Biomaterials for the prevention and treatment of tissue damage

Abstract

The present invention relates to a sterile, dry biomaterial comprising devitalized, differentiated cells with tissue regenerative and / or tissue repair properties and a particulate material, said cells and said particulate material being embedded in an extracellular matrix, preferably gelatin, a ceramic material, or demineralized bone matrix (DBM).

Description

[Technical Field]

[0001] The present invention relates to the field of tissue regeneration and tissue repair, including the prevention and / or treatment of skin, bone and / or cartilage disorders. More particularly, the present invention relates to a sterile, dry biomaterial comprising devitalized, differentiated cells having tissue regenerative and / or tissue repair properties and a particulate material, wherein the cells and particulate material are embedded in an extracellular matrix. [Background technology]

[0002] Tissue reconstruction includes bone reconstruction, cartilage reconstruction, as well as skin reconstruction (including the dermis and epidermis) and muscle reconstruction.

[0003] A bone defect is a loss of bone tissue in a site where bone would normally be present. Bone defects can be treated using a variety of surgical methods. However, there are many factors that can impede bone healing, including diabetes, immunosuppressive therapy, and reduced mobility, which must be considered when planning a procedure. Surgical methods for bone defect reconstruction include decortication, resection and fixation, cancellous bone grafting, and the Ilizarov intercalary bone transport method, among others. However, patients typically experience long-term ambulation problems and achieve suboptimal functional and cosmetic results.

[0004] Tissue engineering uses living cells to restore tissue structure and function. The general process consists of cell isolation and expansion, followed by a reimplantation procedure using a scaffold material. Mesenchymal stem cells (MSCs) offer an excellent alternative to mature tissue cells and have many advantages, for example, as a cell source for the regeneration of skin, bone, and cartilage tissue. By definition, stem cells are characterized by their ability to self-renew and multilineage differentiate, forming terminally differentiated cells. Stem cells for regenerative medicine applications should ideally meet the following criteria: (i) be present in large quantities (millions to billions of cells); be harvestable and obtainable using minimally invasive procedures; (iii) be capable of reproducibly differentiating along multiple cell lineage pathways; and (iv) be capable of safe and effective transplantation into autologous or allogeneic hosts.

[0005] Multiple studies have demonstrated that stem cells have the ability to differentiate into cells of mesodermal, endodermal, and ectodermal origin. The plasticity of MSCs refers to their inherent ability, most often retained within stem cells, to cross lineage barriers and adopt the phenotypic, biochemical, and functional properties of cells native to other tissues. For example, adult mesenchymal stem cells can be isolated from bone marrow and adipose tissue.

[0006] Adipose tissue-derived stem cells are multipotent and have great regenerative potential. Osteogenically differentiated ASCs have been shown in various preclinical models to exhibit significant healing potential when seeded onto various scaffolds, such as β-tricalcium phosphate (β-TCP), hydroxyapatite (HA), type I collagen, polylactic-co-glycolic acid (PLGA), and alginate. Patent Document 1 relates to a bone paste containing a mixture of stem cells and calcium phosphate cements, such as tricalcium phosphate and hydroxyapatite. Patent Document 2 discloses a bone patch containing a scaffold material containing a synthetic ceramic material, mesenchymal stem cells, and signaling molecules. Patent Document 3 discloses a bone regenerator containing a devitalized cell construct containing stem cell-derived cells, minerals, and an extracellular matrix.

[0007] However, despite promising results in small animal models, critical-size bone reconstruction using scaffold-loaded ASCs remains limited by the large size of the bone defect and, therefore, the size of the implants designed. Cell engraftment of seeded cells is also limited by poor oxygen and nutrient diffusion. Furthermore, the location of cells within the scaffold poses a significant constraint on their in vitro and in vivo viability. Bioreactors with scaffold flow perfusion have been designed to improve cell migration within the implant, which distributes cells more uniformly, cell survival by delivering oxygen and nutrients to the center of the implant, and osteogenic cell differentiation (through fluid shear forces). Although these technologies are promising, relevant preclinical and clinical data in large animal models are limited.

[0008] Recently, Patent Document 4 discloses a biomaterial comprising adipose tissue-derived stem cells (ASCs), a biocompatible material, and an extracellular matrix, and the biomaterial secretes osteoprotegerin (OPG).

[0009] Furthermore, Patent Document 5 discloses a biomaterial that has a multidimensional structure containing osteogenic differentiated adipose tissue-derived stem cells (ASCs), a ceramic material, and an extracellular matrix, secretes osteoprotegerin (OPG), and contains insulin-like growth factor (IGF1) and stromal cell-derived factor 1 alpha (SDF-1α). This biomaterial has been shown to be useful for treating bone and cartilage defects.

[0010] Furthermore, International Patent Publication No. 6 discloses a biomaterial having a multidimensional structure containing differentiated adipose tissue-derived stem cells (ASCs), an extracellular matrix, and gelatin. It has been shown that this biomaterial can be used to treat tissue disorders such as those of bone, cartilage, muscle, and skin.

[0011] On the other hand, while both biomaterials may be suitable for autologous grouting, allogeneic or xenogeneic grouting is not feasible because they may induce an immune response and reject the grout, or they may carry adventitious pathogens that could result in infection of the recipient by the biomaterial.[Patent Document 7] discloses a living tissue prosthesis that can manage immunological rejection caused by transplantation and is suitable for long-term storage. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2013 / 059089 Brochure [Patent Document 2] US Patent Application Publication No. 2011 / 104230 [Patent Document 3] US Patent Application Publication No. 2016 / 287753 [Patent Document 4] International Publication No. 2019 / 057861 Brochure [Patent Document 5] International Publication No. 2019 / 057862 Brochure [Patent Document 6] International Publication No. 2020 / 058511 Brochure [Patent Document 7] Japanese Patent Application Laid-Open No. 2004-305259 Summary of the Invention [Problem to be solved by the invention]

[0013] Sterilization processes are often used to mitigate these problems, but these harsh conditions can deteriorate the biological properties of the sterilized materials.

[0014] Thus, there remains a need in the art for tissue engineering materials for tissue reconstruction and / or regeneration that can be used in a wide range of tissues, yet are fully biocompatible and provide appropriate mechanical characteristics for the designated application.

[0015] There is also a need to provide biomaterials for tissue reconstruction and / or regeneration, including the treatment of skin, bone, or cartilage defects, that are compatible with allogeneic or xenogeneic grouts, and that maintain biological properties compared to fresh biomaterials prior to sterilization. [Means for solving the problem]

[0016] A first aspect of the present invention relates to a sterile, dry biomaterial comprising devitalized, differentiated cells having tissue regenerative and / or tissue repair properties and particulate material, wherein said cells and said particulate material are embedded in an extracellular matrix.

[0017] In certain embodiments, the cells are selected from the group consisting of primary cells, stem cells, genetically modified cells, and combinations thereof.

[0018] In some embodiments, at most 10%, preferably at most 1% of the cells are viable.

[0019] In certain embodiments, the particulate material comprises or is selected from the group consisting of: - organic materials, including demineralized bone matrix (DBM), gelatin, agar / agarose, alginate chitosan, chondroitin sulfate, collagen, elastin or elastin-like peptides (ELPs), fibrinogen, fibrin, fibronectin, proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, polysaccharides, laminin, and cellulose derivatives; - ceramic materials comprising calcium phosphate (CaP) particles, calcium carbonate (CaCO3) particles, calcium sulfate (CaSO4) particles, calcium hydroxide (Ca(OH)2) particles, or combinations thereof; polymers, including polyanhydrides, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene oxide / polyethylene glycol (PEO / PEG), poly(vinyl alcohol) (PVA), fumaric acid-based polymers such as poly(propylene fumarate) (PPF) and poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)), oligo(poly(ethylene glycol) fumarate) (OPF), poly(n-isopropylacrylamide) (PNIPPAAm), poly(aldehyde guluronate) (PAG), poly(n-vinylpyrrolidone) (PNVP), or combinations thereof; -gels, including self-assembling oligopeptide gels, microgels, nanogels, particulate gels, hydrogels, thixotropic gels, xerogels, responsive gels, or combinations thereof; -Creamer; and A combination of them.

[0020] In some embodiments, the biomaterial contains an altered factor content compared to the factor content obtained from a corresponding fresh, non-sterile, non-dried biomaterial. In some embodiments, the factor content includes growth factors and / or transcription factors. In some embodiments, the factor content includes IGF-1 and / or VEGF and / or SDF-1α and / or OPG. In some embodiments, the factor content includes RNA content. In some embodiments, the RNA content includes at least one miRNA selected from any one of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, or Table 12. In some embodiments, the dried biomaterial is obtained by lyophilization. In some embodiments, the sterilized biomaterial is obtained by gamma irradiation, preferably at a dose of about 7 kGy to about 45 kGy, more preferably at room temperature.

[0021] Another aspect of the present invention relates to a method for producing a sterile, dry biomaterial comprising devitalized, differentiated cells and particulate material, said cells and said particulate material being embedded in an extracellular matrix, said method comprising the steps of: -(1) contacting differentiable living cells (i) with particulate material (ii) to obtain a first combination; - (2) culturing the first combination obtained in step (1) in a culture medium so that the cells secrete an extracellular matrix and synthesize factor contents to obtain tissue regeneration and / or tissue repair properties, wherein the cells and the particulate material are embedded in the extracellular matrix to form a multidimensional structure; - (3) subjecting the multidimensional structure obtained in step (2) to drying to obtain a dried biomaterial; and - (4) subjecting said dried biomaterial obtained in step (3) to sterilization, preferably by gamma irradiation, to obtain a sterile, dried biomaterial.

[0022] In some embodiments, the differentiable live cells are selected from the group comprising primary cells; stem cells, particularly stem cells from adipose tissue, bone marrow, or umbilical cord blood; genetically modified cells; and mixtures thereof.

[0023] Yet another aspect of the present invention relates to dry, sterile biomaterial obtainable by the method according to the present disclosure.

[0024] Yet another aspect of the present invention relates to a pharmaceutical composition comprising a biomaterial according to the present disclosure and a pharmaceutically acceptable vehicle. In certain embodiments, the composition is in the form of a paste or a film.

[0025] One aspect of the present invention relates to a medical device comprising a biomaterial according to the present disclosure or a pharmaceutical composition according to the present disclosure.

[0026] In one aspect, the present invention also relates to a biomaterial according to the present disclosure or a pharmaceutical composition according to the present disclosure for use as a medicament. In some embodiments, the biomaterial or pharmaceutical composition is used to prevent and / or treat a tissue disorder. In some embodiments, the tissue is selected from the group consisting of bone tissue, cartilage tissue, skin tissue, muscle tissue, epithelial tissue, endothelial tissue, nervous tissue, connective tissue, and adipose tissue. In some embodiments, the tissue disorder is selected from the group comprising: aplasia cutis congenita; burns; cancer, including breast cancer, skin cancer, and bone cancer; compartment syndrome (CS); epidermolysis bullosa; giant congenital nevi; ischemic muscle injuries of the lower extremities; muscle contusions, ruptures, or strains; post-radiation lesions; diabetic ulcers, particularly ulcers including diabetic foot ulcers; arthritis; fractures; bone fragility; Caffey's disease; congenital pseudoarthrosis; cranial deformities; cranial malformations; delayed union; bone infiltration disorders; hyperostosis; decreased bone mineral density; metabolic bone loss; osteogenesis imperfecta; osteomalacia; osteonecrosis; osteopenia; osteoporosis; Paget's disease; pseudoarthrosis; sclerotic lesions; spina bifida; spondylolisthesis; spondylolysis; chondrodysplasia; costochondritis; enchondroma; hallux rigidus; labral tears of the hip; osteochondrosis dissecans; osteochondrodysplasia; polychondritis; and the like. In certain embodiments, the biomaterial or pharmaceutical composition is used to prevent and / or treat bone and / or cartilage disorders. In some embodiments, the biomaterial or pharmaceutical composition is used for tissue reconstruction. In certain embodiments, the biomaterial or pharmaceutical composition is used to compensate for side effects of primary treatments for tissue disorders and / or to enhance primary treatments for tissue disorders. In some embodiments, the biomaterial or pharmaceutical composition is used to compensate for side effects of therapeutic procedures known to have adverse effects on tissues, particularly bone tissue, cartilage tissue, skin tissue, muscle tissue, epithelial tissue, endothelial tissue, nervous tissue, connective tissue, and adipose tissue. (definition)

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the definition of a term in this application shall prevail.

[0028] In the present invention, the following terms have the following meanings:

[0029] The term "about" preceding a value means ±10% of the value in question. It is to be understood that the value to which the term "about" refers is itself also specifically, preferably disclosed.

[0030] The term "comprises" means "contains," "encompasses," and "includes." In some embodiments, the term "comprises" also includes the term "consists of."

[0031] The term "tissue disorder" refers to any disturbance or imbalance in the physiological function of a tissue. Non-limiting examples of symptoms observed in tissue disorders include injury, contusion, infection, sprain, trauma, fissure, swelling, redness, edema, pain, tenderness, soreness, wound, necrosis, or a combination thereof. As used herein, the terms "tissue disorder," "tissue disease," "tissue medical condition," and the like are equivalent.

[0032] The term "regeneration" or "tissue regeneration" includes, but is not limited to, the growth, generation, or reconstruction of new cell types or tissues following treatment with a biomaterial according to the present invention. In one embodiment, these cell types or tissues include, but are not limited to, bone-forming cells (e.g., osteoblasts, osteocytes), chondrocytes, fibroblasts, keratinocytes, endothelial cells, cardiomyocytes, hematopoietic cells, hepatocytes, adipocytes, neurons, and myotubes. As used herein, the term "regeneration" is contemplated as a preventative and / or therapeutic treatment following injury, wound, surgery, congenital, degenerative, traumatic or non-traumatic condition or condition, or other treatment that causes a tissue fissure, opening, depression, wound, etc.

[0033] The term "repair" or "tissue repair" includes, but is not limited to, the healing process of rebuilding healthy tissue from diseased or dysfunctional tissue. In one embodiment, tissue repair includes skin repair, e.g., healing, scar formation, and attenuation. In one embodiment, tissue repair includes bone repair, e.g., fracture reduction. In one embodiment, tissue repair includes cartilage repair. In one embodiment, tissue repair includes filling, plumping, supporting, expanding, stretching, or increasing the size, volume, or mass of a body tissue.

[0034] - As used herein, the term "devitalized cells" refers to cells that do not have an active metabolism and are not capable of dividing when contacted with an appropriate culture medium.

[0035] - As used herein, the term "mesenchymal stem cells" refers to multipotent stem cells that can differentiate into several types of cells belonging to skeletal tissues, such as cartilage, bone; skin tissue; muscle tissue; epithelial and / or endothelial tissue; nervous tissue; connective tissue; and adipose tissue, such as fat.

[0036] The term "adipose tissue" refers to any fat tissue. The adipose tissue may be brown adipose tissue or white adipose tissue derived from subcutaneous, omental / visceral, mammary, gonadal, or other adipose tissue sites. Preferably, the adipose tissue is subcutaneous white adipose tissue. Such cells may comprise primary cell cultures or immortalized cell lines. The adipose tissue may be derived from any organism, living or dead, that possesses adipose tissue. Preferably, the adipose tissue is of animal origin, more preferably of mammalian origin, and most preferably of human origin. A convenient source of adipose tissue is obtained by liposuction, although the source of adipose tissue or the method of isolation of the adipose tissue is not critical to the present invention.

[0037] As used herein, the term "adipose tissue-derived stem cells" "teeth "Adipose tissue-derived stem cells" (ASCs) refer to the "non-adipocyte" fraction of adipose tissue. The cells may be fresh or cultured. "Adipose tissue-derived stem cells" (ASCs) refer to stromal cells that are derived from adipose tissue and can serve as precursors to a variety of different cell types, including but not limited to, adipocytes, osteocytes, chondrocytes, fibroblasts, myoblasts, epithelial cells, endothelial cells, connective cells, neural cells, etc., and their progenitors.

