bone substitutes.
  • exogenous growth factors e.g., BMP2, PDGF, VEGF
  • a third chamber or syringe can comprise exogenous growth factors and additional bone substitutes.
  • a third chamber or syringe can comprise exogenous growth factors.
  • a third chamber or syringe can comprise one or more bone substitutes.
  • a first chamber can comprise isolated whole blood; a second chamber can comprise calcium chloride; thrombin; or thrombin and calcium chloride; and a third chamber can comprise a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • a fourth chamber or syringe can comprise one or more bone substitutes.
  • compositions comprising the ex vivo hematoma can also serve as a scaffold. It some aspects, the compositions comprising the ex vivo hematoma can be combined with a scaffold to form or create a biomimetic scaffold. In some aspects, the methods disclosed herein can be combined. Disclosed herein are methods of promoting bone healing, producing bone replacement material, producing implants, compositions comprising ex vivo hematomas, biomimetic scaffolds or a combination thereof.
  • the methods comprise administering to a subject in need thereof a therapeutically effective amount of any of the compositions disclosed herein.
  • the methods can comprise administering to a subject in need thereof a therapeutically effective amount of a composition comprising the ex vivo hematoma disclosed herein.
  • the compositions can further comprise one or more bone substitutes.
  • the methods comprise implanting any of the biomimetic scaffolds described herein into a site of interest in the subject.
  • the biomimetic scaffold can further comprise one or more bone substitutes.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride; thrombin; or thrombin and calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)- based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) thrombin.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) thrombin; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) thrombin and calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • the ex vivo hematomas can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%.
  • the ex vivo hematomas can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • VML volumetric muscle loss
  • the methods comprise administering to the subject in need thereof a therapeutically effective amount of any of the compositions disclosed herein.
  • the methods can comprise administering to a subject in need thereof a therapeutically effective amount of a composition comprising the ex vivo hematoma disclosed herein.
  • the compositions can further comprise one or more bone substitutes.
  • the methods comprise implanting any of the biomimetic scaffolds described herein into a site of interest in the subject.
  • the biomimetic scaffold can further comprise one or more bone substitutes.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride; thrombin; or thrombin and calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; thrombin; or thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) calcium chloride.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) thrombin.
  • the ex vivo hematomas can comprise:
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) thrombin; and (d) a messenger ribonucleic acid (mRNAj-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNAj-based therapeutic composition a messenger ribonucleic acid (mRNAj-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • RNAj-based therapeutic composition a messenger ribonucleic acid
  • RNA ribonucleic acid
  • the ex vivo hematomas can comprise: (a) isolated whole blood;
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) thrombin and calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • the ex vivo hematomas can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%.
  • the ex vivo hematomas can comprise fibnn fibers having a thickness of 100-400 nm ⁇ 10%.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; (c) calcium chloride; and (d) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) thrombin.
  • the methods comprise administering to a subj ect in need thereof a therapeutically effective amount of any of the compositions disclosed herein.
  • the methods can comprise administering to a subject in need thereof a therapeutically effective amount of a composition comprising the ex vivo hematoma disclosed herein and one or more bone substitutes.
  • the methods comprise implanting any of the biomimetic scaffold described herein into a site of interest in the subject.
  • the biomimetic scaffold can further comprise one or more bone substitutes.
  • the ex vivo hematomas can comprise: (a) isolated whole blood; (b) sodium citrate; and (c) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride.
  • the ex vivo hematomas can comprise: (a) platelet rich plasma, plasma, or plasma with red blood cells; and (b) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride.
  • the ex vivo hematoma can further comprise sodium citrate.
  • the ex vivo hematomas can comprise fibrin fibers having a thickness of at least 1 0-300 nm ⁇ 10%. In some aspects, the ex vivo hematomas can comprise fibrin fibers having a thickness of 100- 400 nm ⁇ 10%. In some aspects, the ecarin; oscutarin 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%.
  • the ecarin; oscutarin 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 100-400 nm ⁇ 10%.
  • the ex vivo hematoma can comprise (a) isolated whole blood and sodium citrate platelet rich plasma, plasma alone, plasma with red blood cells (without platelets) or other blood products; and (b) one or more coagulating factors.
  • the ex vivo hematomas can comprise whole blood and one or more coagulating factors.
  • the whole blood can comprise one or more viable cells.
  • the whole blood can comprise one or more biological factors.
  • the ex vivo hematoma can comprise whole blood, ecarin, and sodium citrate. In some aspects, the ex vivo hematoma can comprise whole blood, calcium chloride, and sodium citrate. In some aspects, the ex vivo hematoma can comprise platelet rich plasma and ecarin. In some aspects, the ex vivo hematoma can comprise platelet rich plasma and calcium chloride. In some aspects, the ex vivo hematoma can comprise plasma and ecarin. In some aspects, the ex vivo hematoma can comprise plasma and calcium chloride. In some aspects, the ex vivo hematoma can comprise plasma with red blood cells and ecarin.
  • the ex vivo hematoma can comprise plasma with red blood cells and calcium chloride. In some aspects, the ex vivo hematoma can comprise plasma with oscutarin and calcium chloride. In some aspects, the ex vivo hematoma can comprise plasma with thrombin and calcium chloride. In some aspects, the ex vivo hematoma can further comprise bone morphogenetic protein 2 (BMP -2). In some aspects, the BMP -2 can be recombinant BMP -2. In some aspects, the recombinant BMP -2 can comprise human BMP -2. In some aspects, the composition can further comprise one or more growth factors, one or more platelets, and one or more cells. In some aspects, the composition can be formulated as a clot or as a scaffold. In some aspects, the scaffold can be chemotactic. In some aspects, the scaffold can attract endogenous grow th factors conducive to bone healing.
  • the one or more bone substitutes can be derived from biological products, be a synthetic bone substitute or a combination thereof.
  • bone substitutes derived from biological products include but are not limited to demineralized bone matrix (DBM), bone morphogenetic proteins (BMPs), hydroxyapatite (HA) and corals, allogeneic cancellous bone chips, or bone marrow aspirate concentrate (BMAC). including bone graft from long bones harvested using the reamer irrigation aspirator (RIA).
  • the one or more bone substitutes can be derived from a biological product, wherein the biological product can be bone morphogenetic proteins (BMPs), platelet derived growth factor (PDGF).
  • the bone substitutes can be synthetic bone substitutes.
  • synthetic bone substitutes can include but not are limited to calcium sulfate, calcium phosphate cements, beta-tri-calcium phosphate (TCP) ceramics, biphasic calcium phosphates (Hydroxyapatite (HA) and Beta-TCP ceramics), bioactive glasses, and polymer-based bone substitutes.
  • synthetic bone substitutes include but are not limited to Calcigen® S Calcium Sulfate Bone Void Filler, STIMULAN® Beads, HydroSet Injectable Bone Substitute (calcium phosphate), Ossilix calcium phosphate cement, Syntoss Synthetic Beta-Tricalcium Phosphate Bone Graft Material, CERASORB® Tri- Calcium Phosphate Bone Graft, GL1894P/-20 58S BIO ACTIVE GLASS, UniGraft Bioactive Glass 200-600um, BonAlive (BonAlive Biomaterials Ltd, Finland), Cerament (bone void filler) and Cerament G (Bonesupport Holding AB, Lund Sweden).