[0038] The term "particulate material" as used herein refers to a solid material having the form of particles. Within the scope of the present invention, "particulate material" includes organic materials such as demineralized bone matrix (DBM) and gelatin; ceramic materials; polymers such as polyanhydrides; gels such as hydrogels; and combinations thereof. Importantly, particulate materials are also characterized by their average diameter.

[0039] As used herein, the term "ceramic material" refers to an inorganic, non-metallic solid material. The ceramic material may include calcium phosphate (CaP), calcium carbonate (CaCO), calcium sulfate (CaSO), calcium hydroxide (Ca(OH)), or a combination thereof. The ceramic material may be in the form of particles. The particulate material, preferably the ceramic material, may be in the form of a powder, beads, or granules. The particulate material, preferably the ceramic material, may be porous.

[0040] As used herein, the term "growth factor" encompasses molecules that promote tissue growth, cell proliferation, angiogenesis, etc. In certain embodiments, the term "growth factor" includes molecules that promote skin, muscle, cartilage, endothelial, epithelial, nerve, connective, adipose, or bone tissue.

[0041] - As used herein, the term "transcription factor" refers to a molecule that controls whether a given gene is transcribed into its corresponding RNA.

[0042] As used herein, the term "differentiated cell" refers to a precursor cell that has evolved from a non-specialized phenotype to a specialized phenotype. For example, stem cells, particularly MSCs such as ASCs, can differentiate into osteogenic cells, chondrogenic cells, adipocytes, epithelial cells, connective cells, neural cells, or endothelial cells.

[0043] The term "differentiation medium" as used herein refers to one of a combination or collection of compounds used in the culture system of the present invention to produce differentiated cells. There is no limitation regarding the mechanism of action of the compound. For example, an agent may aid the differentiation process by inducing or supporting a phenotypic change, by promoting the growth of cells with certain phenotypes or by retarding the growth of others. It may also act as an inhibitor of other factors present in the medium or synthesized by the cell population that would otherwise induce differentiation down a pathway toward an undesired cell type.

[0044] The term "miRNA" or "miR" refers to a non-coding RNA approximately 18 to 25 nucleotides in length. These miRNAs can originate from multiple sources, including individual genes encoding miRNAs, introns of protein-coding genes, or polycistronic transcripts that often encode multiple closely related miRNAs. In the following disclosure, the standard nomenclature is applied, whereby uncapitalized "mir-X" refers to the pre-miRNA (precursor) and capitalized "miR-X" refers to the mature form. When two mature miRNAs are derived from opposite arms of the same pre-miRNA, they are designated with the suffixes -3p or -5p. In the following disclosure, unless otherwise specified, the use of the expression miR-X refers to the mature miRNA, including both -3p and -5p forms, if any. Within the scope of the present invention, the terms microRNA, miRNA, and miR refer to the same compound.

[0045] The term "exosome" refers to extracellular vesicles that are released from cells upon fusion of multivesicular bodies (MVBs), intermediate endocytic compartments, with the plasma membrane. In other words, exosomes correspond to intraluminal vesicles that are released into the extracellular environment.

[0046] The term "secretion" refers to a physiologically active substance that is transported from the cell in which it is synthesized. In one embodiment, the physiologically active substance can be any molecule, particularly a protein (such as a growth factor or transcription factor) or a nucleic acid (such as miRNA). As used herein, the term "secretion" includes both active and passive secretion. The term "active secretion" in the present application refers to the secretion of a physiologically active substance from a cell by a living cell, particularly a mesenchymal stem cell, preferably an adipose tissue-derived stem cell, in response to a stimulus, thereby diffusing into the cell's environment, for example, the extracellular matrix. As used herein, "living cells" refers to cells that exhibit at least one of the following characteristics: growth and development, reproduction (reproduction), homeostasis, response to stimuli, consumption, metabolism, and excretion. The term "passive secretion" in this application refers to a bioactive substance that is released by non-living cells, or fragments thereof, or extracts thereof, in the absence of a stimulus, and thereby diffuses into the environment, such as the original cells, or fragments thereof, or extracts thereof, e.g., extracellular matrix. "Non-living cells" herein refer to cells that do not exhibit any of the following characteristics: growth and development, reproduction, homeostasis, response to stimuli, consumption, metabolism, or excretion (non-living cells, or fragments thereof, or extracts thereof, e.g., dead cells or cell extracts). The actively or passively secreted bioactive substance can then diffuse into tissues or organs to which such extracellular matrix-containing biomaterials are administered.

[0047] The terms "treatment," "treating," or "alleviating" refer to therapeutic procedures aimed at preventing or reducing (alleviating) tissue damage, including skin, bone, and / or cartilage damage. Those in need of treatment include those already suffering from the disorder, as well as those prone to developing the disorder and those in need of preventing tissue damage, including bone and cartilage defects. A subject is successfully "treated" for tissue damage, including skin, bone, and / or cartilage damage, if, after receiving a therapeutic amount of biomaterial according to the methods of the present invention, the subject shows an observable and / or measurable reduction or absence of any one or more of the following: a reduction in tissue damage, including skin, bone, and / or cartilage damage; and / or some alleviation (reduction) of one or more symptoms associated with tissue damage, including skin, bone, and / or cartilage damage; a reduction in morbidity and mortality, and an improvement in quality of life. The above parameters for assessing successful treatment and improvement of a disorder are readily measurable by routine procedures familiar to physicians. In the context of the therapeutic use of the biomaterials of the present disclosure, the term "allogeneic" refers to a treatment that is "allogenic" or "allogeneic." ” therapy In this method, the donor and recipient are different individuals of the same species, whereas in "autologous" therapy, the donor and recipient are the same individual, and in "xenogeneic" therapy, the donor comes from an animal of a different species than the recipient.

[0048] The term "prevention" refers to preventing or avoiding the occurrence of symptoms of tissue damage, including skin, bone, and / or cartilage damage. In the present invention, the term "prevention" may refer to secondary prevention, i.e., preventing the recurrence of symptoms or recurrence of tissue damage, including skin, bone, and / or cartilage damage. It may also refer to the occurrence of metastasis after treatment and / or removal of a tumor, when the disease is cancer, for example, bone cancer.

[0049] The term "effective amount" refers to an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations.

[0050] The term "pharmaceutically acceptable vehicle" refers to a vehicle that does not cause any adverse, allergic, or other undesirable reactions when administered to an animal individual, preferably a human individual. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. For human administration, preparations should meet sterility, pyrogenicity, general safety, quality, and purity standards required by regulatory authorities such as the Food and Drug Administration (FDA) in the United States and the European Medicines Agency (EMA) in the European Union.

[0051] The term "individual" refers to a vertebrate, preferably a mammal, more preferably a human. Examples of individuals include humans, non-human primates, dogs, cats, mice, rats, horses, cattle, sheep, and transgenic species thereof. In one embodiment, the individual is a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting medical attention, undergoing medical attention, or having been / being / being the subject of medical treatment, or being monitored for the development of disease. In one embodiment, the individual is an adult (e.g., a subject aged 18 years or older). In another embodiment, the individual is a child (e.g., a subject under 18 years of age). In one embodiment, the individual is a male. In another embodiment, the individual is a female.

[0052] Other definitions may appear by context throughout this disclosure.

[0053] The present inventors further characterized the biomaterials disclosed in Patent Documents 5 and 6. This further characterization revealed that the cellular content and / or secretory content are of paramount importance in promoting tissue repair, including skin, bone, or cartilage repair. Notably, growth factors, transcription factors, and factors involved in tissue formation, including skin, bone, or chondrogenesis, along with various microRNAs (miRNAs), were shown to potentially represent active agents for promoting, for example, the osteogenic and / or chondrogenic properties of the biomaterial.

[0054] The inventors also observed that the biomaterial can be advantageously freeze-dried and gamma-irradiated and, against all odds, maintain its tissue regeneration and / or tissue repair properties, including its osteogenic and / or chondrogenic properties. Fresh multidimensional biomaterials have been characterized and contain many factors, such as growth factors, transcription factors, including polypeptides and miRNAs. However, the inventors were surprised to find that freeze-drying and gamma-irradiation, which are known to alter biological properties, particularly miRNA properties, did not alter the tissue regeneration and / or tissue repair properties, including the osteogenic and / or chondrogenic properties, of the treated biomaterial, despite significant differences in the relative amounts of various factors compared to the untreated original biomaterial.

[0055] The recitation of embodiments below includes any single embodiment or combination with other embodiments or portions thereof, where the recited embodiment is applicable to one or more of the aspects recited below. Other features and advantages of the invention will become apparent from the detailed description and claims. Accordingly, other aspects and embodiments of the invention are set forth in the following disclosure and are within the scope of the invention.

[0056] The present invention relates to a sterile, dry biomaterial comprising devitalized, differentiated cells having tissue regenerative and / or tissue repair properties and a particulate material, said cells and said particulate material being embedded in an extracellular matrix.

[0057] As used herein, the phrase "differentiated cells with tissue regenerative and / or tissue repair properties" refers to a cell population that has the ability to promote tissue regenerative and / or tissue repair properties and / or maintain existing tissue in a healthy physiological state.

[0058] The present invention also relates to a sterile dry biomaterial comprising devitalized differentiated cells having tissue regenerative and / or tissue repair properties and gelatin, said cells and said gelatin being embedded in an extracellular matrix.

[0059] The present invention further relates to a sterile, dry biomaterial comprising devitalized osteo- and / or chondrogenically differentiated cells and particulate material, said cells and said particulate material being embedded in an extracellular matrix.

[0060] As used herein, the phrase "osteo- and / or chondrogenically differentiated cells" refers to a cell population that has the ability to promote bone formation and / or chondrogenesis and / or maintain existing bone and / or cartilage in a healthy physiological state.

[0061] In certain embodiments, the cells are selected from the group comprising primary cells, stem cells, genetically modified cells, and combinations thereof.

[0062] Indeed, the cells according to the invention may be animal cells, preferably mammalian cells, more preferably human cells.

[0063] In some embodiments, the primary cells are selected from the group comprising or consisting of bone cells, brain cells, skin cells, breast cells, nerve cells, cervical cells, cells of the upper aerodigestive tract, colorectal cells (colon cells), endometrial cells, germ cells, bladder cells, kidney cells, laryngeal cells, liver cells, lung cells, esophageal cells, ovarian cells, pancreatic cells, pleural cells, prostate cells, eye cells, small intestine cells, stomach cells, testicular cells, thyroid cells, etc., and precursors thereof.

[0064] In some embodiments, the primary cells may be selected from the group including osteocytes, osteoblasts, osteoclasts, chondroblasts, chondrocytes, keratinocytes, dermal fibroblasts, fibroblasts, epithelial cells, hematopoietic cells, hepatocytes, neurons, myofibroblasts, endothelial cells, adipocytes, and combinations thereof.

[0065] As used herein, the term "neuronal cell" includes cells of the central nervous system, such as, for example, neuronal cells and glial cells.

[0066] In some embodiments, the primary cells may be selected from the group including osteocytes, osteoblasts, osteoclasts, chondroblasts, chondrocytes, and combinations thereof. Because primary cells are differentiated cells, they can be cultured in any suitable culture medium for maintenance or proliferation. In some embodiments, the primary cells may be cultured in a culture medium (also called proliferation medium (MP)) suitable for allowing cell proliferation or maintenance.

[0067] In one embodiment, the growth medium can be any culture medium designed to support cell growth known to those skilled in the art. As used herein, the growth medium is also referred to as a "growth medium." Examples of growth media include, but are not limited to, RPMI, MEM, DMEM, IMDM, RPMI 1640, FGM or FGM-2, 199 / 109 medium, HamF 10 / HamF 12, or McCoy's 5A. In a preferred embodiment, the growth medium is DMEM.

[0068] In certain embodiments, the stem cells may be selected from the group including osteoprogenitor cells, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), pluripotent stem cells (pSCs), and induced pluripotent stem cells (ipSCs).

[0069] As used herein, the term "embryonic stem cells" (ESCs) generally refers to embryonic cells that can differentiate into cells of any one of the three embryonic germ layers, i.e., endoderm, ectoderm, or mesoderm, or can be maintained in an undifferentiated state. Such cells may include cells (e.g., blastocysts) obtained from embryonic tissue formed after conception before implantation of the embryo (i.e., preimplantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at the late implantation / pregastrulation stage (see WO 2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during gestation, preferably before 10 weeks of gestation, and before other methods using unfertilized eggs, such as parthenogenesis or nuclear transfer.

[0070] In one embodiment, the ESCs according to the present invention are animal ESCs, preferably mammalian ESCs, more preferably human ESCs (hESCs).

[0071] In practice, suitable ESCs can be obtained using known cell culture methods. For example, ESCs can be isolated from blastocysts. Blastocysts are typically obtained from in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell embryos can be propagated to the blastocyst stage. For details on the preparation of ESCs, please refer to U.S. Patent No. 5,843,780.

[0072] In some embodiments, human embryonic stem cells (hESCs) are generated without embryo destruction, as described in Chung et al. (2008). In some embodiments, hESCs are advantageously obtained from embryos harvested or isolated less than 14 days after fertilization. In some embodiments, the ESCs are not human ESCs.

[0073] As used herein, the term "mesenchymal stem cells" (MSCs) generally refers to stromal cells that are obtained from specialized tissues (also called differentiated tissues), are capable of self-renewal (i.e., making identical copies of themselves) for the life of the organism, and have multipotent differentiation potential.

[0074] In some embodiments, the MSCs according to the present invention are animal MSCs, preferably mammalian MSCs, more preferably human MSCs (hMSCs). Indeed, hMSCs suitable for the practice of the present invention include any suitable human pluripotent stem cells derived from any suitable tissue, using any suitable isolation method.

[0075] For example, hMSCs have been shown to be a safe and readily available source of such cells for transplantation. These cells are derived from adult multilineage-induced (MIAMI) cells (D'Ippolito et al., 2004), umbilical cord blood-derived stem cells (Koegler et al., 2004), mesoangioblasts (Sampaolesi et al., 2006, Dellavalle et al., 2007), and amniotic stem cells (De Coppi et al., 2007). Furthermore, umbilical cord blood banks (e.g., Etablissement Français du Sang, France) provide a safe and readily available source of such cells for transplantation.

[0076] In some embodiments, the MSCs are pre-osteoblasts, pre-chondroblasts, pre-keratinocytes, or pre-fibroblasts. In some embodiments, the MSCs according to the present invention are pre-osteoblasts or pre-chondroblasts.

[0077] In some embodiments, the mesenchymal stem cells are adipose tissue-derived stem cells (ASCs). As used herein, the following terms are considered to refer to ASCs: adipose-derived stem / stromal cells (ASCs); adipose-derived adult stem cells (ADAS cells), adipose-derived adult stromal cells, adipose-derived stromal cells (ADSCs), adipose stromal cells (ASCs), adipose mesenchymal stem cells (AdMSCs), lipoblasts, pericytes, preadipocytes, processed lipoaspirate cells (PLA cells).

[0078] In one embodiment, the ASC is of animal origin, preferably of mammalian origin, more preferably of human origin.Therefore, in one embodiment, the ASC is an animal ASC, preferably a mammalian ASC, more preferably a human ASC.In a preferred embodiment, the ASC is a human ASC.

[0079] Methods for isolating stem cells from adipose tissue are known in the art and are disclosed, for example, in Zuk et al. (Tissue Engineering. 2001, 7:211-228). In one embodiment, ASCs are isolated from adipose tissue by liposuction.

[0080] For example, adipose tissue can be collected by needle biopsy or liposuction. ASCs can be isolated from adipose tissue by first thoroughly washing the tissue sample with phosphate-buffered saline (PBS) containing antibiotics (e.g., 1% penicillin / streptomycin (P / S)) if necessary. The sample is then placed in a sterile tissue culture plate or sterile tube with collagenase (e.g., collagenase type I prepared in PBS containing 2% P / S) for tissue digestion and incubated in a water bath at 37°C and 5% CO2 for 60 minutes, with manual shaking every 20 minutes. Collagenase activity can be neutralized by adding medium (e.g., DMEM containing 10% human platelet lysate (hPL)). Upon disintegration, the sample can be transferred to a tube. The stromal vascular fraction (SVF), which contains ASCs, can be obtained by centrifuging the sample (e.g., at 2000 rpm for 5 minutes). To completely separate stromal cells from primary adipocytes, the sample can be shaken vigorously to completely disrupt the pellet and mix the cells. The centrifugation step can be repeated. After spinning and aspirating the collagenase solution, the pellet is resuspended in lysis buffer, incubated on ice (e.g., for 10 minutes), washed (e.g., with PBS containing 2% P / S), and centrifuged (e.g., at 2000 rpm for 5 minutes). The supernatant is then aspirated, and the cell pellet is resuspended in medium (e.g., stromal medium, i.e., α-MEM supplemented with 20% FBS, 1% L-glutamine, and 1% P / S). The cell suspension can then be filtered (e.g., through a 70 μm cell strainer). The cell-containing sample can finally be plated onto a culture plate and incubated at 37°C and 5% CO2.

[0081] In one embodiment, ASCs of the present invention are isolated from the stromal vascular fraction of adipose tissue. In one embodiment, lipoaspirate is kept at room temperature for several hours, or at +4°C for 24-72 hours before use, or at 0°C or below, e.g., -18°C or -80°C, for long-term storage.

[0082] In one embodiment, the ASCs may be fresh or frozen. Fresh ASCs are isolated ASCs that have not been frozen. Frozen ASCs are isolated ASCs that have been frozen. In one embodiment, freezing refers to treatment at 0°C or below. In one embodiment, freezing may be performed at about -18°C, -80°C, or -180°C. In a specific embodiment, freezing may be cryopreservation.

[0083] As an example of refrigeration, ASCs can be harvested at 80-90% confluence. After washing and removal from the dish, the cells can be pelleted and placed in vials at 20°C in refrigerated storage medium. In one embodiment, the refrigerated storage medium contains 80% fetal bovine serum or human serum, 10% dimethyl sulfoxide (DMSO), and 10% DMEM / Ham's F-12. The vials can then be stored overnight at -80°C. For example, the vials can be placed in an alcohol freezing container, which slowly cools them at approximately 1°C per minute until they reach -80°C. Finally, the frozen vials can be transferred to a liquid nitrogen container for long-term storage.

[0084] In one embodiment, the ASCs are differentiated ASCs. In some embodiments, the differentiated cells are differentiated adipose-derived stem cells (ASCs), preferably ASCs differentiated into cells selected from the group including osteoblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, connective cells, or neurons and adipocytes.

[0085] In preferred embodiments, the ASCs are osteogenic differentiated ACS. In other words, in preferred embodiments, the ASCs differentiate into bone-forming cells. In certain embodiments, the ASCs differentiate into osteoblasts.

[0086] In another embodiment, the ASCs are chondrogenic differentiated ACS. In other words, in one embodiment, the ASCs differentiate into chondrogenic cells. In a specific embodiment, the ASCs differentiate into chondrocytes.

[0087] In another embodiment, the ASCs are keratinic differentiated ACS. In other words, in one embodiment, the ASCs differentiate into keratinocyte-forming cells. In a specific embodiment, the ASCs differentiate into keratinocytes.

[0088] In another embodiment, the ASCs are myofibroblastic differentiated ACS. In other words, in one embodiment, the ASCs differentiate into myofibroblastic cells. In a specific embodiment, the ASCs differentiate into myofibroblasts.

[0089] In another embodiment, the ASCs are endothelial differentiated ACS. In other words, in one embodiment, the ASCs differentiate into endothelial cells. 。

[0090] In another embodiment, the ASCs are epithelial differentiated ACS. In other words, in one embodiment, the ASCs differentiate into epithelial cells. 。

[0091] In another embodiment, the ASCs are adipogenic differentiated ACS. In other words, in one embodiment, the ASCs differentiate into adipogenic cells. In a specific embodiment, the ASCs differentiate into adipocytes.

[0092] In another embodiment, the ASCs are neuronally differentiated ASCs. In other words, in one embodiment, the ASCs differentiate into neural cells.

[0093] As used herein, the term "pluripotency" refers to cells that, under appropriate conditions, have the ability to give rise to cellular progeny capable of differentiating into cell types that exhibit a combination of characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to tissues in prenatal, postnatal, or adult organisms. Standard technology-acceptable tests, such as the ability to form teratomas in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population. However, identification of various pluripotent stem cell characteristics can also be used to identify pluripotent cells. In some embodiments of the present invention, the pluripotent stem cells are animal pluripotent stem cells, preferably mammalian pluripotent stem cells, and more preferably human pluripotent stem cells.

[0094] As used herein, the term "induced pluripotent stem cells" (iPSCs) refers to pluripotent stem cells artificially induced from non-pluripotent cells. Non-pluripotent cells may have lower self-renewal and differentiation capacity (or potency) than pluripotent stem cells. Cells with lower potency may be, but are not limited to, somatic stem cells, tissue-specific progenitor cells, primary cells, or secondary cells. In some embodiments, iPSCs are human iPSCs (hiPSCs).

[0095] Conversely, stem cells and genetically modified cells are not differentiated cells and may therefore undergo differentiation processes, such as osteogenic and / or chondrogenic differentiation processes.

[0096] In some embodiments, the cells are genetically modified cells, in effect engineered to synthesize factors and nucleic acids that promote tissue regeneration and / or tissue repair, including osteogenic and / or chondrogenic.

[0097] Within the scope of the present invention, the expression "genetically modified" refers to a cell that has one or more nucleotide substitutions, additions or deletions in its genome and / or contains one or more additional chromosomal nucleic acids that encode one or more factors that interfere with the physiological outcome of the cell's fate. In one embodiment, the genetically modified cell is of animal origin, preferably of mammalian origin, more preferably of human origin.

[0098] In some embodiments, the genetically modified cells are engineered to enable the synthesis of one or more growth factors, transcription factors, or RNA involved in tissue regeneration and / or tissue repair, including osteogenesis and / or chondrogenesis.

[0099] In one embodiment, osteogenic differentiation of stem cells or genetically modified cells, particularly ASCs, is carried out by culturing the cells in osteogenic differentiation medium (MD). In one embodiment, the osteogenic differentiation medium comprises human serum. In a specific embodiment, the osteogenic differentiation medium comprises human platelet lysate (hPL). In one embodiment, the osteogenic differentiation medium does not contain other animal serum, preferably does not contain serum other than human serum.

[0100] Methods for controlling and assessing osteogenic differentiation are known in the art. For example, osteogenic differentiation of the cells or tissues of the present invention can be assessed by staining for osteocalcin and / or phosphate (e.g., von Kossa), staining for calcium phosphate (e.g., Alizarin Red), magnetic resonance imaging (MRI), measuring mineralized matrix formation, or measuring alkaline phosphatase activity.

[0101] In one embodiment, the osteogenic differentiation medium comprises or consists of growth medium supplemented with dexamethasone, ascorbic acid, and sodium phosphate. In one embodiment, the osteogenic differentiation medium further comprises antibiotics such as penicillin, streptomycin, gentamicin, and / or amphotericin B. In one embodiment, all media are animal protein-free.

[0102] In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine (Aladdin, also known as "Glutamax®" or "Ultraglutamine®"), hPL, dexamethasone, ascorbic acid, and sodium phosphate. In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL, dexamethasone, ascorbic acid, and sodium phosphate, and antibiotics, preferably penicillin, streptomycin, gentamicin, and / or amphotericin B.

[0103] In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 mM), ascorbic acid (about 0.25 mM), and sodium phosphate (about 2.93 mM). In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 pM), ascorbic acid (about 0.25 mM), sodium phosphate (about 2.93 mM), penicillin (about 100 U / mL), and streptomycin (about 100 μg / mL). In one embodiment, the osteogenic differentiation medium further comprises amphotericin B (about 0.1%).

[0104] In one embodiment, the osteogenic differentiation medium consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 pM), ascorbic acid (about 0.25 mM), and sodium phosphate (about 2.93 mM). In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 mM), ascorbic acid (about 0.25 mM), sodium phosphate (about 2.93 mM), penicillin (about 100 U / mL), streptomycin (about 100 μg / mL), and amphotericin B (about 0.1%).

[0105] In another embodiment, the cells, particularly ASCs, are differentiated chondrogenically. In other words, in a preferred embodiment, the cells, particularly ASCs, are differentiated into chondrogenic cells. In yet another embodiment, the cells, particularly ASCs, are differentiated in a chondrogenic medium. In a specific embodiment, the cells, particularly ASCs, are differentiated into chondrocytes.

[0106] The method of controlling and evaluating chondrogenic differentiation is known in the art.For example, the chondrogenic differentiation of the cells or tissue of the present invention can be evaluated by Alcian blue staining, by measuring the expression level of chondrocyte-specific genes such as aggrecan, collagen II and SOX-9.Methods include but are not limited to real-time PCR or histological analysis.

[0107] In one embodiment, chondrogenic differentiation is performed by culturing the cells, particularly ASCs, in chondrogenic differentiation medium.

[0108] In one embodiment, the chondrogenic differentiation medium comprises or consists of DMEM, hPL, sodium pyruvate, ITS, proline, TGF-β1 and dexamethasone, hi one embodiment, the chondrogenic differentiation medium further comprises an antibiotic such as penicillin, streptomycin, gentamicin and / or amphotericin B.

[0109] In one embodiment, the chondrogenic differentiation medium comprises or consists of growth medium supplemented with sodium pyruvate, ascorbic acid, and dexamethasone. In one embodiment, the chondrogenic differentiation medium further comprises antibiotics such as penicillin, streptomycin, gentamicin, and / or amphotericin B. In one embodiment, the chondrogenic differentiation medium further comprises growth factors such as IGF and TGF-β. In one embodiment, all media are animal protein-free.

[0110] In one embodiment, the chondrogenic differentiation medium comprises or consists of DMEM supplemented with hPL, dexamethasone, ascorbic acid, and sodium pyruvate. In one embodiment, the chondrogenic differentiation medium may further comprise proline and / or growth factors and / or antibiotics.

[0111] In one embodiment, the chondrogenic differentiation medium comprises or consists of DMEM, hPL (about 5%, v / v), dexamethasone (about 1 mM), sodium pyruvate (about 100 μg / mL), ITS (about 1×), proline (about 40 μg / mL), and TGF-β1 (about 10 ng / mL).

[0112] In another embodiment, the cells, particularly ASCs, undergo keratinocyte-forming differentiation. In other words, in a preferred embodiment, the cells, particularly ASCs, differentiate into keratinocyte-forming cells. In yet another word, in a preferred embodiment, the cells, particularly ASCs, differentiate in a keratinocyte-forming medium. In a specific embodiment, the cells, particularly ASCs, differentiate into keratinocytes.

[0113] Methods for controlling and assessing keratinogenic differentiation are known in the art. For example, keratinogenic differentiation of the cells or tissues of the invention can be assessed by staining for pankeratin or CD34.

[0114] In one embodiment, differentiation into keratinocytes is achieved by culturing the cells, particularly ASCs, in a keratinocyte-forming differentiation medium.

[0115] In one embodiment, the keratinocyte differentiation medium comprises or consists of DMEM, hPL, insulin, KGF, hEGF, hydrocortisone, and CaCl2, hi one embodiment, the keratinocyte differentiation medium further comprises an antibiotic such as penicillin, streptomycin, gentamicin, and / or amphotericin B.

[0116] In one embodiment, the keratinocyte-forming differentiation medium comprises or consists of DMEM, hPL (about 5%, v / v), insulin (about 5 μg / mL), KGF (about 10 ng / mL), hEGF (about 10 ng / mL), hydrocortisone (about 0.5 μg / mL), and CaCl2 (about 1.5 mM).

[0117] In another embodiment, the cells, particularly ASCs, undergo endothelial differentiation. In other words, in a preferred embodiment, the cells, particularly ASCs, differentiate in an endothelial medium. In a specific embodiment, the cells, particularly ASCs, differentiate into endothelial cells.

[0118] Methods for controlling and assessing endothelial differentiation are known in the art. For example, endothelial differentiation of the cells or tissues of the present invention can be assessed by staining for CD34.

[0119] In one embodiment, differentiation into endothelial cells is Endothelial differentiation medium This is done by culturing ASCs in

[0120] In one embodiment, the endothelial differentiation medium comprises or consists of EBMTM-2 medium, hPL, hEGF, VEGF, R3-IGF-1, ascorbic acid, hydrocortisone, and hFGFb, hi one embodiment, the endothelial differentiation medium further comprises an antibiotic such as penicillin, streptomycin, gentamicin, and / or amphotericin B.

[0121] In one embodiment, the endothelial differentiation medium comprises or consists of EBMTM-2 medium, hPL (about 5%, v / v), hEGF (about 0.5 mL), VEGF (about 0.5 mL), R3-IGF-1 (about 0.5 mL), ascorbic acid (about 0.5 mL), hydrocortisone (about 0.2 mL), and hFGFb (about 2 mL), reagents from the kit Clonetics™ EGM™-2MV BulletKit™ CC-3202 (Lonza).

[0122] In another embodiment, the cells, particularly ASCs, undergo myogenic differentiation. In other words, in a preferred embodiment, the cells, particularly ASCs, differentiate into myogenic cells. In yet another word, in a preferred embodiment, the cells, particularly ASCs, differentiate in a myogenic medium. In a specific embodiment, the cells, particularly ASCs, differentiate into myofibroblasts.

[0123] Methods for controlling and assessing myogenic differentiation are known in the art. For example, myogenic differentiation of the cells or tissues of the present invention can be assessed by staining for α-SMA.

[0124] In one embodiment, differentiation into myogenic cells is achieved by culturing the cells, particularly ASCs, in a myogenic differentiation medium.

[0125] In one embodiment, the myogenic differentiation medium comprises or consists of DMEM:F12, sodium pyruvate, ITS, RPMI1640 vitamins, TGF-β1, glutathione, MEM, and further comprises an antibiotic such as penicillin, streptomycin, gentamicin, and / or amphotericin B.

[0126] In one embodiment, the myogenic differentiation medium comprises or consists of DMEM:F12, sodium pyruvate (about 100 μg / mL), ITS (about 1×), RPMI1640 vitamins (about 1×), TGF-β1 (about 1 ng / mL), glutathione (about 1 μg / mL), MEM (about 0.1 mM).

[0127] In another embodiment, the cells, particularly ASCs, are differentiated adipogenically. In other words, in a preferred embodiment, the cells, particularly ASCs, are differentiated into adipogenic cells. In yet another word, in a preferred embodiment, the cells, particularly ASCs, are differentiated in an adipogenic medium. In a specific embodiment, the cells, particularly ASCs, are differentiated into adipocytes.

[0128] Methods for controlling and assessing adipogenic differentiation are known in the art. For example, adipogenic differentiation of the cells or tissues of the present invention can be assessed by staining with Oil Red.

[0129] In one embodiment, differentiation into adipocytes is achieved by culturing the cells, particularly ASCs, in an adipogenic differentiation medium.

[0130] In one embodiment, the adipogenic differentiation medium comprises or consists of DMEM, hPL, dexamethasone, insulin, indomethacin, and IBMX. In one embodiment, the adipogenic differentiation medium further comprises an antibiotic such as penicillin, streptomycin, gentamicin, and / or amphotericin B.