  • polymers include but are not limited to collagen, gelatin, chitosan, and synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly caprolactone (PCL) - GalaFIex P4HB biopolymer, and the like.
  • PLA poly(lactic acid)
  • PGA poly(glycolic acid)
  • PCL poly caprolactone
  • the bone substitutes can be available in a variety of forms including but not limited to dry. moldable or injectable forms, and pastes, powders, putty, granules, gels, sponges, or strips.
  • the bone substitute can be a commercially available product.
  • the bone substitute can be demineralized bone matrix (DBX; MTF Biologies, Edison, NJ), RegenaVate DBM, Puros DBM, StaGraft DBM, or FiberStack DBM (Zimmer Biomet; Warsaw, IN).
  • the DBM can be an allograft cancellous or cortical bone that has been decalcified to produce a product of collagen and non-collagenous protein. Examples of DBMs include but are not limited to Grafton DBM (Osteotech, Inc. Eatontown, New Jersey), Allosource (Denver, Colorado).
  • DBX Musculoskeletal Transplant Foundation and Synthes, Paoli, Pennsylvania
  • Osteofil Medtronic Sofamor Danek, Minneapolis, Minnesota
  • corals include but are not limited to animalia, coelenterate, scleractenia, poratidae. porites species, and gonioporas species; each of which can be used for developing coralline hydroxyapatite (CHA) bone substitute.
  • the bone substitute is not BMP, rhBMP-2 or BMP-2.
  • the subject can be a human. In some aspects, the subject has a skeletal defect. In some aspects, the skeletal defect can be a large segmental bone defect. In some aspects, the skeletal defect can be a nonunion. In some aspects, the skeletal defect can be a small segmental bone defect. In some aspects, the skeletal defect can be independent of the size or volume of the defect regardless of whether the defect is complete or incomplete. In some aspects, the subject has a dental bone defect. In some aspects, the subject has a musculoskeletal injury. In some aspects, the subject has a volumetric muscle loss injury. In some aspects, the subject has one or more bone fractures. In some aspects, the subject has one or more bone injuries. In some aspects, the subject has one or more muscle injuries.
  • the subject has one or more bone injuries and one or more muscle injuries.
  • muscle injuries include but are not limited to volumetric muscle loss (e.g., loss of a significant volume of muscle tissue due to trauma, surgery', blast injuries or other causes); large muscle defects (e.g., injuries that result in the removal or loss of a substantial portion of a muscle; extensive muscle trauma (e.g., injuries involving severe damage to a wide area of muscle tissue; complex muscle injuries (e.g., injuries that affect multiple layers and regions of a muscle, requiring a comprehensive approach to repair; severe contusions or crush injuries (e.g., high-impact injuries that cause extensive damage to the muscle structure); extensive surgical resections (e.g., removal of a significant portion of muscle tissue during surgery, often to address medical conditions such as tumors); and degloving injuries (e.g., injuries that involve the separation of skin and subcutaneous tissue from the underlying muscle, leading to volumetric muscle loss).
  • volumetric muscle loss e.g., loss of a significant volume of muscle tissue due
  • the composition can be formulated as a clot or a scaffold. In some aspects, the composition can be formulated for local administration and combined with any of the scaffolds disclosed herein. In some aspects, the composition can be administered locally via a carrier or a scaffold. In some aspects, the composition can be administered locally without a carrier or a scaffold. In some aspects, the composition can be implanted or delivered percutaneously. In some aspects, the composition can be implanted. In some aspects, the composition can be implanted either directly or indirectly. In some aspects, the composition can be delivered by a surgeon or by any autonomous or semi-autonomous delivery' device acting on behalf of a human or robotic/semi-autonomous agent. In some aspects, the composition can be delivered percutaneously.
  • the methods can comprise: a) dimensioning a depot implant in at least one of a shape and a size that facilitates implantation of the depot implant into a bone defect; and b) structuring the depot implant to have a scaffold by introducing: (i) isolated whole blood and sodium citrate; or platelet rich plasma, plasma, or plasma with red blood cells; (ii) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride; and (iii) a bone substitute to create the scaffold.
  • the scaffold can have a porosity of 55 to 75%.
  • the scaffold can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%. In some aspects, the scaffold can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • the shape of the depot implant can be that of a cylinder or a sphere or any other shape. In some aspects, the scaffold can be constructed as a clot. In some aspects, one or more grow th factors can be introduced into the scaffold. In some aspects, the one or more growth factors can be bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4, BMP-6, BMP-9, BMP- 14, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF).
  • BMP-2 bone morphogenetic protein 2
  • BMP-7 bone morphogenetic protein 2
  • BMP-4 platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • the BMP-2 can be introduced into the scaffold.
  • the amount of ecarin present in the scaffold can be at least 0.05 U/mL; and the amount of BMP -2 present in the scaffold can be at least 0.01 mg.
  • the ratio of the ex vivo hematoma to bone substitute can be from 1000: 1 to 1: 1000.
  • the scaffold can resemble the size and shape of a given bone defect.
  • the scaffold can be chemotactic.
  • the scaffold can comprise viable blood cells and appropriate biological factors.
  • the bone substitute can be demineralized bone matrix.
  • the bone substitute can be derived from a biological product, a synthetic bone substitute or a combination thereof.
  • the biological product can be a demineralized bone matrix, hydroxyapatite, or a coral.
  • the synthetic bone substitute can be calcium sulfate, a calcium phosphate cement, P-tri-calcium phosphate ceramics, bioactive glasses, or a polymer.
  • the one or more growth factors is not BMP, rhBMP-2, BMP-2, BMP-7, BMP-4. BMP-6, BMP-9, or BMP- 14.
  • the methods can comprise: a) dimensioning a depot implant in at least one of a shape and a size that facilitates implantation of the depot implant into a bone defect; and b) structuring the depot implant to have a scaffold by introducing: (i) isolated whole blood and sodium citrate; or platelet rich plasma, plasma, or plasma with red blood cells; (ri) ecarin; oscutann and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride; and (iii) a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition to create the scaffold.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • the step b) can further comprise a bone substitute to create the scaffold.
  • the scaffold can have a porosity of 55 to 75%.
  • the scaffold can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%.
  • the scaffold can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • the shape of the depot implant can be that of a cylinder or a sphere or any other shape.
  • the scaffold can be constructed as a clot.
  • one or more growth factors can be introduced into the scaffold.
  • the one or more growth factors can be bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4, BMP-6, BMP-9, BMP-14, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF- 2), or a combination thereof.
  • BMP-2 can be introduced into the scaffold.
  • the amount of ecarin present in the scaffold can be at least 0.05 U/mL; and the amount of BMP-2 present in the scaffold can be at least 0.01 mg.
  • the ratio of the ex vivo hematoma to bone substitute can be from 1000: 1 to 1 : 1000.
  • the scaffold can resemble the size and shape of a given bone defect.
  • the scaffold can be chemotactic.
  • the scaffold can comprise viable blood cells and appropriate biological factors.
  • the bone substitute can be demineralized bone matrix.
  • the bone substitute can be derived from a biological product, a synthetic bone substitute or a combination thereof.
  • the biological product can be a demineralized bone matrix, hydroxyapatite, or a coral.
  • the synthetic bone substitute can be calcium sulfate, a calcium phosphate cement, P-tri-calcium phosphate ceramics, bioactive glasses, or a polymer.
  • the one or more growth factors is not BMP, rhBMP-2, BMP -2, BMP-7, BMP -4, BMP-6, BMP -9, or BMP- 14.
  • the mRNA-loaded lipid nanoparticles can be combined with a mineral coated microparticle.
  • the mRNA-loaded lipid nanoparticles can comprise mRNA.
  • the messenger ribonucleic acid (mRNA)-based therapeutic composition can comprise mRNA.
  • the mRNA can encode a protein of interest.
  • the mRNAs can encode any protein of interest.
  • the mRNA can encode roof plate-specific sondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2), transforming growth factors (e.g., TGF-a and TGF-[3), and platelet-derived growth factors (e.g., PDGF-AA and PDGF-BB), insulin-like growth factor-1 (IGF-1), tumor necrosis factor-a (TNF-a), vascular endothelial growth factor (e.g., VEGF A and B), or basic fibroblast growth factor (bFGF).
  • RSPO-2 roof plate-specific sondin-2
  • NRF2 nuclear factor erythroid 2-related factor 2
  • TGF-a and TGF-[3 transforming growth factors
  • platelet-derived growth factors e.g., PDGF-AA and PDGF-BB
  • IGF-1 insulin-like growth factor-1
  • TNF-a tumor necrosis factor-a
  • VEGF A and B vascular endothelial growth factor
  • bFGF basic
  • the mRNA can encode bone morphogenetic protein-2 (BMP-2), BMP-2/BMP- 4, BMP-2/BMP-7, BMP-4/BMP-7, BMP-6, or BMP-9.
  • BMP-2 bone morphogenetic protein-2
  • BMP-2/BMP- 4 BMP-2/BMP-7
  • BMP-4/BMP-7 BMP-6
  • BMP-9 BMP-9
  • the mRNA can encode roof plate-specific spondin-2 (RSPO-2).
  • RSPO-2 roof plate-specific spondin-2
  • the mRNA can encode a growth factor.
  • the growth factor is roof plate-specific spondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2), a transforming grow th factors (TGF-a and TGF-P), and a platelet-derived growth factor (PDGF-AA and PDGF-BB), insulin-like growth factor- 1 (IGF-1), tumor necrosis factor-a [TNF-a]), a vascular endothelial growth factor (VEGF A and B), basic fibroblast growth factor (bFGF; known as FGF-2) bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4, BMP-6, BMP -9, BMP-14, or BMP-2/-7.
  • the growth factor can be BMP-2 or BMP-2/-7.
  • the mRNA can comprise the any of nucleic acid sequences of SEQ ID NOs: 3, 4. 5, 6, 7, 8, 9, 10, or 11.
  • the methods can comprise: a) dimensioning a scaffold in at least one of a shape and a size that facilitates implantation of the scaffold into a bone defect; and b) combining the scaffold in a) with an ex vivo hematoma comprising: (i) isolated whole blood and sodium citrate; or platelet rich plasma, plasma, or plasma with red blood cells; and (ii) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride to create the biomimetic scaffold.
  • the scaffold can have a porosity of 55 to 75%.
  • the scaffold can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%. In some aspects, the scaffold can comprise fibrin fibers having a thickness of 100-400 nm ⁇ 10%.
  • the shape of the scaffold can be that of a cylinder or a sphere or any other shape. In some aspects, the shape of the scaffold can be that any and all other geometric shape or shapes that could theoretically occupy a discrete subset or volume within Euclidean space.
  • the scaffold can be collagen, chitins, bioabsorbable polymers, nonabsorbable polymers such as PEEK, or titanium or a metallic alloy.
  • one or more growth factors can be introduced into the scaffold or ex vivo hematoma.
  • the one or more grow th factors can be bone morphogenetic protein 2 (BMP -2), BMP-7, BMP -4, BMP-6, BMP-9, BMP-14, platelet-derived growth factor (PDGF). vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF- 2), or a combination thereof.
  • BMP-2 bone morphogenetic protein 2
  • BMP-7 bone morphogenetic protein 2
  • BMP-4 BMP-6
  • BMP-9 platelet-derived growth factor
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • FGF- 2 fibroblast growth factor 2
  • the amount of ecarin present in the scaffold or ex vivo hematoma can be at least 0.05 U/mL; and the amount of BMP -2 present in the scaffold or ex vivo hematoma can be at least 0.01 mg.
  • the ratio of the ex vivo hematoma to bone substitute can be from 1000: 1 to 1 : 1000.
  • the ex vivo hematoma can comprise viable blood cells and appropriate biological factors.
  • the bone substitute can be demineralized bone matrix.
  • the bone substitute can be derived from a biological product, a synthetic bone substitute or a combination thereof.
  • the biological product can be a demineralized bone matrix, hydroxyapatite, or a coral.
  • the synthetic bone substitute can be calcium sulfate, a calcium phosphate cement, P-tri-calcium phosphate ceramics, bioactive glasses, or a polymer.
  • the scaffold can resemble the size and shape of a given bone defect.
  • the scaffold can be chemotactic. Additionally, the scaffold can be biodegradable such that it degrades without the necessity of surgical removal.
  • the one or more growth factors is not BMP, rhBMP-2, BMP-2, BMP-7, BMP-4. BMP-6, BMP-9, or BMP-14.
  • the methods can comprise: a) dimensioning a scaffold in at least one of a shape and a size that facilitates implantation of the scaffold into a bone defect; b) combining the scaffold in a) with an ex vivo hematoma comprising: (i) isolated whole blood and sodium citrate; or platelet rich plasma, plasma, or plasma with red blood cells; and (ii) ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride to create the biomimetic scaffold; c) combining the biomimetic scaffold in b) with a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • the scaffold can have a porosity of 55 to 75%.
  • the scaffold can comprise fibrin fibers having a thickness of at least 150-300 nm ⁇ 10%.
  • the scaffold can comprise fibrin fibers having a thickness of 100- 400 nm ⁇ 10%.
  • the shape of the scaffold can be that of a cylinder or a sphere or any other shape.
  • the shape of the scaffold can be that any and all other geometric shape or shapes that could theoretically occupy a discrete subset or volume within Euclidean space.
  • the scaffold can be collagen, chitins, bioabsorbable polymers, nonabsorbable polymers such as PEEK, or titanium or a metallic alloy.
  • one or more growth factors can be introduced into the scaffold or ex vivo hematoma.
  • the one or more growth factors can be bone morphogenetic protein 2 (BMP- 2), BMP-7, BMP -4, BMP-6, BMP-9, BMP- 14, platelet-derived growth factor (PDGF), vascular endothelial grow th factor (VEGF), fibroblast growth factor 2 (FGF-2), or a combination thereof.
  • BMP-2 bone morphogenetic protein 2
  • BMP-7 bone morphogenetic protein 2
  • BMP-7 bone morphogenetic protein 2
  • BMP-4 platelet-derived growth factor
  • VEGF vascular endothelial grow th factor
  • FGF-2 fibroblast growth factor 2
  • the amount of ecarin present in the scaffold or ex vivo hematoma can be at least 0.05 U/mL; and the amount of BMP -2 present in the scaffold or ex vivo hematoma can be at least 0.01 mg.