[0131] In one embodiment, the adipogenic differentiation medium comprises or consists of DMEM, hPL (about 5%), dexamethasone (about 1 mM), insulin (about 5 μg / mL), indomethacin (about 50 pM), and IBMX (about 0.5 mM).

[0132] In another embodiment, the cells, particularly ASCs, undergo neural differentiation. In other words, in a preferred embodiment, the cells, particularly ASCs, differentiate into neural cells. . Ingredients In one specific embodiment, the cells, particularly ASCs, differentiate into neurons. In another specific embodiment, the cells, particularly ASCs, differentiate into glial cells.

[0133] In one embodiment, differentiation into neural cells is achieved by culturing the cells, particularly ASCs, in neuronal or glial cell differentiation medium.

[0134] Methods for controlling and evaluating neural differentiation are known in the art.For example, the neural differentiation of the cells or tissues of the present invention can be evaluated according to morphology, physiology, or overall gene expression pattern.For example, the neural differentiation of the cells or tissues of the present invention can be evaluated by cell elongation, growth cone development, and / or staining of neuroectodermal stem cell markers, including NESTIN, PAX6, and SOX2.Another method for controlling and evaluating neural differentiation is to evaluate the electrophysiological profile of differentiated cells.

[0135] In one embodiment, the cells, particularly ASCs, are late-passage adipose tissue-derived stem cells. As used herein, the term "late passage" refers to adipose tissue-derived stem cells that have differentiated at least at passage 4 or later. As used herein, "passage 4" refers to the fourth passage, i.e., the fourth time the cells are separated by detaching them from the surface of the culture vessel and then resuspended in fresh medium. In one embodiment, the late-passage adipose tissue-derived stem cells are differentiated at passage 4, passage 5, passage 6 or later. In a preferred embodiment, the cells, particularly ASCs, are differentiated at passage 4 or later.

[0136] As used herein, vessel "vessel" refers to any cell culture surface, such as a flask or well plate.

[0137] The initial passage of primary cells was called passage 0 (P0). According to the present invention, passage P0 refers to seeding a cell suspension of pelleted stromal vascular fraction (SVF) into a culture vessel. Therefore, passage P4 means that the cells were detached from the surface of the culture vessel (e.g., by digestion with trypsin) four times (P1, P2, P3, and P4) and resuspended in fresh medium.

[0138] In one embodiment, the cells of the present invention, particularly ASCs, are cultured in a proliferation medium until the fourth passage. In one embodiment, the cells of the present invention, particularly ASCs, are cultured in a differentiation medium from the fourth passage onwards. Thus, in one embodiment, at passages P1, P2 and P3, the cells of the present invention, particularly ASCs, are detached from the surface of the culture vessel and then diluted to an appropriate cell density with a proliferation medium. Further in this embodiment, at passage P4, the cells, particularly ASCs, are detached from the surface of the culture vessel and then diluted to an appropriate cell density with a differentiation medium. Thus, according to this embodiment, at P4, the cells of the present invention, particularly ASCs, are directly resuspended and cultured in a differentiation medium until confluent, rather than being resuspended and cultured in a proliferation medium until confluent before differentiation (i.e., culturing in a differentiation medium).

[0139] In one embodiment, the cells are maintained in differentiation medium until they reach at least confluence, preferably 70% to 100% confluence, and more preferably 80% to 95% confluence. In one embodiment, the cells are maintained in differentiation medium for at least 5 days, preferably at least 10 days, and more preferably at least 15 days. In one embodiment, the cells are maintained in differentiation medium for 5 to 30 days, preferably 10 to 25 days, and more preferably 15 to 20 days. In one embodiment, the differentiation medium is changed every 2 days. However, as is known in the art, cell growth rates may vary slightly depending on the donor.

[0140] Therefore, the duration of differentiation and the number of medium changes may vary depending on the donor.

[0141] In one embodiment, the cells are maintained in differentiation medium at least until characteristic tissues are formed depending on the differentiation medium used.

[0142] In one embodiment, the cells are maintained in osteogenic differentiation medium at least until osteoid (ie, the non-mineralized organic portion of the bone matrix that forms before maturation of bone tissue) is formed.

[0143] In one embodiment, the cells are maintained in chondrogenic differentiation medium at least until immature or mature cartilage having viscoelastic properties is formed.

[0144] In some embodiments, at most 10%, preferably at most 1% of the cells are viable.

[0145] Within the scope of the present invention, the expression "up to 10%" includes 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, and 0%. Cell viability according to the present invention can be assessed by any suitable method known in the art or adapted therefrom. For example, reference may be made to "Mammalian Cell Viability: Methods and Protocols" (2011; editor: MJ Stoddart). Exemplarily, cells can be harvested upon hydration of dried biomaterials and contacted with a suitable culture medium under suitable culture conditions. Cell viability can be assessed by trypan blue exclusion staining. Alternatively, cell viability can be assessed by measuring the consumption of a carbon source, particularly glucose, in the culture medium.

[0146] In some embodiments, the biomaterial comprises substantially non-viable cells, in which the biomaterial comprises undetectable levels of viable cells, in which the biomaterial may be referred to as devitalized.

[0147] In one embodiment, the particulate material of the present invention is in the form of particles, which may be beads, powders, spheres, microspheres, and the like.

[0148] In some embodiments, the particulate material of the present invention is formed from a material that provides structural support for cell growth and proliferation, hi one embodiment, the particulate material is biocompatible and comprises a natural or synthetic material, or a chemical derivative thereof.

[0149] Within the scope of the present invention, "biocompatibility" refers to the quality of not having toxic or harmful effects on the body.

[0150] In one embodiment, the particulate material of the present invention is not structured to form a predetermined 3D shape or scaffold, such as a cube. In one embodiment, the particulate material of the present invention does not have a predetermined shape or scaffold. In one embodiment, the particulate material of the present invention does not have a cubic morphology. In one embodiment, the particulate material is not a 3D scaffold. In one embodiment, the biomaterial of the present invention does not include a scaffold.

[0151] In certain embodiments, the particulate material comprises or is selected from the group consisting of: - organic materials, including demineralized bone matrix (DBM), gelatin, agar / agarose, alginate chitosan, chondroitin sulfate, collagen, elastin or elastin-like peptides (ELPs), fibrinogen, fibrin, fibronectin, proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, polysaccharides, laminin, and cellulose derivatives; - ceramic materials comprising calcium phosphate (CaP) particles, calcium carbonate (CaCO3) particles, calcium sulfate (CaSO4) particles, calcium hydroxide (Ca(OH)2) particles, or combinations thereof; polymers, including polyanhydrides, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene oxide / polyethylene glycol (PEO / PEG), poly(vinyl alcohol) (PVA), fumaric acid-based polymers such as poly(propylene fumarate) (PPF) and poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)), oligo(poly(ethylene glycol) fumarate) (OPF), poly(n-isopropylacrylamide) (PNIPPAAm), poly(aldehyde guluronate) (PAG), poly(n-vinylpyrrolidone) (PNVP), or combinations thereof; -gels, including self-assembling oligopeptide gels, microgels, nanogels, particulate gels, hydrogels, thixotropic gels, xerogels, responsive gels, or combinations thereof; -Creamer; and A combination of them.

[0152] In some preferred embodiments, the particulate material is gelatin.

[0153] In one embodiment, the gelatin of the present invention is animal gelatin, preferably mammalian gelatin, more preferably porcine gelatin. As used herein, the term "porcine gelatin" may be interchangeable with "pork gelatin" or "pig gelatin." In one embodiment, the gelatin is pigskin gelatin.

[0154] In one embodiment, the gelatin is in the form of particles, preferably particles having an average diameter of about 50 μm to about 1,000 μm.

[0155] Within the scope of the present invention, the expression "about 50 μm to about 1,000 μm" includes 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, and 1,000 μm.

[0156] In one embodiment, the gelatin of the present invention is in the form of particles, beads, spheres, microspheres, and the like.

[0157] In one embodiment, the gelatin of the present invention is not structured to form a predetermined 3D shape or scaffold, such as a cube. In one embodiment, the gelatin of the present invention does not have a predetermined shape or scaffold. In one embodiment, the gelatin of the present invention does not have a cubic morphology. In one embodiment, the gelatin, preferably porcine gelatin, is not a 3D scaffold. In one embodiment, the gelatin of the present invention is a macroporous microcarrier.

[0158] Examples of porcine gelatin particles include, but are not limited to, Cultispher® G, Cultispher® S, Spongostan, and Cutanplast. In one embodiment, the gelatin of the present invention is Cultispher® G or Cultispher® S.

[0159] In one embodiment, the gelatin of the present invention, preferably porcine gelatin, has an average diameter of at least about 50 μm, preferably at least about 75 μm, more preferably at least about 100 μm, more preferably at least about 130 μm. In one embodiment, the gelatin of the present invention, preferably porcine gelatin, has an average diameter of at most about 1000 μm, preferably at most about 750 μm, more preferably at most about 500 μm. In another embodiment, the gelatin of the present invention, preferably porcine gelatin, has an average diameter of at most about 450 μm, preferably at most about 400 μm, more preferably at least at most about 380 μm.

[0160] In one embodiment, the gelatin of the present invention, preferably porcine gelatin, has an average diameter of about 50 μm to about 1000 μm, preferably about 75 μm to about 750 μm, and more preferably about 100 μm to about 500 μm. In another embodiment, the gelatin of the present invention, preferably porcine gelatin, has an average diameter of about 50 μm to about 500 μm, preferably about 75 μm to about 450 μm, and more preferably about 100 μm to about 400 μm. In another embodiment, the gelatin of the present invention, preferably porcine gelatin, has an average diameter of about 130 μm to about 380 μm.

[0161] Methods for assessing the average diameter of gelatin particles according to the present invention are known in the art, examples of such methods include, but are not limited to, particle size distribution measurements, especially using appropriate sieves, sedimentation methods, centrifugation techniques, laser diffraction and image analysis, especially using high performance cameras with telecentric lenses.

[0162] In one embodiment, the gelatin is mixed with 150 cm 2 Approximately 0.1 cm relative to the container 3 ~about 5cm 3 , preferably about 0.5 cm 3 ~about 4cm 3 , more preferably about 0.75 cm 3 ~about 3cm 3 In one embodiment, gelatin is added at a concentration of 150 cm 2 Approximately 1cm from the container 3 ~approx. 2cm 3 In one embodiment, gelatin is added at a concentration of 150 cm 2 Approximately 1cm from the container 3 , 1.5cm 3 or 2 cm 3 Within the scope of the present invention, the expression "0.1 cm 3 ~about 5cm 3 " is 0.1 cm 3 , 0.2cm 3 , 0.3cm 3 , 0.4cm 3 , 0.5cm 3 , 0.6cm 3 , 0.7cm 3 , 0.8cm 3 , 0.9cm 3 , 1.0cm 3 , 1.5cm 3 , 2.0cm 3 , 2.5cm 3 , 3.0cm 3 , 3.5cm 3 , 4.0cm 3 , 4.5cm 3 , and 5.0 cm 3 Includes.

[0163] In one embodiment, the gelatin is mixed with 150 cm 2 The gelatin is added to the container at a concentration of about 0.1 g to about 5 g, preferably about 0.5 g to about 4 g, and more preferably about 0.75 g to about 3 g. 2 In one embodiment, gelatin is added to the container at a concentration of about 1 g to about 2 g. 2 The gelatin is added to the culture medium at a concentration of about 1 g, 1.5 g, or 2 g per container. Within the scope of the present invention, the expression "0.1 g to about 5 g" includes 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g, and 5.0 g. In one embodiment, the gelatin of the present invention is added to the culture medium after cell differentiation. In one embodiment, the gelatin of the present invention is added to the culture medium when the cells are subconfluent. In one embodiment, the gelatin of the present invention is added to the culture medium when the cells are overconfluent. In one embodiment, the gelatin of the present invention is added to the culture medium when the cells reach confluence after differentiation. In other words, in one embodiment, the gelatin of the present invention is added to the culture medium when the cells reach confluence in the differentiation medium. In one embodiment, the gelatin of the present invention is added to the medium at least 5 days after P4, preferably 10 days after P4, and more preferably 15 days after P4. In one embodiment, the gelatin of the present invention is added to the medium 5 to 30 days after P4, preferably 10 to 25 days after P4, and more preferably 15 to 20 days after P4.

[0164] In some preferred embodiments, the particulate material is a ceramic material.

[0165] In one embodiment, the ceramic material of the present invention is calcium phosphate (CaP) particles, calcium carbonate (CaCO3) particles, calcium sulfate (CaSO4) particles, or calcium hydroxide (Ca(OH)2) particles, or a combination thereof.

[0166] Examples of calcium phosphate particles include hydroxyapatite (HA, Ca 10 (PO4)6(OH)2), tricalcium phosphate (TCP, Ca3(PO4)2), α-tricalcium phosphate (α-TCP, (α-Ca3(PO4)2), β-tricalcium phosphate (β-TCP, β-Ca3(PO4)2), tetracalcium phosphate (TTCP, Ca4(PO4)2O), octacalcium phosphate (Ca8H2(PO4)6.5H2O), amorphous calcium phosphate (Ca3(PO4)2), hydroxyapatite / β-tricalcium phosphate (HA / β-TCP), hydroxyapatite / tetracalcium phosphate (HA / TTCP), and the like.

[0167] In one embodiment, the ceramic material of the present invention comprises or consists of hydroxyapatite (HA), tricalcium phosphate (TCP), hydroxyapatite / β-tricalcium phosphate (HA / β-TCP), calcium sulfate (CaSO4), or a combination thereof. In one embodiment, the ceramic material of the present invention comprises or consists of hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), hydroxyapatite / β-tricalcium phosphate (HA / β-TCP), α-tricalcium phosphate (α-TCP), calcium sulfate (CaSO4), or a combination thereof.

[0168] In some embodiments, the particulate material preferably comprises a ceramic material comprising particles of calcium phosphate, preferably hydroxyapatite (HA) and / or β-tricalcium phosphate (β-TCP), more preferably calcium phosphate.

[0169] In one embodiment, the ceramic material comprises particles of calcium phosphate, preferably hydroxyapatite (HA) and / or β-tricalcium phosphate (β-TCP), more preferably calcium phosphate.

[0170] In one embodiment, the ceramic particles of the present invention are particles of hydroxyapatite (HA). In another embodiment, the ceramic particles of the present invention are particles of β-tricalcium phosphate (β-TCP). In another embodiment, the ceramic particles of the present invention are particles of hydroxyapatite and β-tricalcium phosphate (HA / β-TCP). In other words, in one embodiment, the ceramic particles of the present invention are a mixture of hydroxyapatite and β-tricalcium phosphate particles (referred to as HA / β-TCP particles). In one embodiment, the ceramic particles of the present invention consist of hydroxyapatite particles and β-tricalcium phosphate particles (referred to as HA / β-TCP particles).

[0171] In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is in the form of granules, powder, or beads. In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is in the form of porous granules, powder, or beads. In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is a porous ceramic material. In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is a powder particle. In a specific embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is in the form of porous granules. In another specific embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is in the form of a powder.

[0172] In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, are not structured to form a predetermined 3D shape or scaffold, such as a cube. In one embodiment, the particulate material, preferably the ceramic material of the present invention, is not a 3D scaffold. In one embodiment, the particulate material, preferably the ceramic material, does not have a predetermined shape or scaffold. In one embodiment, the particulate material, preferably the ceramic material of the present invention, does not have a cubic morphology.

[0173] In one embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter greater than about 50 μm, preferably greater than about 100 μm. In one embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter greater than about 50 μm, preferably greater than about 100 μm.

[0174] In one embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter of at least about 50 μm, preferably at least about 100 μm, more preferably at least about 150 μm. In another embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter of at least about 200 μm, preferably at least about 250 μm, more preferably at least about 300 μm.