  • the ratio of the ex vivo hematoma to bone substitute can be from 1000: 1 to 1 :1000.
  • the ex vivo hematoma can comprise viable blood cells and appropriate biological factors.
  • the bone substitute can be demineralized bone matrix.
  • the bone substitute can be derived from a biological product, a synthetic bone substitute or a combination thereof.
  • the biological product can be a demineralized bone matrix, hydroxyapatite, or a coral.
  • the synthetic bone substitute can be calcium sulfate, a calcium phosphate cement, P-tri-calcium phosphate ceramics, bioactive glasses, or a polymer.
  • the scaffold can resemble the size and shape of a given bone defect.
  • the scaffold can be chemotactic.
  • the scaffold can be biodegradable such that it degrades without the necessity of surgical removal.
  • the one or more growth factors is not BMP, rhBMP-2, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, or BMP-14.
  • the mRNA-loaded lipid nanoparticles can be combined with a mineral coated microparticle.
  • the mRNA-loaded lipid nanoparticles can comprise mRNA.
  • the messenger ribonucleic acid (mRNA)-based therapeutic composition can comprise mRNA.
  • the mRNA can encode a protein of interest.
  • the mRNAs can encode any protein of interest.
  • the mRNA can encode roof plate-specific sondin-2 (RSPO-2), nuclear factor erythroid 2-related factor 2 (NRF2).
  • the mRNA can encode bone morphogenetic protein-2 (BMP-2), BMP-2/BMP- 4, BMP-2/BMP-7, BMP-4/BMP-7, BMP-6, or BMP-9.
  • the mRNA can encode roof plate-specific spondin-2 (RSPO-2).
  • the mRNA can encode a grow th factor.
  • the growth factor is roof plate-specific spondin-2 (RSPO-2), nuclear factor ery throid 2-related factor 2 (NRF2), a transforming growth factors (TGF-a and TGF-P), and a platelet-derived growth factor (PDGF-AA and PDGF-BB), insulin-hke growth factor-1 (IGF-1), tumor necrosis factor-a [TNF-a]), a vascular endothelial growth factor (VEGF A and B), basic fibroblast growth factor (bFGF; known as FGF-2) bone morphogenetic protein 2 (BMP-2), BMP-7, BMP-4.
  • RSPO-2 roof plate-specific spondin-2
  • NRF2 nuclear factor ery throid 2-related factor 2
  • TGF-a and TGF-P transforming growth factors
  • PDGF-AA and PDGF-BB platelet-derived growth factor
  • IGF-1 insulin-hke growth factor-1
  • TGF-a tumor necros
  • the growth factor can be BMP-2 or BMP-2/-7.
  • the mRNA can comprise the any of nucleic acid sequences of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the composition can be implanted as all or part of a biomimetic scaffold.
  • the composition can be injected with a syringe into a carrier or scaffold.
  • the amount of the 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 in between.
  • the amount of the 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 pg or any amount in between.
  • the ratio of the ex vivo hematoma to bone substitute can be from 1000: 1 to 1: 1000 or any ratio in between.
  • the treatment regimen can be a standard treatment regimen for treating any bone defect.
  • the defect wound can be debrided, fixed with an internal plate, external fixator or intramedullary nail.
  • the compositions; compositions and scaffolds; and biomimetic scaffolds and ex vivo hematomas described herein can then be inserted as a unit into the skeletal defect before closing the wound.
  • the components of the compositions; the components of the compositions and the scaffolds; and biomimetic scaffolds and the components of the ex vivo hematomas described herein can then be inserted as separately into the skeletal defect before closing the wound.
  • a multicompartment device comprising two or more chambers or two or more syringes can be used to deliver components of the ex vivo hematoma and the bone substitutes separately.
  • a first chamber or syringe can comprise the coagulants (e.g., calcium and thrombin, or ecarin) at pre-determined concentrations.
  • a second chamber or syringe can comprise whole blood alone.
  • a second chamber or syringe can comprise whole blood in combination with exogenous growth factors (e.g., BMP2, PDGF, VEGF).
  • a second chamber or syringe can comprise whole blood in combination with bone substitutes (e.g., DBM, allogeneic cancellous bone chips).
  • a second chamber or syringe can comprise whole blood in combination with exogenous grow th factors (e.g.. BMP2. PDGF. VEGF) bone substitutes.
  • a third chamber or synnge can comprise exogenous growth factors and additional bone substitutes.
  • a third chamber or syringe can comprise exogenous growth factors.
  • a third chamber or syringe can comprise one or more bone substitutes.
  • a fourth chamber or syringe can comprise one or more bone substitutes.
  • the one or more growth factors is not BMP, rhBMP-2, BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, or BMP- 14.
  • the treatment regimen can be consistent and invariant provided there is no infection present and the defect is otherwise ready for definitive treatment.
  • the compositions or biomimetic scaffold (or implant) disclosed herein can be inserted into bone areas by entering the body through the skin or through a body cavity or an anatomical opening to minimize any additional damage to nearby structures.
  • Selection of the type, including size and shape, of the compositions, biomimetic scaffold, scaffold or implant can be based upon many factors, including, but not limited to, the shape and/or size of the bone into which the compositions, biomimetic scaffold, scaffold or implant is to be implanted; the percentage of bone density (i.e., the porousness of the remaining bone); and/or the desired rate and distribution of diffusion of the scaffold or implant into the bone; or a combination of such factors.
  • the shape of the compositions, biomimetic scaffold, scaffold or implant can be constructed to match the shape of the bone or vertebral body and thus allow for a more uniform distribution of the compositions, biomimetic scaffold, implant or ex vivo hematoma or the components present in the composition, scaffold, biomimetic scaffold, implant or ex vivo hematoma.
  • Application of the composition, biomimetic scaffold or implant can occur at the time of surgery or in any other suitable manner.
  • the shape of the depot implant or scaffold can be that of a sphere or cylinder. In some aspects, the shape of the depot implant or scaffold can be of any shape. In some aspects, the shape of the depot implant or scaffold can be that of a sphere or any other patient specific geometries, forms, or shapes as dictated by clinical exigency. In some aspects, the cylinder shape can be at least 5 mm to about 30 cm (or more) in length. In some aspects, the cylinder shape can be at least 1 mm to about 60 mm (or more) in diameter. In some aspects, the cylinder shape can be straight, and/or curved. In some aspects, the cylinder shape can be a straight rod or a curved rod.
  • the cylinder or rod shape can be any shape with a longitudinal axis that can be longer along one direction than in other directions.
  • the cross- sectional shape of the depot across the longitudinal axis can be any shape.
  • the cross-section shape can be elliptical, circular, trefoil, or any other shape.
  • the depot or scaffold can be either straight or curved in such longitudinal direction.
  • the end surface of the depot or scaffold can be shaped such that it is either flat, rounded or convoluted in shape.
  • the dimensions of the implant depot or scaffold or ex vivo hematoma can depend on the size of the bone defect and the anatomical site treated.