[0175] In another embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter of at most about 2500 μm, preferably at most about 2000 μm, more preferably at most about 1500 μm. In one embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter of at most about 1000 μm, 900 μm, 800 μm, 700 μm, or 600 μm.

[0176] In one embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter of about 50 μm to about 1500 μm, preferably about 50 μm to about 1250 μm, more preferably about 100 μm to about 1000 μm.In one embodiment, the particulate material, preferably the ceramic particles of the present invention, more preferably the HA, β-TCP and / or HA / β-TCP particles, has an average diameter of about 100 μm to about 800 μm, preferably 150 μm to about 700 μm, more preferably about 200 μm to about 600 μm.

[0177] In one embodiment, the HA / β-TCP particles have an average diameter of about 50 μm to about 1500 μm, preferably about 50 μm to about 1250 μm, and more preferably about 100 μm to about 1000 μm.In one embodiment, the HA and β-TCP particles have an average diameter of about 100 μm to about 800 μm, preferably about 150 μm to about 700 μm, and more preferably about 200 μm to about 600 μm.

[0178] In practice, the average particle size and diameter can be measured by any suitable method known in the art, or adapted therefrom, including, but not limited to, atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS).

[0179] In one embodiment, the ratio of HA to β-TCP (HA / β-TCP ratio) in the particles is in the range of about 0 / 100 to about 100 / 0, preferably about 10 / 90 to about 90 / 10, and more preferably about 20 / 80 to about 80 / 20. In one embodiment, the ratio of HA to β-TCP in the particles is in the range of about 30 / 70 to about 70 / 30, about 35 / 65 to about 65 / 35, or about 40 / 60 to about 60 / 40.

[0180] In one embodiment, the HA / β-TCP ratio in the particles is 0 / 100, i.e., the particles are β-tricalcium phosphate particles. In another embodiment, the HA / β-TCP ratio in the particles is 100 / 0, i.e., the particles are hydroxyapatite particles. In one embodiment, the HA / β-TCP ratio in the particles is about 10 / 90. In another embodiment, the HA / β-TCP ratio in the particles is about 90 / 10. In one embodiment, the HA / β-TCP ratio in the particles is about 20 / 80. In another embodiment, the HA / β-TCP ratio in the particles is about 80 / 20. In one embodiment, the HA / β-TCP ratio in the particles is about 30 / 70. In another embodiment, the HA / β-TCP ratio in the particles is about 70 / 30. In another embodiment, the HA / β-TCP ratio in the particles is about 35 / 65. In another embodiment, the HA / β-TCP ratio in the particles is about 65 / 35. In one embodiment, the HA / β-TCP ratio in the particles is about 40 / 60. In another embodiment, the ratio of HA / β-TCP in the particles is about 60 / 40. In another embodiment, the ratio of HA / β-TCP in the particles is about 50 / 50.

[0181] In one embodiment, the HA / b-TCP ratio in the particles is 100 / 0, 99 / 1, 98 / 2, 97 / 3, 96 / 4, 95 / 5, 94 / 6, 93 / 7, 92 / 8, 91 / 9, 90 / 10, 89 / 11, 88 / 12, 87 / 13, 86 / 14, 85 / 15, 84 / 16, 83 / 17, 82 / 18, 81 / 19, 80 / 20, 79 / 21, 78 / 22, 77 / 23, 76 / 24, 75 / 25, 74 / 26, 73 / 27, 72 / 28, 71 / 29, 70 / 30, 69 / 31, 68 / 32, 67 / 33, 66 / 34, 65 / 35, 64 / 36, 63 / 37, 62 / 38, 61 / 39, 60 / 40, 59 / 41, 58 / 42, 57 / 43, 56 / 44, 55 / 45, 54 / 46, 53 / 47, 52 / 48 , 51 / 49, 50 / 50, 49 / 51, 48 / 52, 47 / 53, 46 / 54, 45 / 55, 44 / 56, 43 / 57, 42 / 58, 41 / 59, 40 / 60, 39 / 61, 38 / 62, 37 / 63, 36 / 64, 35 / 65, 34 / 66, 33 / 67, 32 / 68, 31 / 69, 30 / 70, 29 / 71, 28 / 72, 27 / 73, 26 / 74 4, 25 / 75, 24 / 76, 23 / 77, 22 / 78, 21 / 79, 20 / 80, 19 / 81, 18 / 82, 17 / 83, 16 / 84, 15 / 85, 14 / 86, 13 / 87, 12 / 88, 11 / 89, 10 / 90, 9 / 91, 8 / 92, 7 / 93, 6 / 94, 5 / 95, 4 / 96, 3 / 97, 2 / 98, 1 / 99, or 0 / 100.

[0182] According to one embodiment, the amount of particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is optimal to provide a 3D structure to the biomaterial. In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is added to a volume of 150 cm 2 Approximately 0.1 cm relative to the container 3 ~about 5cm 3 , preferably about 0.5 cm 3 ~about 3cm 3 , more preferably about 1 cm 3 ~about 3cm 3In a preferred embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is added at a concentration of 150 cm 2 Approximately 1.5cm from the container 3 ~about 3cm 3 Add at a concentration of

[0183] In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is added at a concentration of about 7×10 per mL of medium. 3 ~7×10 2 cm 3 In one embodiment, the particulate material, preferably ceramic particles, more preferably HA, β-TCP and / or HA / β-TCP particles, is added to a volume of 1 cm 2 Approximately 3.3 x 10 3 ~3.3×10 2 cm 3 Add at a concentration of

[0184] In one embodiment, the particulate material, preferably ceramic material, of the present invention is added to the culture medium after cell differentiation. In one embodiment, the particulate material, preferably ceramic material, of the present invention is added when the cells are subconfluent. In one embodiment, the particulate material, preferably ceramic material, of the present invention is added when the cells are overconfluent. In one embodiment, the particulate material, preferably ceramic material, of the present invention is added when the cells reach confluence after differentiation. In other words, in one embodiment, the particulate material, preferably ceramic material, of the present invention is added when the cells reach confluence in the differentiation medium. In one embodiment, the particulate material, preferably ceramic material, of the present invention is added at least 5 days after P4, preferably 10 days after P4, and more preferably 15 days after P4. In one embodiment, the particulate material, preferably ceramic material, of the present invention is added 5 to 30 days after P4, preferably 10 to 25 days after P4, and more preferably 15 to 20 days after P4.

[0185] In another embodiment, the particulate material of the present invention is demineralized bone matrix (DBM).

[0186] In one embodiment, the DBM is of animal origin, preferably of mammalian origin, and more preferably of human origin. In a specific embodiment, human DBM is obtained by grinding cortical bone from a human donor.

[0187] Methods for obtaining DBM are known in the art. For example, human bone tissue may first be degreased overnight in an acetone (e.g., about 99%) bath and then washed with demineralized water for about 2 hours. Decalcification may be performed by immersion in HCl (e.g., about 0.6 N) for about 3 hours (20 mL of solution per gram of bone) at room temperature with stirring. The demineralized bone powder is then rinsed with demineralized water for about 2 hours, and the pH is adjusted. If the pH is too acidic, the DBM may be buffered with a phosphate solution (e.g., about 0.1 M) with stirring. Finally, the DBM may be dried and weighed. The DBM may be sterilized by gamma irradiation, for example, at about 25 kGray, based on techniques known in the field.

[0188] In one embodiment, the DBM is allogeneic. In one embodiment, the DBM is homogenous. In another embodiment, the DBM is heterogeneous.

[0189] In one embodiment, the DBM is in the form of particles, referred to herein as demineralized bone matrix particles or DBM particles. In one embodiment, the DBM particles have an average diameter of about 50 to about 2500 μm, preferably about 50 μm to about 1500 μm, and more preferably about 50 μm to about 1000 μm. In one embodiment, the DBM particles have an average diameter of about 100 μm to about 1500 μm, and more preferably about 150 μm to about 1000 μm. In one embodiment, the DBM particles have an average diameter of about 200 μm to about 1000 μm, preferably about 200 μm to about 800 μm, and more preferably about 300 μm to about 700 μm.

[0190] In one embodiment, the biomaterial of the present invention comprises an extracellular matrix. In one embodiment, the extracellular matrix of the present invention is derived from cells, preferably ASCs. In one embodiment, the extracellular matrix of the present invention is produced by cells, preferably ASCs. In one embodiment, the extracellular matrix of the biomaterial of the present invention is derived from differentiated cells, preferably differentiated ASCs.

[0191] As used herein, the term "extracellular matrix" (ECM) refers to a non-cellular, three-dimensional, macromolecular network. The matrix components of the ECM bind to each other as well as to cell adhesion receptors, thereby forming a complex network in which cells reside in the tissue or biomaterial of the present invention.

[0192] In one embodiment, the extracellular matrix of the present invention comprises collagen, proteoglycans / glycosaminoglycans, elastin, fibronectin, laminin, and / or other glycoproteins. In a specific embodiment, the extracellular matrix of the present invention comprises collagen. In another specific embodiment, the extracellular matrix of the present invention comprises proteoglycans. In another specific embodiment, the extracellular matrix of the present invention comprises collagen and proteoglycans. In one embodiment, the extracellular matrix of the present invention comprises growth factors, proteoglycans, secreted factors, extracellular matrix regulators, and glycoproteins.

[0193] In one embodiment, the cells, preferably ASCs, and particulate material, preferably gelatin, DBM or a ceramic material of the invention, are embedded in an extracellular matrix.

[0194] In some embodiments, the biomaterial comprises an altered factor content compared to the factor content obtained from a corresponding fresh, non-sterile, non-dried biomaterial.

[0195] Within the scope of the present invention, the term "altered factors content" refers to a distinct amount of the content of factors in a biomaterial when compared with a reference value, where, considering a specific factor, the altered content of this factor refers either to an increase in the relative amount of said factor or to a decrease in the relative amount of said factor compared with the reference value.

[0196] In some embodiments, the reference value is the factor content obtained from a corresponding fresh, non-sterile, non-dried biomaterial.

[0197] The term "altered" as used herein means "in substantially different relative amounts." In other words, "altered content of ingredients" refers to a variation in the relative amounts of ingredients from the biomaterial according to the present invention by at least about 10% compared to the relative amounts of ingredients from the reference biomaterial. Within the scope of the present invention, the expression "at least about 10%" can be used to refer to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950 ...0%, 1050%, 1050%, 1050%, 1050%, 1050%, 1050%, 1050%, 1050%, 1050%, 1050%, 1050%, 1 Including 50%, 900%, 950%, 1,000%, 1,250%, 1,500%, 1,750%, 2,000%, 2,250%, 2,500%, 2,750%, 3,000%, 3,250%, 3,500%, 3,750%, 4,000%, 4,250%, 4,500%, 4,750%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, and 10,000%.

[0198] Within the scope of the present invention, the term "varies" means "decreases" or "increases".

[0199] In some embodiments, the average relative amount of a component present in a biomaterial according to the present invention varies by at least about 10% relative to the average relative amount of said component obtained from a corresponding fresh, non-sterile, non-dried biomaterial.

[0200] In some embodiments, the average relative amount of a content contained in a biomaterial according to the present invention varies by at least about 10% relative to the average relative amount of said content obtained from viable differentiated cells, including osteo- and / or chondrocyte-differentiated cells, prior to being subjected to the step of producing the sterile, dry biomaterial.

[0201] In some embodiments, the factor content includes proteins such as growth factors, transcription factors, osteogenic factors and chondrogenic factors, and nucleic acids such as RNA, particularly miRNA.

[0202] In some embodiments, the factor content includes growth factors and / or transcription factors.

[0203] In some embodiments, the factor content includes growth factors, and / or transcription factors, and / or osteogenic factors, and / or chondrogenic factors.

[0204] As used herein, "growth factor" refers to a polypeptide that regulates many aspects of cell function, including survival, proliferation, migration, and differentiation. Non-limiting examples of growth factors according to the present invention include BMP, EGF, FGF, HGF, IGF-1, OPG (osteoprotegerin), SDF-1α, TGFB-1, TGFB-3, VEGF (including VEGFA and VEGFB).

[0205] As used herein, "transcription factor" refers to a polypeptide that controls whether a given gene is transcribed into corresponding RNA. In some embodiments, transcription factors according to the present invention include, but are not limited to, SMAD-2, SMAD-3, SMAD-4, and SMAD-5. In certain embodiments, transcription factors according to the present invention include, but are not limited to, AKT, ANG, ANGPT1, ANGPTL4, ANPEP, COL18A1, CTGF, CXCL1, EDN1, EFNA1, EFNB2, ENG, EPHB4, F3, FGF1, FGF2, FN1, HIFIA, ID1, IL6, ITGAV, JAG1, LEP, MMP14, MMP2, NRP1, PTGS1, SERPINE1, SERPINF1, TGFB1, TGFBR1, THBS1, THBS2, TIMP1, TIMP2, TIMP3, VEGFA, VEGFB, and VEGFC.

[0206] As used herein, "osteogenic factor" refers to a polypeptide that promotes bone formation and / or inhibits bone destruction. In some embodiments, osteogenic factors according to the present invention are involved in skeletal development. Non-limiting examples of osteogenic factors according to the present invention include OPG, SDF-1α, BMPR-1A, BMPR-2, FGFR-1, FGFR-2, TWIST-1, CSF-1, IGFR, RUNX2, and TGFBR-1.

[0207] In some embodiments, the factor content includes VEGF and / or IGF-1 and / or SDF-1α. Without wishing to be bound by a particular theory, the inventors believe that factors such as VEGF and / or IGF-1 and / or SDF-1α are involved in the healing process. VEGF promotes angiogenesis, IGF-1 has a positive correlation with the wound healing process, such as recruiting myofibroblasts, promoting collagen synthesis, and stimulating fibroblasts and keratinocytes, and SDF-1α promotes stem cell recruitment for wound healing.

[0208] In some embodiments, the factor content includes IGF-1 and / or VEGF and / or SDF-1α and / or (OPG).

[0209] As can be seen from the Examples section below, the average relative amounts of the factors OPG, SDF-1α, BMPR-1A, BMPR-2, CSF-1, IGF1R, TWIST-1, SMAD-2, SMAD-3, SMAD-4, SMAD-5 in the dry sterilized biomaterial according to the invention are increased by more than 10% with respect to the average relative amounts of said contents obtained from the corresponding fresh non-sterilized non-dried biomaterial.

[0210] In some embodiments, the biomaterial according to the present invention comprises from about 0.1 ng to about 200 ng of IGF-1 per gram (w / w) of biomaterial, preferably from about 1 ng to about 150 ng of IGF-1 per gram of biomaterial, and more preferably from about 10 ng to about 80 ng of IGF-1 per gram of biomaterial. Within the scope of the present invention, the expression "about 0.1 ng to about 200 ng per gram" includes about 0.1 ng / g, about 0.2 ng / g, about 0.3 ng / g, about 0.4 ng / g, about 0.5 ng / g, about 0.6 ng / g, about 0.7 ng / g, about 0.8 ng / g, about 0.9 ng / g, about 1 ng / g, about 2 ng / g, about 3 ng / g, about 4 ng / g, about 5 ng / g, about 6 ng / g, about 7 ng / g, about 8 ng / g, about 9 ng / g, about 10 ng / g, about 12 ng / g, about 14 ng / g, about 16 ng / g, about 18 ng / g, about 20 ng / g, about 22 ng / g, about 24 ng / g, about 26 ng / g, about 28 ng / g, about 30 ng / g, about 32 ng / g, about 34 ng / g, about 35 ng / g, about 36 ng / g, about 37 ng / g, about 38 ng / g, about 39 ng / g, about 40 ng / g, about 41 ng / g, about 42 ng / g, about 43 ng / g, about 44 ng / g, about 45 ng / g, about 46 ng / g, about 47 ng / g, about 48 ng / g, about 49 ng / g, about 50 ng / g, about 51 ng / g, about 52 ng / g, about 53 ng / g, about 54 ng / g, about 55 ng / g, about 56 ng / g, about 5 ng / g, about 26 ng / g, about 28 ng / g, about 30 ng / g, about 35 ng / g, about 40 ng / g, about 45 ng / g, about 50 ng / g, about 55 ng / g, about 60 ng / g, about 65 ng / g, about 70 ng / g, about 75 ng / g, about 80 ng / g, about 85 ng / g, about 90 ng / g, about 95 ng / g, about 100 ng / g, about 110 ng / g, about 120 ng / g, about 130 ng / g, about 140 ng / g, about 150 ng / g, about 160 ng / g, about 170 ng / g, about 180 ng / g, about 190 ng / g, and about 200 ng / g.