  • the scaffold can be approximately 20% longer than the actual size of the defect, so that it tightly fits and completely fills the volume of the missing bone.
  • the implant depot or scaffold or ex vivo hematoma will likely need to be constructed with dimensions, for example, that are about 3-4 cm in diameter and 3.6 cm in length.
  • the implant depot or scaffold or ex vivo hematoma can resemble the size and shape of a given bone defect.
  • the implant depot or scaffold can be chemotactic.
  • compositions and biomimetic scaffolds described herein to treat fractures at risk (e.g., in the osteoporotic, diabetic, elderly, or smokers). Also disclosed herein are methods of using any of the biomimetic scaffolds and compositions described herein to treat fractures at risk using ecarin to initiate the normal fracture healing cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous growth factors locally.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat atypical femur fractures. Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat atypical femur fractures percutaneously using ecarin to create an ex vivo hematoma that initiates the normal fracture healing cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous growth factors locally.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat minimally displaced femoral neck fractures. Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat minimally displaced femoral neck fractures percutaneously using ecarin to create an ex vivo hematoma that initiates the normal fracture healing cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous grow th factors locally.
  • osteoporotic insufficiency fractures e.g., pelvis, spine
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat delayed union of long bone fractures (percutaneously or open). Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat delayed union of long bone fractures (percutaneously or open) using ecarin to create an ex vivo hematoma, that initiates a fracture healing cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous growth factors locally.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat established non-unions of long bone fractures (percutaneously or open). Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat established non-unions of long bone fractures (percutaneously or open) using ecarin to create an ex vivo hematoma, that initiates a fracture healing cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous growth factors locally.
  • compositions and biomimetic scaffolds described herein to improve (e.g. accelerate) healing of long bone fractures.
  • compositions and biomimetic scaffolds described herein to accelerate healing of long bone fractures in selected veterinary candidates (such as thoroughbred racehorses). Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to accelerate healing of long bone fractures in selected veterinary candidates (such as thoroughbred racehorses) to facilitate more rapid recovery, by using ecarin to create an ex vivo hematoma, that initiates a fracture healing cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous growth factors locally.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to facilitate more rapid and predictable dental and maxilla-facial reconstructions. Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to facilitate more rapid and predictable dental and maxilla-facial reconstructions by using ecarin to create an ex vivo hematoma that initiates a bone formation cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous growth factors locally.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to and reverse conditions resulting in spontaneous jaw bone resorption. Also disclosed herein are methods of using any of the compositions described herein to and reverse conditions resulting in spontaneous jaw bone resorption, using ecarin to create an ex vivo hematoma to regenerate bone locally.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat and/or reverse conditions resulting in spontaneous avascular necrosis of the femoral head where the femoral head has collapsed. Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat and/or reverse conditions resulting in spontaneous avascular necrosis of the femoral head where the femoral head has collapsed, using ecarin to create an ex vivo hematoma delivered in an open procedure following surgical dislocation of the hip.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat osteonecrosis resulting from chemotherapy, alcoholism, smoking, or other exogenous agents. Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to treat osteonecrosis resulting from chemotherapy, alcoholism, smoking, or other exogenous agents, using ecarin to create an ex vivo hematoma delivered percutaneously or open.
  • compositions and biomimetic scaffolds described herein are methods of using any of the compositions and biomimetic scaffolds described herein to augment any standard fusion procedures. Also disclosed herein are methods of using any of the compositions and biomimetic scaffolds described herein to augment any standard fusion procedures (e.g., hip, knee, ankle, wrist, elbow, shoulder, subtalar joint, any of the limited fusions in the carpus or midfoot, fusions in any of the smaller joints such as the hallux, pollex, or lesser digits, either toes or fingers), using ecarin to create an ex vivo hematoma, that initiates a bone formation cascade by delivering catalytic amounts of BMP or one or more bone substitutes that then hyperactivates endogenous growth factors locally.
  • standard fusion procedures e.g., hip, knee, ankle, wrist, elbow, shoulder, subtalar joint, any of the limited fusions in the carpus or midfoot, fusions in any of the smaller joints such as the hallux, pollex
  • compositions and biomimetic scaffolds described herein in joint arthroplasty components w ith specially adapted bone ingrow th surfaces augmented with ecarin to induce local formation of an ex vivo hematoma embedded on a structural substrate, that more rapidly initiates a bone healing cascade.
  • compositions and biomimetic scaffolds described herein with osseointegration stems and components with specialty adapted bone ingrowth surfaces augmented with ecarin to induce local formation of an ex vivo hematoma embedded on a structural substrate, that more rapidly initiates a bone healing cascade.
  • the one or more bone substitutes is not BMP. rhBMP-2, BMP-2. BMP-7, BMP-4, BMP-6, BMP-9, or BMP-14.
  • biomimetic hematoma can be used to refer to an “ex vivo hematoma”.
  • any of the compositions described herein can be formulated to be sprayed on topically as an aqueous aerosol (using an atomizer for ecarin distribution to affected areas).
  • any of the compositions described herein can be administered on a bead (e g. magnetic bead).
  • any of the compositions described herein can be applied as a clamp/clamshell on the end of a vessel to simultaneously clamp off and deliver ecarin locally, restricting the application to the specific injured vessel end.
  • the clamp or clamp element can constrict the adjacent injured vessel adjacent, and can eliminate or minimize the risk of systemic administration of the compositions.
  • any of the compositions described herein can be delivered via an interventional radiologist to one or more targeted blood vessels to manage or stop intra-pelvic/intra- abdominal/oesophageal/intra-cranial bleeding using a long radiographically directed catheter that then allows selective and highly specific administration of ecarin limited to discrete pathology as indicated (e.g., similar to methods carried out using angiographic coils).
  • compositions described herein can be used to direct the installation or placement of any of the compositions described herein into the uterus in affected women.
  • ecarin can be formulated to be delivered as part of a bio-degradable collagen bead(s).
  • ecarin can be formulated to be delivered as part of a bio-degradable collagen bead(s).
  • compositions described herein can be formulated to be delivered as part of a biodegradable collagen bead(s).
  • compositions described herein can be used as a selective embolization.
  • compositions described herein can be used as a selective embolization.
  • ecarin can be formulated to be delivered as part of a bio-degradable collagen bead(s) or nanoparticle(s).
  • the bio-degradable collagen bead(s) or nanoparticle(s) can be delivered or sprinkled liberally into the joint immediately prior to closure of the wound.
  • ecarin can be formulated to be delivered as part of a biodegradable collagen bead(s).
  • the bio-degradable collagen bead(s) can be embedded in a fabric packing material or enclosed within a fabric sheath to limit their distribution and contain them locally.
  • ecarin can be delivered in the form of a nasal pack, such that the ecarin is formulated to be a part of a bio-degradable collagen bead(s) that is embedded in a fabric packing material or enclosed within a fabric sheath.
  • any of the compositions described herein can be used as a selective embolization.
  • ecarin can be formulated to be delivered as part of a bio-degradable collagen bead(s) or nanoparticle(s).
  • the bio-degradable collagen bead or nanoparticle formulation can be used to create a Velcro-type effect by creating a self-adherent geometry' to minimize the risk of recurrence and actively address retinal detachment.