[0211] In one embodiment, the biomaterial according to the present invention comprises about 0.1 ng to about 100 ng of OPG per gram (w / w) of biomaterial, preferably about 1 ng to about 50 ng of OPG per gram of biomaterial. Within the scope of the present invention, the expression "about 0.1 ng to about 100 ng per gram" includes about 0.1 ng / g, about 0.2 ng / g, about 0.3 ng / g, about 0.4 ng / g, about 0.5 ng / g, about 0.6 ng / g, about 0.7 ng / g, about 0.8 ng / g, about 0.9 ng / g, about 1 ng / g, about 2 ng / g, about 3 ng / g, about 4 ng / g, about 5 ng / g, about 6 ng / g, about 7 ng / g, about 8 ng / g, about 9 ng / g, about 10 ng / g, about 12 ng / g, and the like. g, about 14 ng / g, about 16 ng / g, about 18 ng / g, about 20 ng / g, about 22 ng / g, about 24 ng / g, about 26 ng / g, about 28 ng / g, about 30 ng / g, about 35 ng / g, about 40 ng / g, about 45 ng / g, about 50 ng / g, about 55 ng / g, about 60 ng / g, about 65 ng / g, about 70 ng / g, about 75 ng / g, about 80 ng / g, about 85 ng / g, about 90 ng / g, about 95 ng / g, and about 100 ng / g.

[0212] In some embodiments, the biomaterial according to the present invention comprises about 0.1 ng to about 200 ng of VEGF per gram (w / w) of biomaterial, preferably about 1 ng to about 150 ng of VEGF per gram of biomaterial, and more preferably about 20 ng to about 100 ng of VEGF per gram of biomaterial. Within the scope of the present invention, the expression "about 0.1 ng to about 200 ng per gram" means about 0.1 ng / g, about 0.2 ng / g, about 0.3 ng / g, about 0.4 ng / g, about 0.5 ng / g, about 0.6 ng / g, about 0.7 ng / g, about 0.8 ng / g, about 0.9 ng / g, about 1 ng / g, about 2 ng / g, about 3 ng / g, about 4 ng / g, about 5 ng / g, about 6 ng / g, about 7 ng / g, about 8 ng / g, about 9 ng / g, about 10 ng / g, about 11 ng / g, about 12 ng / g, about 13 ng / g, about 14 ng / g, about 15 ng / g, about 16 ng / g, about 17 ng / g, about Including 18ng / g, about 19ng / g, about 20ng / g, about 25ng / g, about 30ng / g, about 35ng / g, about 40ng / g, about 45ng / g, about 50ng / g, about 55ng / g, about 60ng / g, about 65ng / g, about 70ng / g, about 75ng / g, about 80ng / g, about 85ng / g, about 90ng / g, about 95ng / g, about 100ng / g, about 110ng / g, about 120ng / g, about 130ng / g, about 140ng / g, about 150ng / g, about 160ng / g, about 170ng / g, about 180ng / g, about 190, and about 200ng / g.

[0213] In some embodiments, the biomaterial according to the present invention comprises about 0.1 ng to about 20 ng of VEGF per gram (w / w) of biomaterial, and preferably about 1 ng to about 15 ng of VEGF per gram of biomaterial. Within the scope of the present invention, the expression "about 0.1 ng to about 20 ng per gram" includes about 0.1 ng / g, about 0.2 ng / g, about 0.3 ng / g, about 0.4 ng / g, about 0.5 ng / g, about 0.6 ng / g, about 0.7 ng / g, about 0.8 ng / g, about 0.9 ng / g, about 1 ng / g, about 2 ng / g, about 3 ng / g, about 4 ng / g, about 5 ng / g, about 6 ng / g, about 7 ng / g, about 8 ng / g, about 9 ng / g, about 10 ng / g, about 11 ng / g, about 12 ng / g, about 13 ng / g, about 14 ng / g, about 15 ng / g, about 16 ng / g, about 17 ng / g, about 18 ng / g, about 19, and about 20 ng / g.

[0214] In one embodiment, the biomaterial according to the present invention contains about 0.1 ng to about 400 ng of SDF-1α per gram (w / w) of biomaterial, preferably about 1 ng to about 250 ng of SDF-1α per gram of biomaterial, and more preferably about 10 ng to about 200 ng of SDF-1α per gram of biomaterial. Within the scope of the present invention, the expression "about 0.1 ng to about 400 ng per gram" includes about 0.1 ng / g, about 0.2 ng / g, about 0.3 ng / g, about 0.4 ng / g, about 0.5 ng / g, about 0.6 ng / g, about 0.7 ng / g, about 0.8 ng / g, about 0.9 ng / g, about 1 ng / g, about 2 ng / g, about 3 ng / g, about 4 ng / g, about 5 ng / g, about 6 ng / g, and about 7 ng / g. g, about 8ng / g, about 9ng / g, about 10ng / g, about 12ng / g, about 14ng / g, about 16ng / g, about 18ng / g, about 20ng / g, about 22ng / g, about 24ng / g, about 26ng / g, about 28ng / g, about 30ng / g, about 35ng / g, about 40ng / g, about 45ng / g, about 50ng / g, about 55ng / g, about 60ng / g, about 65ng / g, about 70 ng / g, about 75ng / g, about 80ng / g, about 85ng / g, about 90ng / g, about 95ng / g, about 100ng / g, about 110ng / g, about 120ng / g, about 130ng / g, Approximately 140ng / g, approximately 150ng / g, approximately 160ng / g, approximately 170ng / g, approximately 180ng / g, approximately 190ng / g, approximately 200ng / g, approximately 210ng / g, approximately 220ng / g, approximately Including 230ng / g, about 240ng / g, about 250ng / g, about 260ng / g, about 270ng / g, about 280ng / g, about 290ng / g, about 300ng / g, about 310ng / g, about 320ng / g, about 330ng / g, about 340ng / g, about 350ng / g, about 360ng / g, about 370ng / g, about 380ng / g, about 390, and about 400ng / g.

[0215] Indeed, under appropriate culture conditions, cells secrete extracellular matrix and synthesize factors, including polypeptides and nucleic acids, that promote tissue regeneration and / or tissue repair, which may be considered biomarkers for tissue regeneration and / or tissue repair.

[0216] In practice, the content of factors as polypeptides in biomaterials according to the present invention can be assessed by any suitable method known in the art or adapted therefrom. Illustratively, the expression or non-expression of these biomarkers may be monitored at the nucleic acid level or polypeptide level. Non-limiting examples of methods for monitoring biomarkers at the nucleic acid level include RT-PCR (qPCR) analysis of RNA extracted from cultured cells using specific primers. Non-limiting examples of methods for monitoring biomarkers at the polypeptide level include immunofluorescence analysis using marker-specific antibodies, such as Western blotting and ELISA, fluorescence-activated cell sorting (FACS), mass spectrometry, and enzymatic assays.

[0217] In one embodiment, the factor content comprises RNA content.

[0218] In some embodiments, the mean relative expression of an RNA contained in a biomaterial according to the invention varies by at least about 10% relative to the mean relative expression of said RNA from a corresponding fresh, non-sterile, non-dried biomaterial.

[0219] In one embodiment, the RNA content includes one or more miRNAs.

[0220] Within the scope of the present invention, the criteria and rules for identification and naming of miRNAs are as set out in Ambros et al. (A uniform system for microRNA annotation.RNA 2003 9(3):277-279). miRNA sequences can be easily obtained from the miRbase database (http: / / www.mirbase.org / ) or the miRDB database (http: / / www.mirdb.org / ).

[0221] In certain embodiments, the RNA content comprises at least one miRNA selected from any one of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, or Table 12.

[0222] As used herein, the term "at least one" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or more.

[0223] [Table 1-1]

[0224] [Table 1-2]

[0225] In some embodiments, at least one miRNA is hsa-let-7a-5p, hsa-miR-199a-3p, hsa-miR-10a-5p, hsa-miR-41l-5p, hsa-let-7b-5p, hsa-miR-145-5p, hsa-miR-495-3p, hsa-miR-505-5p, hsa-let-7f-5p, hsa-miR-30a-3p, hsa-miR-425-5p, hsa-miR-664a-3p, hsa-miR-24-3p, hsa-miR-382-5p, hsa-miR-2053, hsa-miR-26a-5p, hsa-miR-...hsa-miR-99b-5p、hsa-miR-99b-3p、hsa-miR-126-3p、hsa-let-7c-5p、hsa-miR-625-3p、hsa-miR-127-3p、hsa-miR-149-5p、hsa-miR-199b-3p、hsa-miR-4668-5p、hsa-miR-134-5p、hsa-miR-193b-3p、hsa-miR-191-5p、hsa-miR-29b-3p、hsa-miR-324-5p、hsa-miR-223-3p、hsa-miR-574-5p、hsa-miR-423-3p、hsa-miR-3605-3p、hsa-miR-340-3p、hsa-miR-424-3p、hsa-miR-376c-3p、hsa-miR-101-3p、hsa-miR-369-5p、hsa-miR-423-5p、hsa-let-7b-3p、hsa-miR-103a-3p、hsa-miR-6724-5p、hsa-miR-342-3p、hsa-miR-3074-5p、hsa-miR-1246、hsa-miR-7847-3p、hsa-let-7d-3p、hsa-miR-98-5p、hsa-miR-138-5p、hsa-miR-874-3p、hsa-miR-130a-3p、hsa-miR-185-5p、hsa-miR-190a-5p、hsa-miR-3653-5p、hsa-miR-3184-3p、hsa-miR-19a-3p、hsa-miR-24-2-5p、hsa-miR-664b-3p、hsa-miR-222-3p、hsa-miR-34a-5p、hsa-miR-26a-2-3p、hsa-miR-664b-5p、hsa-let-7g-5p、hsa-miR-374c-3p、hsa-miR-301a-3p、hsa-miR-6516-3p、hsa-miR-125a-5p、hsa-miR-181a-5p、hsa-miR-98-3p、hsa-let-7i-3p、hsa-let-7d-5p、hsa-miR-328-3p、hsa-miR-1273a、hsa-miR-154-5p、hsa-miR-29a-3p、hsa-miR-92b-3p、hsa-miR-28-5p、hsa-miR-664a-5p、hsa-let-7i-5p、hsa-miR-335-5p、hsa-miR-34a-3p、hsa-miR-1291, hsa-miR-146b-5p, hsa-let-7f-l-3p, hsa-miR-425-3p, hsa-miR-140-5p, hsa-miR-4454, hsa-miR-196b-5p, hsa-miR-505-3 p, hsa-miR-3609, hsa-miR-28-3p, hsa-miR-3613-3p, hsa-miR-34b-3p, hsa-miR-4461, hsa-miR-92a-3p, hsa-miR-23a-5p, hsa-miR-361-3p, Selected from the group consisting of hsa-miR-3613-5p, hsa-miR-125b-5p, hsa-miR-374b-5p, hsa-miR-10b-5p, hsa-miR-663b, hsa-miR-337-3p, hsa-miR-660-5p, hsa-miR-1306-5p, hsa-miR-378a-3p, hsa-miR-93-5p, hsa-miR-186-5p, hsa-miR-22-5p, hsa-miR-454-3p, hsa-miR-409-3p, and combinations thereof.

[0226] [Table 2-1]

[0227] [Table 2-2]

[0228] In some embodiments, at least one miRNA is hsa-let-7a-5p, hsa-miR-30a-3p, hsa-miR-103a-3p, hsa-miR-542-3p, hsa-let-7b-5p, hsa-miR-320b, hsa-miR-19a-3p, hsa-miR-663a, hsa-miR-24-3p, hsa-miR-193a-5p, hsa-miR-126-5p, hsa-miR-101-3p, hsa-miR-21-5p, hsa-miR-382-5p, hsa-miR-2053, hsa-miR-143-3p, hsa-let-7f-5p, hsa-miR-423-3p, hsa-miR-29b-l-5p, hsa-miR-21-3p, hsa-miR-574-3p, hsa-miR-17-5p, hsa-miR-3648, hsa-miR-224-5p, hsa-miR-23b-3p, hsa-miR-19b-3p, hsa-miR-374a-3p, hsa-miR-26a-5p, hsa-miR-1273g-3p, hsa-miR-92b-3p, hsa-miR-454-3p, hsa-miR-27a-5p, hsa-miR-25-3p, hsa-miR-320a, hsa-miR-532-3p, hsa-miR-324-5p, hsa-miR-199a-5p, hsa-miR-3074-5p, hsa-miR-136-3p, hsa-miR-340-3p, hsa-miR-196a-5p, hsa-miR-376c-3p, hsa-miR-361-3p, hsa-miR-379-5p, hsa-miR-214-3p, hsa-let-7b-3p, hsa-miR-1246, hsa-miR-409-5p, hsa-miR-125a-5p, hsa-miR-625-3p, hsa-miR-130b-3p, hsa-miR-543, hsa-miR-221-3p, hsa-miR-99b-5p, hsa-miR-134-5p, hsa-miR-5787, hsa-miR-222-3p, hsa-miR-34a-5p, hsa-miR-154-5p, hsa-miR-6089, hsa-let-7e-5p, hsa-miR-5096, hsa-miR-34a-3p, hsa-miR-127-3p, hsa-miR-191-5p, hsa-miR-30e-3phsa-miR-576-5p, hsa-miR-149-5p, hsa-miR-199b-3p, hsa-miR-22-3p, hsa-miR-874-3p, hsa-miR-181c-5p, hsa-miR-342-3p, hsa-miR-151a-3p, hsa-m iR-100-5p, hsa-miR-193b-3p, hsa-miR-23a-3p, hsa-miR-186-5p, hsa-miR-103b, hsa-miR-222-5p, hsa-miR-424-3p, hsa-miR-193b-5p, hsa-miR-1273 a, hsa-miR-3613-5p, hsa-miR-28-3p, hsa-miR-328-3p, hsa-miR-1306-5p, hsa-miR-365b-3p, hsa-let-7g-5p, hsa-miR-4449, hsa-miR-138-5p, hsa-miR-3960, hsa-miR-92a-3p, hsa-miR-27a-3p, hsa-miR-15b-3p, hsa-m iR-485-3p, hsa-miR-424-5p, hsa-miR-30c-5p, hsa-miR-26b-3p, hsa-miR-6087, hsa-let-7d-3p, hsa-miR-494-3p, hsa-miR-10b-5p, hsa-miR-92a-l-5p, hsa-miR-4454, hsa-miR-98-5p, hsa-miR-22-5p, hsa-miR-3607-5p hsa-miR-146b-5p, hsa-miR-10a-5p, hsa-miR-3613-3p, hsa-miR-3653-5p, hsa-miR-423-5p, hsa-miR-29b-3p, hsa-miR-655-3p, hsa-miR-664b-5p, hsa-miR-29a-3p, hsa-miR-374b-5p, hsa-miR-7-l-3p, hsa-miR-664b-3 p、hsa-miR-574-5p、hsa-miR-335-5p、hsa-miR-23a-5p、hsa-miR-6516-3p、hsa-miR-199b-5p、hsa-miR-374c- 3p、hsa-miR-24-2-5p、hsa-miR-1291、hsa-miR-125b-5p、hsa-miR-425-5p、hsa-miR-3605-3p、hsa-let-7i-3p、hsa-miR-3184-3p, hsa-miR-181a-5p, hsa-miR-6832-3p, hsa-miR-455-3p, hsa-let-7c-5p, hsa-miR-196b-5 p, hsa-miR-146a-5p, hsa-miR-671-5p, hsa-miR-337-3p, hsa-let-7f-l-3p, hsa-miR-16-2-3p, hsa-miR-127l -5p, hsa-let-7d-5p, hsa-miR-4668-5p, hsa-miR-18lb-5p, hsa-miR-4461, hsa-miR-145-5p, hsa-miR-660-5p , hsa-miR-26a-2-3p, hsa-miR-6724-5p, hsa-miR-93-5p, hsa-miR-664a-3p, hsa-miR-376a-3p, hsa-miR-190a -5p, hsa-miR-619-5p, hsa-miR-185-5p, hsa-miR-539-5p, hsa-miR-3609, hsa-miR-130a-3p, hsa-miR-3651, hsa-miR-708-5p, hsa-miR-41l-5p, hsa-let-7i-5p, hsa-miR-495-3p, hsa-miR-98-3p, hsa-miR-425-3p, hsa- miR-409-3p, hsa-let-7a-3p, hsa-miR-1237-5p, hsa-miR-4485-3p, hsa-miR-210-3p, hsa-miR-28-5p, hsa-miR-223-3p, hsa-miR-532-5p, hsa-miR-199a-3p, hsa-miR-99b-3pd, and combinations thereof.

[0229] [Table 3-1]

[0230] [Table 3-2]

[0231] In some embodiments, at least one miRNA is hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-24-2-5p, hsa-let-7b-5p, hsa-miR-125b-5p, hsa-miR-335-5p, hsa-miR-26a-2-3p, hsa-let-7f-5p, hsa-miR-337-3p, hsa-let-7f-l-3p, hsa-miR-301a-3p, hsa-miR-24-3p, hsa-miR-93-5p, hsa-miR-196b-5p, hsa-miR-98-3p, hsa-miR-21-5p, hsa-miR-409-3p, hsa-miR-3613-3p, hsa-miR-1273a, hsa-miR-23b-3p, hsa-miR-199a-3p, hsa-miR-23a-5p, hsa-miR-28-5p, hsa-miR-1273g-3p, hsa-miR-145-5p, hsa-miR-374b-5p, hsa-miR-34a-3p, hsa-miR-574-3p, hsa-miR-30a-3p, hsa-miR-660-5p, hsa-miR-425-3p, hsa-miR-25-3p, hsa-miR-382-5p, hsa-miR-186-5p, hsa-miR-505-3p, hsa-let-7e-5p, hsa-miR-19b-3p, hsa-miR-454-3p, hsa-miR-34b-3p, hsa-miR-214-3p, hsa-miR-210-3p, hsa-miR-10a-5p, hsa-miR-361-3p, hsa-miR-199a-5p, hsa-miR-619-5p, hsa-miR-495-3p, hsa-miR-10b-5p, hsa-miR-196a-5p, hsa-miR-17-5p, hsa-miR-425-5p, hsa-miR-1306-5p, hsa-miR-199b-5p, hsa-miR-193a-5p, hsa-miR-2053, hsa-miR-22-5p, hsa-miR-221-3p, hsa-miR-320b, hsa-miR-5096, hsa-miR-378a-3p, hsa-miR-424-5p, hsa-miR-193b-5p, hsa-miR-494-3p, hsa-miR-41l-5p,hsa-miR-23a-3p、hsa-miR-320a、hsa-miR-27a-3p、hsa-miR-505-5p、hsa-let-7c-5p、hsa-miR-151a-3p、hsa-miR-4449、hsa-miR-664a-3p、hsa-miR-199b-3p、hsa-let-7a-3p、hsa-miR-532-3p、hsa-miR-26a-5p、hsa-miR-191-5p、hsa-miR-30e-3p、hsa-miR-532-5p、hsa-miR-377-3p、hsa-miR-574-5p、hsa-miR-22-3p、hsa-miR-126-5p、hsa-miR-485-3p、hsa-miR-424-3p、hsa-miR-99b-5p、hsa-miR-30c-5p、hsa-miR-590-3p、hsa-miR-423-5p、hsa-miR-625-3p、hsa-miR-130b-3p、hsa-miR-99a-3p、hsa-miR-342-3p、hsa-miR-4668-5p、hsa-miR-136-3p、hsa-miR-143-3p、hsa-let-7d-3p、hsa-miR-29b-3p、hsa-miR-15b-3p、hsa-miR-26b-3p、hsa-miR-130a-3p、hsa-miR-423-3p、hsa-miR-29b-1-5p、hsa-miR-3607-5p、hsa-miR-3184-3p、hsa-miR-376c-3p、hsa-miR-99b-3p、hsa-miR-3651、hsa-miR-222-3p、hsa-let-7b-3p、hsa-miR-127-3p、hsa-miR-374a-3p、hsa-let-7g-5p、hsa-miR-3074-5p、hsa-miR-134-5p、hsa-miR-376a-3p、hsa-miR-125a-5p、hsa-miR-98-5p、hsa-miR-324-5p、hsa-miR-485-5p、hsa-let-7d-5p、hsa-miR-185-5p、hsa-miR-3605-3p、hsa-miR-103b、hsa-miR-29a-3p、hsa-miR-19a-3p、hsa-miR-101-3p、hsa-miR-126-3p、hsa-let-7i-5p、hsa-miR-34a-5p、The miR-103a-3p, the miR-149-5p, the miR-146b-5p, the miR-374c-3p, the miR-1246, the miR-193b-3p, the miR-4454, the miR-181a-5p, the miR-138-5p, the miR-223-3p, the miR-28-3p, the miR-328-3p, the miR-190a-5p, the miR-340-3p, the miR-874-3p, the miR-7847-3p, the miR-6724-5p, the miR-369-5p, and a combination thereof. ,

[0232] [Table 4-1]

[0233] [Table 4-2]

[0234] In certain embodiments, at least one miRNA is hsa-let-7a-5p, hsa-let-7i-5p, hsa-miR-660-5p, hsa-miR-6832-3p, hsa-let-7b-5p, hsa-miR-409-3p, hsa-miR-664a-3p, hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-210-3p, hsa-miR-185-5p, hsa-miR-16-2-3 p, hsa-miR-21-5p, hsa-miR-199a-3p, hsa-miR-3651, hsa-miR-18 lb-5p, hsa-let-7f-5p, hsa-miR-30a-3p, hsa-miR-495-3p, hsa-miR-26a-2-3p, hsa-miR-574-3p, hsa-miR-320b, hsa-let-7a-3p, hsa-miR-376a-3 p, hsa-miR-23b-3p, hsa-miR-193 a-5p, hsa-miR-28-iR-663a、hsa-miR-222-3p、hsa-miR-376c-3p、hsa-miR-374a-3p、hsa-miR-101-3p、hsa-let-7e-5p、hsa-let-7b-3p、hsa-miR-454-3p、hsa-miR-143-3p、hsa-miR-191-5p、hsa-miR-625-3p、hsa-miR-532-3p、hsa-miR-21-3p、hsa-miR-199b-3p、hsa-miR-99b-5p、hsa-miR-136-3p、hsa-miR-224-5p、hsa-miR-342-3p、hsa-miR-34a-5p、hsa-miR-361-3p、hsa-miR-26a-5p、hsa-miR-23a-3p、hsa-miR-5096、hsa-miR-1246、hsa-miR-27a-5p、hsa-miR-424-3p、hsa-miR-30e-3p、hsa-miR-130b-3p、hsa-miR-324-5p、hsa-miR-28-3p、hsa-miR-22-3p、hsa-miR-134-5p、hsa-miR-340-3p、hsa-let-7g-5p、hsa-miR-15 la-3 p、hsa-miR-154-5p、hsa-miR-379-5p、hsa-miR-92a-3p、hsa-miR-186-5p、hsa-miR-34a-3p、hsa-miR-409-5p、hsa-miR-424-5p、hsa-miR-193b-5p、hsa-miR-576-5p、hsa-miR-543、hsa-let-7d-3p、hsa-miR-328-3p、hsa-miR-874-3p、hsa-miR-5787、hsa-miR-4454、hsa-miR-4449、hsa-miR-100-5p、hsa-miR-6089、hsa-miR-146b-5p、hsa-miR-27a-3p、hsa-miR-103b、hsa-miR-127-3p、hsa-miR-423-5p、hsa-miR-30c-5p、hsa-miR-1273 a、hsa-miR-149-5p、hsa-miR-29a-3p、hsa-miR-494-3p、hsa-miR-1306-5p、hsa-miR-18lc-5p, hsa-miR-574-5p, hsa-miR-98-5p, hsa-miR-138-5p, hsa-miR-193b-3p, hsa-miR-199b-5p, hsa-miR-10a-5p, hsa-miR-15b-3p, h sa-miR-222-5p, hsa-miR-125b-5p, hsa-miR-29b-3p, hsa-miR-26b-3p, hsa-miR-3613-5p, hsa-miR-3184-3p, hsa-miR-374b-5p, hsa-m iR-10b-5p, hsa-miR-365b-3p, hsa-let-7c-5p, hsa-miR-335-5p, hsa-miR-22-5p, hsa-miR-3960, hsa-miR-337-3p, hsa-miR-374c-3p, hsa-miR-3613-3p, hsa-miR-485-3p, hsa-let-7d-5p, hsa-miR-425-5p, hsa-miR-655-3p, hsa-miR-6087, hsa-miR-145-5p, hsa-miR-18 and combinations thereof.

[0235] [Table 5]

[0236] In some embodiments, at least one miRNA is hsa-let-7a-5p, hsa-miR-210-3p, hsa-miR-29b-3p, hsa-miR-30e-3p, hsa-let-7b-5p, hsa-miR-3184-3p, hsa-miR-92a-3p, hsa-miR-320a, hsa-miR-24-3p, hsa-let-7d-5p, hsa-miR-193b-5p, hsa-miR-361-3p, hsa-miR-199a-5p, hsa-miR-25-3p, hsa-miR-181a-5p, hsa-miR-151a-3p, hsa-miR-214-3p, hsa-miR-193 a-5p, hsa-miR-30c-5p, hsa-miR-154-5p, hsa-let-7f-5p, hsa-miR-199a-3p, hsa-miR-664b-3p, hsa-miR-664a-5p, hsa-miR-3607-5p, hsa-miR-29a-3p, hsa-miR-27a-3p, hsa-miR-92b-3p, hsa-miR-199b-3p, hsa-miR-342-3p, hsa-miR-320b, hsa-miR-1291, hsa-let-7e-5p, hsa-miR-130a-3p, hsa-miR-3651, hsa-miR-103b, hsa-miR-1273g-3p, hsa-miR-30a-3p, hsa-miR-664b-5p, hsa-miR-34a-3p, hsa-miR-125a-5p, hsa-miR-145-5p, hsa-miR-664a-3p, hsa-miR-140-5p, hsa-miR-21-5p, hsa-miR-28-3p, hsa-miR-98-5p, hsa-miR-3609, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-146b-5p, hsa-miR-374c-3p, hsa-miR-125b-5p, hsa-miR-34a-5p, hsa-miR-337-3p, hsa-miR-10a-5p, hsa-lSelected from the group consisting of et-7g-5p, hsa-miR-222-3p, hsa-miR-4449, hsa-miR-22-3p, hsa-miR-191-5p, hsa-miR-3074-5p, hsa-miR-6516-3p, hsa-miR-4668-5p, hsa-miR-574-3p, hsa-miR-424-5p, hsa-let-7i-3p, hsa-miR-24-2-5p, hsa-miR-199b-5p, hsa-miR-424-3p, hsa-miR-103a-3p, hsa-miR-29b-l-5p, hsa-miR-423-5p, hsa-miR-328-3p, hsa-miR-324-5p, hsa-miR-335-5p, hsa-miR-574-5p, hsa-miR-17-5p, hsa-miR-660-5p, hsa-miR-425-5p, hsa-miR-23b-3p, hsa-miR-23a-3p, hsa-miR-185-5p, hsa-miR-4461, hsa-miR-196a-5p, hsa-let-7d-3p, hsa-miR-374b-5p, hsa-miR-127-3 p, hsa-let-7c-5p, hsa-miR-423-3p, hsa-miR-409-3p, hsa-miR-196b-5p, hsa-miR-221-3p, hsa-miR-382-5p, hsa-miR-619-5p, hsa-miR-3613-5p, hsa-miR-3653-5p, hsa-miR-19b-3p, hsa-miR-99b-5p, hsa-miR-376c-3p, hsa-miR-99b-3p, hsa-miR-66-3b, hsa-miR-495-3p, hsa-miR-454-3p, and combinations thereof.

[0237]

Table 6

[0238] In some embodiments, at least one miRNA is hsa-let-7a-5p, hsa-miR-3653-5p, hsa-miR-98-5p, hsa-miR-28-5p, hsa-let-7b-5p, hsa-miR-342-3p, hsa-miR-664a-3p, hsa-miR-10a-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-92b-3p, hsa-miR-151a-3p, hsa-let-7f-5p, hsa-miR-23b-3p, hsa-miR-4449, hsa-miR-30e-3p, hsa-miR-199a-5p, hsa-let-7c-5p, hsa-miR-320a, hsa-miR-324-5p, hsa-miR-214-3p, hsa-miR-222-3p, hsa-miR-181a-(-5)p, hsa-miR-495-3p, hsa-miR-3607-5p, hsa-miR-29a-3p, hsa-miR-3651, hsa-miR-576-5p, hsa-miR-125a-5p, hsa-miR-92a-3p, hsa-miR-185-5p, hsa-miR-625-3p, hsa-miR-199b-3p, hsa-miR-30a-3p, hsa-miR-664b-(-5)p, hsa-miR-671-5p, hsa-miR-125b-5p, hsa-miR-424-3p, hsa-miR-196b-5p, hsa-miR-127l-5p, hsa-miR-21-5p, hsa-miR-423-3p, hsa-miR-27a-3p, hsa-miR-186-5p, hsa-let-7e-5p, hsa-miR-34a-5p, hsa-miR-29b-3p, hsa-miR-23a-5p, hsa-let-7i-5p, hsa-miR-424-5p, hsa-miR-664b-3p, hsa-miR-361(-3)p, hsa-let-7g-5p, hsa-miR-145-5p, hsa-miR-99b-5p, hsa-miR-376c-3p, hsa-miR-574-3p, hsa-miR-328-3p, hsa-miR-103a-3p, hsa-miR-409-3p, hsa-miR-574-5p, hsa-miR-3074-5p, hsa-miR-6516-3p, hsa-miR-4461, hsa-miR-191-5p It should be noted that there seems to be a small error in the original text where "hsa-miR-181a-5p" has an extra "-" in the middle, and "hsa-miR-664b-5p" has an extra "-" in the middle, and "hsa-miR-3613-5p" is written as "hsa-miR-361(-3)p" in the translation. These have been translated as accurately as possible based on the provided text.hsa-let-7d-3p, hsa-miR-22-3p, hsa-miR-454-3p, hsa-miR-196a-5p, hsa-miR-93-5p, hsa-miR-26a-5 p, hsa-miR-6724-5p, hsa-miR-221-3p, hsa-miR-23a-3p, hsa-miR-103b, hsa-let-7b-3p, hsa-miR-25-3 p, hsa-miR-19b-3p, hsa-miR-1291, hsa-miR-1990a-5p, hsa-miR-423-5p, hsa-miR-146b-5p, hsa-miR-4 25-5p, hsa-miR-26b-3p, hsa-miR-210-3p, hsa-miR-320b, hsa-miR-22-5p, hsa-miR-3609, hsa-miR-127 3g-3p, hsa-miR-337-3p, hsa-miR-374c-3p, hsa-miR-41l-5p, hsa-let-7d-5p, hsa-miR-17-5p, hsa-let -7i-3p, hsa-miR-425-3p, hsa-miR-199b-5p, hsa-miR-130a-3p, hsa-miR-374b-5p, hsa-miR-4485-3p, h The miR-199a-3p, hsa-miR-193b-5p, hsa-miR-455-3p, hsa-miR-30c-5p, hsa-miR-193a-5p, hsa-miR-382-5p, hsa-miR-532-3p, hsa-miR-619-5p, hsa-miR-3184-3p, and combinations thereof.