  • bleeding can be a hemorrhage.
  • blood can be escaping the circulatory system from one or more damaged blood vessels.
  • bleeding can be internal or external.
  • compositions and biomimetic scaffolds described herein can be packaged in a suitable container labeled, for example, for use as a therapy to treat bone defects or any of the methods disclosed herein.
  • the composition comprising the ex vivo hematomas described herein can be packaged in a suitable container labeled, for example, for use as a therapy to treat bone defects or any of the methods disclosed herein, and can be packaged separately from the scaffold portion of the biomimetic scaffold.
  • packaged products e.g., scaffolds, sterile containers containing the compositions including the individual components of any of the compositions or ex vivo hematomas described herein and packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations
  • kits including at least isolated whole blood and sodium citrate; or platelet rich plasma, plasma, or plasma with red blood cells; and ecarin; oscutarin and calcium chloride; calcium chloride; thrombin; or thrombin and calcium chloride; and a messenger ribonucleic acid (mRNA)-based therapeutic composition or a ribonucleic acid (RNA)-based therapeutic composition as described herein and instructions for use, are also w ithin the scope of the disclosure.
  • mRNA messenger ribonucleic acid
  • RNA ribonucleic acid
  • a product can include a container (e g., a vial, jar, bottle, bag. or the like) containing the biomimetic scaffold or composition or ex vivo hematomas described herein.
  • a container e g., a vial, jar, bottle, bag. or the like
  • an article of manufacture further may include, for example, packaging materials, instructions for use, syringes, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required.
  • the product may also include a legend (e.g., a printed label or insert or other medium describing the product's use (e.g., an audio- or videotape).
  • the legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the biomimetic scaffold, compositions or ex vivo hematomas therein should be administered (e.g., the frequency and route of administration), indications therefore, and other uses.
  • the biomimetic scaffolds or compositions or ex vivo hematomas can be ready for administration (e.g., present in dose- appropriate units), and may include a pharmaceutically acceptable adjuvant, carrier or other diluent.
  • the compounds can be provided in a concentrated form with a diluent, with accompanying instructions for dilution.
  • Example 1 Ex-vivo Hematoma for Musculoskeletal Regeneration.
  • the disclosed ex vivo hematomas or biomimetic hematoma scaffolds can be used to deliver growth factors for VML regeneration. Both muscle and bone inj uries begin with hematoma formation.
  • Experiments were performed to investigate whether the ex vivo hematoma disclosed herein can act as a delivery vehicle for mRNA, for example mRNA encoding roof plate-specific spondin-2 (RSPO-2), to regenerate volumetric muscle defects.
  • mRNA lipid nanoparticles encoding for RSPO-2 w ere delivered via a mineral-coated microparticle (MCM) system to ensure efficient, localized delivery.
  • MCM mineral-coated microparticle
  • LGRs leucine-rich repeat-containing G-protein coupled receptors
  • mRNA enables modular and straightforward co-deli very of multiple growth factors with native folding, post-translational modifications, and translation of multiple protein isoforms.
  • MCM-mediated delivery of growth factors within the ex vivo hematoma disclosed herein can further amplify myogenic and neurogenic signaling, enhancing muscle regeneration.
  • an added benefit of the ex vivo hematoma is that the delivered RSPO-2 mRNA LNPs + MCMs is securely contained within the scaffold, eliminating and/or minimizing adverse effects, such as immune rejection, infection, and poor tissue integration, as it constitutes an autologous scaffold.
  • RSPO-2 mRNA LNPs Determine the biological activity of RSPO-2 mRNA LNPs within an ex vivo hematoma to enhance regeneration in a volumetric muscle defect model in rat. It will be tested whether RSPO-2 mRNA LNPs will be superior to an ex vivo hematoma alone to promote muscle regeneration. The minimal dose of RSPO-2 mRNA LNPs +/- MCMs to initiate muscle defect regeneration will also be determined, and RSPO-2 mRNA LNPs gene transfection levels will be evaluated over time in vivo in rats. The impact of RSPO-2 mRNA LNPs on changes of the ex vivo hematoma on architecture and gene expression will also be determined.
  • RSPO-2 roof plate-specific spondin 2
  • RSPO-2 was selected as the bioactive molecule because R- Spondins are implicated in myogenic differentiation (Han XH, et al. 2011. The Journal of Biological Chemistry:286: 10649-59; Kazanskaya O, et al. 2004. Dev Cell:7:525-34; and Knight MN, Hankenson KD. 2014. Matrix Biol:37: 157-61) and neuromuscular junction formation (Li J, et al. 2018.
  • R- Spondins activate and amplify WNT signaling by blocking the degradation of the WNT ligand receptor the lipoprotein receptor-related proteins (LRPs) by sequestering the ubiquitin ligases ZNRF3 and RNF43 (Zebisch M, et al. 2013. Nat Commun:4:2787).
  • WNT/p-catenin signaling is implicated in muscle formation and regeneration (Girardi F, Le Grand F. Chapter Five - Wnt Signaling in Skeletal Muscle Development and Regeneration. In: Larrain J, Olivares G, Larrain J, Olivares G, editors. Progress in Molecular Biology and Translational Science 2018. p. 157-79), however, many WNT agonists are highly hydrophobic making them difficult therapeutic targets whereas R-Spondins are hydrophilic along with other advantages (Dhamdhere GR, et al. 2014. PLoS One:9). This establishes RSPO-2 as a suitable target molecule for muscle regeneration delivered within the ex vivo hematoma.
  • mRNA delivery is a method for non-viral delivery of nucleic acids allowing for higher efficacy than recombinant proteins and with improved safety over viral delivery (Chen C-Y, et al. 2020. Mol Ther Nucleic Acids:20:534-44; and Wadhwa A, et al. 2020. Pharmaceutics: 12).
  • mRNA lipid nanoparticles (LNP) have been shown to be highly efficacious with vaccination against SARS-CoV-2 globally (Baden LR, et al. 2021. New England Journal of Medicine:384:403- 16; and Polack FP, et al. 2020. New England Journal of Medicine:383:2603-15).
  • MCMs Mineral coated microparticles
  • MCMs extend the biological effect of produced proteins through sequestration and have shown to be therapeutically effective with a single dose in a diabetic wound healing and spinal cord injury model. MCMs are also biomimetic and dissolve overtime (Choi S, Murphy WL. 2010. Acta Biomater: 6: 3426-35; Khalil AS, et al. 2022. Adv Healthc Mater: e2200206; Choi S, et al. 2013. Sci Rep: 3: 1567-93; Hellenbrand DJ, et al. 2019. J Neuroinflammation: 16:93; Khalil AS, et al. 2020. Science Advances:6:eaba2422; Yu X. 2017. Adv Mater:l l; and Yu X, et al. 2014.
  • ex vivo hematomas disclosed herein comprising MCMs can be used to deliver RSPO-2 encoding mRNA and will be superior in to at least an ex vivo hematoma alone to promote functional muscle regeneration in a rat model of volumetric muscle loss.
  • VML volumetric muscle loss
  • a BT et al. 2015. J Rehabil Res Dev:52:785-92; Grogan BF, Hsu JR. 2011. J Am Acad Orthop Surg: 19 Suppl LS35-7; Anderson SE, et al. 2019. Tissue Eng Part C Methods:25:59-70).
  • Biomaterial scaffolds may recapitulate the biological and physical properties of the extracellular matrix to assist with muscle regeneration, support cellular infiltration, proliferation, and differentiation, and promote the distribution of nutrients and oxygen (Mostafavi A, et al. 2021. Appl Phys Rev:8:041415; Mulbauer GD. Matthew HWT. 2019.
  • Musculoskeletal injuries including muscle, commence with hematoma formation, an important step that initiates the biological cascade of the healing process.
  • blood vessels contract to prevent sustained blood loss, followed by a coagulation cascade that leads to the formation of a hematoma, or blood clot, which serves as a natural scaffold.
  • the structural parameters in fibrin clots can be characterized by the fiber diameter, density, the number of branch points, distances between branch points, and dimension of the pores (Weisel JW, Litvinov RI. 2013. Blood: 121 : 1712-9).
  • the diameter and density of fibers has an impact on the porosity and surface area of fibrin clots (Pham QP, et al. 2006. Biomacromolecules:7:2796-805) and is responsible for the biological functions of stem cells, such as adhesion, proliferation, and differentiation (Badami AS. et al. 2006. Biomaterials:27:596-606).
  • low thrombin concentrations ⁇ 1 nM
  • high concentrations of thrombin result in thin fibers that form a poorly permeable fibrin network that is relatively resistant to fibrinolysis (Gabriel DA, et al. 1992.
  • the ex vivo hematoma will deliver mRNA RSPO-2 LNPs to the injury site and be expressed or translated in the desired cell type or target site, initiating muscle regeneration. Therefore, it will be tested whether RSPO-2 encoding mRNA will activate cells at the target site when delivered to an injury’ site as part of an ex vivo hematoma to promote muscle regeneration.
  • TA muscle will be dissected from fixed, Microfil perfused rats and imaged using MicroCT.
  • a desktop MicroCT imaging system (Bruker Skyscan 1173, Belgium) equipped with a microfocus X-ray tube with a ⁇ 5 pm spot size will be used, and the samples will be scanned at 5 pm isotropic voxel size using 60 kV, 167 pA, 0.5 mm.
  • the vessel volume fraction (VV/TV, %), vessel thickness (Vs. Th, pm), vessel number (Vs.N, mm’ 1 ), vessel separation (Tb.Sp, mm’ 1 ) and connectivity density (ConnD, l/mm3) will be used.
  • vWF von Willebrand factor
  • CD68 macrophage marker
  • eMHC embryonic myosin heavy chain
  • Pax7 myogenic progenitor cells
  • transfection kinetics of translated mRNA will be determined by in vivo imaging using an IVIS® Spectrum machine (PerkinElmer, Waltham, USA).
  • Reporter (Firefly Luciferase or eGFP) mRNA LNP functionalized clots, labeled with DiR, will be implanted into the rat VML model. DiR fluorescence images will be captured under the excitation and emission w avelengths of 745 and 800 nm, respectively.
  • animals will receive an IP injection of 150 mg/kg of D-Luciferin, and bioluminescence activity of the Firefly Luciferase will be recorded with IVIS on days 1, 3, 5, 7, and 14 following implantation. Peak luminescent activity' over 20 minutes will be recorded and used for subsequent experiments. The luminescence or fluorescence intensities in each region of interest (ROIs) will be quantified using Living Image 3.0 software (PerkinElmer, Waltham. USA).
  • ROIs region of interest
  • RSPO-2 mRNA LNPs will be delivered within an ex vivo hematoma disclosed herein into the defect site. The ex vivo hematoma will be recovered after 1, 3, and 7 days, and tissue explants will be homogenized.
  • RSPO-2 will then be detected using a commercially available ELISA kit (Thermo Fisher Scientific, Waltham, USA). Determination of eGFP mRNA transfected cell types will be done by flow cytometry after explanation of the ex vivo hematoma on day 1 following transfection. The following markers will be used to distinguish blood cells (CD45+) and muscle cells: leukocytes (CD32), T-cells (CD3), neutrophils (CD43), B-cells (CD45R), muscle stem cells (VCAM-1), skeletal muscle (FABP3).
  • fibrin matrix affects its biological function (Woloszy k A, et al. 2022. Biomater Adv: 139:213027; and Laurens N, et al. 2006. Journal of Thrombosis and Haemostasis:4:932-9).
  • the binding of fibrin(ogen) to hemostasis proteins and platelets as well as to several different cells such as endothelial cells, smooth muscle cells, fibroblasts, leukocytes, and keratinocytes is indispensable during the process of wound repair (Laurens N, et al. 2006. Journal of Thrombosis and Haemostasis:4:932-9).
  • the muscle tissue slices will be dried using a Critical Point Dry' er and will be sputter-coated with gold525 palladium before imaging using a scanning electron microscopy (SEM) at a magnification of 10,000x.
  • SEM scanning electron microscopy
  • SEM images of the hematomas, such as thickness, density’, and porosity of the muscle fibers will be used for quantitative image analysis, in ImageJ, to turn micrographs into quantitative measurements.
  • RNA-sequencing will be performed at the Genome Sequencing Core Facility (GSCF) of the Greehey Children’s Cancer Research Institute at UT Health San Antonio.
  • GSCF Genome Sequencing Core Facility
  • the RNA data will be analyzed using Qiagen’s Ingenuity Pathway Analysis.
  • the CIBERSORT tool Newman AM, et al. 2015. Nat Methods: 12:453-7 will be utilized.
  • Upstream regulator analyses will be generated from the differential expression gene data set acquired from RNA-sequencing analysis using Ingenuity 7 Pathway Analysis (IP A, Qiagen Inc, Germantown, MD. Furthermore, histology 7 and immunohistochemistry (IHC) will also be performed to characterize the tissues and confirm the presence of important proteins involved in the initiation of the repair process, as described herein.
  • IP A Ingenuity 7 Pathway Analysis
  • IHC immunohistochemistry
  • RSPO-2 will play an important role in myogenesis and reinnervation. The time for functional muscle regeneration using mRNA encoding RSPO-2 delivered within the ex vivo hematoma will be determined.
  • Neurovascular regeneration during remodeling phase will also be characterized. Muscle strength and function recovery will be determined using biomechanical studies.
  • the sequential phases of tissue healing using the disclosed ex vivo hematoma will be carried out.
  • the ex vivo hematoma disclosed herein will be assessed in a VML model for its ability to provide functional muscle regeneration. Therefore, it will be tested whether the ex vivo hematoma disclosed herein comprising mRNA encoding R-Spondin-2 can mitigate inflammation and facilitate functional muscle regeneration in a volumetric muscle defect.
  • R- Spondin-2 will play an important role in myogenesis and reinnervation.
  • the time required for functional muscle regeneration using mRNA encoding RSPO-2 delivered within the ex vivo hematoma will be determined using the same VML rat model described herein The optimal dose of mRNA encoding RSPO-2 will be delivered either with or without MCMs.
  • animals will be euthanized for various outcome measures to determine the time required for the muscle to regain full function. The selection of the 4-week time point is based on the understanding that scar tissue (fibrosis) typically begins around the third week post-injury and increases in size over time. If fibrosis is present at 4 weeks. the muscle is unlikely to regain function. By 8 weeks, the muscle defects should show almost complete muscle revascularization and reinnervation.
  • the set of samples from each group will be utilized as described herein.
  • the tibialis anterior muscle will be dissected from fixed, Mi crofil -perfused rats and imaged using MicroCT. Following the MicroCT analysis, muscle samples will undergo analysis for vasculature and neuromuscular regeneration using histology' and IHC.
  • mRNA RSPO-2 + MCMs + ex vivo hematoma will achieve functional muscle regeneration at the end of 8 weeks. Additionally, it is expected that regenerated muscle will have similar neurovascular characteristics and seamless reintegration with the native muscle. If muscle regeneration is not be complete by 8 weeks, the study endpoint will be extended to 12 weeks. If natural regeneration in an empty VML defect, representing a 30% resection of the tibialis anterior (TA) full muscle, occurs by 8 w eeks, defect size will be increased to a 50% resection of the TA.
  • VML defect representing a 30% resection of the tibialis anterior (TA) full muscle
  • R-Spondins are WNT agonists involved in myogenic and osteogenic regeneration.
  • RSPO-2 but not RSPO-1 contains a BMP-R inhibitory domain in the TSP-1 domain (Nat Commun 1 1, 5570 (2020). However, RSPO-1 does not impact BMP-R activity but has reduced potency (Lee, H., et al. Nat. Commun. 11, 5570 (2020)).
  • Mutant RSPO-2 with the TSP-1 domain from RSPO-1 is functionally active ((Lee, H., et al. Nat. Commun. 11, 5570 (2020)) and may promote BMP signaling when combined with BMPs.
  • RSPO-2 myogenic RSPO-2
  • RSPO-2dTSP-l RSPO-1
  • WT-RSPO-2 can inhibit heterotopic ossification from the underlying regenerating bone while actively inhibiting heterotopic ossification.
  • Mineral coated microparticles improve mRNA delivery and prolong the expression of the protein product and can be injected (Adv Mater. 2017 Sep;29(33)).
  • the ex vivo hematomas disclosed herein can be used as scaffolds for MSK repair (Biomater Adv. 2024 May;148:213).
  • Described herein is the development of biomaterial and a therapeutic mRNA approach to promote regeneration of composite muscle and bone defects. It was tested whether that a single dose of mRNA for R-Spondins using mineral-coated microparticles to deliver the mRNA via an ex vivo hematoma can be used promote myogenesis and osteogenesis.
  • Synthetic mRNA for RSPO-2, RSPO-1, RSPO-2dTSP-l (RSPO-1) and BMP-2 were created by cloning the coding sequences into a plasmid DNA expression vector flanked by 5’ and 3' untranslated regions from the beta-globin gene. DNA templates were created using PCR with an appended 120 nt long polydT tail in the 3’ primer. mRNA were synthesized with co-transcriptional capping using CleanCap-AG (Trilink Biotechnologies) and uridine were fully substituted with N1 -methylpseudouridine. LipofectamineTM Messenger Max (Thermofisher Scientific) was used as the transfection reagent.
  • MCM Mineral-coated microparticles
  • Myogenesis Muman H9 embry onic stem cells or murine C2C12 myoblasts were cultured in growth media (DMEM, 1% Pen/Strep. 10% FBS) until 75% confluence, then were cultured in low serum media: DMEM, 1% Pen/Strep, 2% B-27 supplement (Gibco).
  • RSPO-2 mRNA +/- MCMs were delivered two days after myogenic induction and were fixed on d7 or dl4 and stained for myosin heavy chain expression (MYHC). On d7 mRNA was extracted for gene expression for myog, myf5. myostatin or axin2.
  • RSPO-2 mRNA delivery increased myf5 expression in differentiating C2C12 cells after 7 days (FIG. I ll) but only RSPO-2 mRNA delivered with MCMs decreased expression myostatin (FIG. 11 J).
  • RSPO-2 mRNA + MCMs enhanced myotube formation, cell fusion and MYHC staining in C2C12 cells (FIG. 8). MCMs alone improved myotube formation (FIG. 8B).
  • RSPO-2 mRNA+MCMs increased axin2 and myf5 gene expression and decreased myostatin expression, an inhibitory gene (FIG. 9).
  • Calcium phosphates are known to be osteoconductive and sequester proteins, which may help to override any inhibitory effects of WT-RSPO-2.
  • RSPO-2 has been shown to enhance myogenesis in C2C12 cells.
  • a mRNA delivery strategy that promoted myogenesis in C2C12 cells as assessed by MYHC expression and gene expression of the pro-myogenic gene myf5.
  • MCMs alone improved myotube formation without RSPO-2.
  • RSPO-2 or MCM delivery seemed to promote alignment of the myotubes relative to the untreated control. Furthermore, RSPO-2 mRNA with MCMs reduced myostatin, an inhibitory protein for myogenesis, while MCMs alone increased myostatin expression.
  • compositions and methods disclosed herein can be used to promote healing of muscle and bone defects while inhibiting heterotopic ossification.

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Abstract

L'invention concerne des compositions et des échafaudages biomimétiques et des procédés d'utilisation pour traiter, apporter des améliorations à, favoriser et accélérer la cicatrisation, la régénération osseuse, la régénération musculaire, ou une combinaison de ceux-ci chez un sujet. Les procédés comprennent l'implantation desdites compositions et des échafaudages biomimétiques chez un sujet.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050255546A1 (en) * 2002-04-26 2005-11-17 Kirin Beer Kabushiki Kaisha Polypeptide having an activity to support proliferation or survival of hematopoietic stem cell or hematopoietic progenitor cell, and dna coding for the same
US20160151540A1 (en) * 2014-12-02 2016-06-02 Denver M. Lough Methods for Development and Use of Minimally Polarized Function Cell Micro-Aggregate Units in Tissue Applications Using LGR4, LGR5 and LGR6 Expressing Epithelial Stem Cells
WO2023034451A1 (fr) * 2021-08-31 2023-03-09 Board Of Regents, The University Of Texas System Compositions et méthodes destinées au traitement de fractures osseuses
WO2023133595A2 (fr) * 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Méthodes de dosage et d'administration ex vivo de particules lipidiques ou de vecteurs viraux ainsi que systèmes et utilisations associés

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050255546A1 (en) * 2002-04-26 2005-11-17 Kirin Beer Kabushiki Kaisha Polypeptide having an activity to support proliferation or survival of hematopoietic stem cell or hematopoietic progenitor cell, and dna coding for the same
US20160151540A1 (en) * 2014-12-02 2016-06-02 Denver M. Lough Methods for Development and Use of Minimally Polarized Function Cell Micro-Aggregate Units in Tissue Applications Using LGR4, LGR5 and LGR6 Expressing Epithelial Stem Cells
WO2023034451A1 (fr) * 2021-08-31 2023-03-09 Board Of Regents, The University Of Texas System Compositions et méthodes destinées au traitement de fractures osseuses
WO2023133595A2 (fr) * 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Méthodes de dosage et d'administration ex vivo de particules lipidiques ou de vecteurs viraux ainsi que systèmes et utilisations associés

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