[0239] [Table 7]

[0240] In some embodiments, the at least one miRNA is hsa-miR-3687, hsa-miR-619-5p, hsa-let-7e-5p, hsa-miR-24-3p, hsa-miR-664b-5p, hsa-miR-181a-5p, hsa-miR-25-3p, hsa-miR-382-5p, hsa-miR-210-3p, hsa-miR-409-3 p, hsa-miR-374c-3p, hsa-miR-214-3p, hsa-miR-4449, hsa-let-7a-3p, hsa-miR-29b-3p, hsa-miR-199b -5p, hsa-miR-3651, hsa-miR-4454, hsa-let-7b-3p, hsa-miR-199a-5p, hsa-miR-663a, hsa-let-7i-5p, hsa-miR-23b-3p, hsa-miR-3074-5p, hsa-miR-664b-3p, hsa-miR-335-5p, hsa-miR-3613-3p, hsa-miR- 361-3p, hsa-miR-3653-5p, hsa-miR-1246, hsa-miR-138-5p, hsa-miR-6723-5p, hsa-miR-664a-3p, hsa- miR-6516-5p, hsa-miR-6516-3p, hsa-miR-130a-3p, hsa-miR-3648, hsa-miR-3607-5p, hsa-miR-4485-3p, hsa-miR-660-5p, hsa-miR-196b-5p, hsa-miR-342-3p, hsa-miR-221-3p, and combinations thereof.

[0241] [Table 8]

[0242] In one embodiment, at least one miRNA is hsa-miR-210-3p, hsa-miR-409-3p, hsa-miR-219, hsa-miR-29b, hsa-miR-4454, hsa-miR-3607-5p, hsa-miR-299-5p, has-miR-140-5p, hsa-miR-619-5p, hsa-miR-3609, hsa-miR-302b, hsa-miR-31, hsa-miR-1246, hsa-miR-663a, has-miR-221, hsa-miR-30, hsa-miR-222-3p, hsa-miR-19a-3p, hsa-miR-155, hsa-miR-30e, hsa-miR-181a-5p, hsa-miR-3651, hsa-miR-885-5p, hsa-miR-17, hsa-miR-6832-3p, hsa-miR-4668-5p, hsa-miR-181a, hsa-miR-433, hsa-miR-335-5p, hsa-miR-301a-3p, hsa-miR-320c, hsa-miR-486-5p, hsa-let-7a-3p, hsa-miR-664a-3p, hsa-miR-548d-5p, hsa-miR-335, hsa-miR-28-3p, hsa-miR-485-5p, hsa-miR-34a, hsa-miR-106a, hsa-miR-125a-5p, hsa-miR-382-5p, hsa-miR-378, hsa-miR-21-3p, hsa-miR-374c-3p, hsa-miR-4449, hsa-346, hsa-miR-26a-5p, hsa-miR-181c-5p, hsa-miR-138-5p, hsa-10a, let-7a-5p, hsa-miR-374b-5p, let-7a, hsa-125b, hsa-miR-10a, hsa-miR-3687, hsa-miR-199b, hsa-miR-322, hsa-miR-148-a, hsa-miR-3653-5p, hsa-miR-218, hsa-miR-21, hsa-miR-31-5p, hsa-miR-664b-5p, hsa-miR-148a, hsa-miR-96, hsa-miR-486-5p, hsa-miR-664b-3p, hsa-miR-135b, hsa-miR-22, hsa-miR-24-3phsa-miR-3613-3p, hsa-miR-203, hsa-miR-27, hsa-let-7i-5p, hsa-miR-3074-5p, hsa-miR-4485-3p, hsa-let-7c-5p, hsa-miR-6723-5p, hsa-miR-671-5p, hsa-miR-93-5p, hsa-miR-154-5p, and combinations thereof.

[0243] [Table 9]

[0244] In some embodiments, the at least one miRNA is hsa-miR-210-3p, hsa-let-7i-5p, hsa-miR-29b-3p, hsa-miR-199a-5p, hsa-miR-619-5p, hsa-miR-335-5p, hsa-miR-23b-3p, hsa-miR-3074-5p, hsa-miR-181a-5p, hsa-miR-1246, hsa-miR-24-3 p, hsa-miR-361-3p, hsa-let-7a-3p, hsa-let-7e-5p, hsa-miR-214-3p, hsa-miR-130a-3p, hsa-miR-4454, hsa-miR-374c-3p, hsa-miR-199b-5p, hsa-miR-3607-5p, hsa-miR-660-5p, hsa-miR-342-3p, and combinations thereof.

[0245] [Table 10]

[0246] In certain embodiments, the at least one miRNA is selected from the group consisting of hsa-miR-210-3p, hsa-miR-125a-5p, hsa-miR-219, hsa-miR-21, hsa-miR-4454, hsa-miR-374c-3p, hsa-miR-299-5p, hsa-miR-96, hsa-miR-619-5p, hsa-miR-181c-5p, hsa-miR-302b, hsa-miR-22, hsa-miR-1246, hsa-miR-374b-5p, hsa-miR-548d-5p, hsa-miR-27, hsa-miR-222-3p, let-7a, hsa-miR-34a ...374d-5p, hsa-miR-374c-3p, hsa-miR-374d-5p, hsa-miR-374c-3p, hsa-miR-374c-3p, hsa-miR-374c-3p, hsa-miR-374c-3p, hsa-miR-3 -29b, hsa-miR-181a-5p, hsa-miR-199b, hsa-miR-378, hsa-miR-24-3p, hsa-miR-6832-3p, hsa-miR-218, hsa-346, hsa-let-7i-5p, hsa-miR-335-5p, hsa-miR-148a, hsa-10a, hsa-miR-3074-5p, hsa-let-7a-3p, hsa-miR-135b, hsa-125b, hsa-miR-671-5p, hsa-miR-28-3p, hsa-miR-203, hsa-miR-322, and combinations thereof.

[0247] [Table 11]

[0248] In some embodiments, the at least one miRNA is hsa-miR-3687, hsa-miR-664b-3p, hsa-miR-6516-5p, hsa-miR-138-5p, hsa-miR-664b-5p, hsa-miR-3653-5p, hsa-miR-3607-5p, hsa-miR-6516-3p, hsa-miR-4449, hsa-miR-664a-3p, hsa-miR-25-3p, hsa-miR-4485-3p, hsa-miR-3 651, hsa-miR-3648, hsa-let-7b-3p, hsa-miR-382-5p, hsa-miR-663a, hsa-miR-409-3p, hsa-miR-3613-3p, hsa-miR-6723-5p, hsa-miR-3687, hsa-miR-664b-3p, hsa-miR-6516-5p, hsa-miR-138-5p, hsa-miR-196b-5p, hsa-miR-221-3p, and combinations thereof.

[0249] [Table 12]

[0250] In certain embodiments, the at least one miRNA is selected from the group consisting of hsa-miR-3687, hsa-miR-19a-3p, has-miR-221, hsa-miR-17, hsa-miR-3653-5p, hsa-miR-3651, hsa-miR-155, hsa-miR-433, hsa-miR-664b-5p, hsa-miR-4668-5p, hsa-miR-885-5p, hsa-miR-486-5p, hsa-miR-664b-3p, hsa-miR-301a-3p, hsa-miR-181a, hsa-miR-335, hsa-miR-3613-3p, hsa-miR-664a-3p, and hsa-miR-32 miR-3609, hsa-miR-4449, hsa-miR-30, hsa-let-7c-5p, hsa-miR-663a, hsa-miR-138-5p, hsa-miR-30e, hsa-miR-154-5p, hsa-miR-6723-5p, and combinations thereof.

[0251] In certain embodiments, the at least one miRNA is selected from the group consisting of hsa-miR-210-3p, hsa-miR-409-3p, hsa-miR-361-3p, hsa-miR-130a-3p, hsa-miR-660-5p, hsa-miR-199b-5p, hsa-miR-3074-5p, hsa-let-7i-5p, hsa-miR-24-3p, and hsa-miR-342-3p. , hsa-miR-214-3p, hsa-miR-199a-5p, hsa-miR-3607-5p, hsa-miR-221-3p, hsa-miR-4449, hsa-miR-382-5p, hsa-miR-196b-5p, hsa-miR-663a, hsa-miR-4485-3p, hsa-miR-6723-5p, and combinations thereof.

[0252] In some embodiments, the at least one miRNA is selected from the group consisting of hsa-miR210-3p, hsa-miR-409-3p, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-382-5p, hsa-miR-4485-3p, and combinations thereof. In certain embodiments, the at least one miRNA is selected from the group consisting of hsa-miR210-3p, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-382-5p, and combinations thereof.

[0253] In one embodiment, the at least one miRNA is hsa-miR210-3p and / or hsa-miR-409-3p.

[0254] In one embodiment, a pharmaceutical composition of the invention comprises a therapeutically effective amount of at least two miRNAs selected from Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, or Table 12. In another embodiment, a pharmaceutical composition of the invention comprises a therapeutically effective amount of at least three, four, five, six, seven, eight, nine, ten, twelve, fourteen, sixteen, eighteen, twenty miRNAs selected from Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, or Table 12.

[0255] In some embodiments, the composition comprises a combination of hsa-miR-210-3p, hsa-miR-361-3p, hsa-miR-130a-3p, hsa-miR-660-5p, hsa-miR-199b-5p, hsa-miR-3074-5p, hsa-let-7i-5p, hsa-miR-24-3p, hsa-miR-342-3p, hsa-miR-214-3p, hsa-miR-199a-5p, and hsa-miR-3607-5p.

[0256] In some embodiments, the RNA content comprises or consists of hsa-miR210-3p, hsa-miR-409-3p, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-382-5p, and hsa-miR-4485-3p. In some preferred embodiments, the RNA content comprises or consists of hsa-miR210-3p, hsa-let-7i-5p, hsa-miR-93-5p, and hsa-miR-382-5p.

[0257] In some embodiments, the composition comprises a combination of hsa-miR210-3p, hsa-miR-409-3p, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-382-5p, and hsa-miR-4485-3p. In some preferred embodiments, the composition comprises a combination of hsa-miR210-3p, hsa-let-7i-5p, hsa-miR-93-5p, and hsa-miR-382-5p. In some preferred embodiments, the composition comprises at least hsa-miR210-3p.

[0258] In certain embodiments, a combination of miRNAs may be referred to as an miRNA cocktail.

[0259] In practice, the RNA content of biomaterials according to the present invention can be assessed by any suitable method known in the art or adapted therefrom. Illustratively, RNA can be extracted using a commercially available kit (such as the Qiagen® miRNeasy kit) and then sequenced using a high-throughput sequencing system (such as the Illumina® NextSeq 500 system). Illustratively, Qiazol lysis reagent (Qiagen®, Hilden, Germany) and a PreCellys homogenizer (Bertin® instruments, Montigny-le-Bretonneux, France) can be used. RNA can be purified using an Rneasy Mini Kit (Qiagen®, Hilden, Germany) with on-column DNase digestion according to the manufacturer's instructions. RNA quality and quantity can be measured using a spectrophotometer (Spectramax 190, Molecular Devices, California, USA). Customized PCR arrays (Customized Human Osteogenic and Angiogenic RT-PCR) can also be used. 2 RT for gene expression profiling via Profiler Assay - Qiagen®, Hilden, Germany 2 cDNA can be synthesized from 0.5 μg of total RNA using an RNA First Strand Kit (Qiagen®, Hilden, Germany). Amplified products can be detected using the ABI Quantstudio 5 system (Applied Biosystems®) and SYBR Green ROX Mastermix (Qiagen®, Hilden, Germany). Quantification can be performed using the ΔΔCT (delta-delta Ct) method. The final results for each sample can be normalized to the average expression level of three housekeeping genes (e.g., ACTB, B2M, GAPDH). In some embodiments, factor contents, including proteins and nucleic acids, are derived from cells.

[0260] In some embodiments, at least a portion of the factor content is cellular. In some embodiments, at least a portion of the factor content is cellular miRNA.

[0261] In practice, cellular miRNA can be isolated by any suitable method known in the art or adapted therefrom. For example, see Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells: A Laboratory Manual (Russell and Sambrook, 2001; Cold Spring Harbor Laboratory). Exemplarily, miRNA can be isolated using commercially available kits, such as the RNeasy Mini Kit (Qiagen®) or the MagMAX mirVana Total RNA Isolation Kit (Applied Biosystems®), according to the manufacturer's instructions. RNA concentration can be measured using a Nanodrop (ThermoFisher®, Waltham, MA, USA).

[0262] In some embodiments, at least one miRNA is hsa-let-7a-5p, hsa-miR-210-3p, hsa-miR-29b-3p, hsa-miR-30e-3p, hsa-let-7b-5p, hsa-miR-3184-3p, hsa-miR-92a-3p, hsa-miR-320a, hsa-miR-24-3p, hsa-let-7d-5p, hsa-miR-193b-5p, hsa-miR-361-3p, hsa-miR-199a-5p, hsa-miR-25-3p, hsa-miR-181a-5p, hsa-miR-151a-3p, hsa-miR-2l4-3p, hsa-miR-193a-5p, hsa-miR-30c-5p, hsa-miR-154-5p, hsa-let-7f-5p, hsa-miR-199a-3p, hsa-miR-664b-3p, hsa-miR-664a-5p, hsa-miR-3607-5p, hsa-miR-29a-3p, hsa-miR-27a-3p, hsa-miR-92b-3p, hsa-miR-199b-3p, hsa-miR-342-3p, hsa-miR-320b, hsa-miR-1291, hsa-let-7e-5p, hsa-miR-130a-3p, hsa-miR-3651, hsa-miR-103b, hsa-miR-1273g-3p, hsa-miR-30a-3p, hsa-miR-664b-5p, hsa-miR-34a-3p, hsa-miR-125a-5p, hsa-miR-145-5p, hsa-miR-664a-3p, hsa-miR-140-5p, hsa-miR-21-5p, hsa-miR-28-3p, hsa-miR-98-5p, hsa-miR-3609, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-146b-5p, hsa-miR-374c-3p, hsa-miR-125b-5p, hsa-miR-34a-5p, hsa-miR-337-3p, hsa-miR-10a-5p, hsa-let-7g-5p, hsa-miR-222-3p, hsa-miR-4449, hsa-miR-22-3p, hsa-miR-191-5p, hsa-miR-3074-5p, hsa-miR-6516-3p, hsa-miR-4668-5p, hsa-miR-574-3p,hsa-miR-424-5p, hsa-let-7i-3p, hsa-miR-24-2-5p, hsa-miR-199b-5p, hsa-miR-424-3p, hsa-miR-103a-3p, hsa-miR-29b-l-5p, hsa-miR-423-5p, hsa-miR-328-3p, hsa-miR-324-5p , hsa-miR-335-5p, hsa-miR-574-5p, hsa-miR-17-5p, hsa-miR-660-5p, hsa-miR-425-5p, h sa-miR-23b-3p, hsa-miR-23a-3p, hsa-miR-185-5p, hsa-miR-4461, hsa-miR-196a-5p, hsa- let-7d-3p, hsa-miR-374b-5p, hsa-miR-127-3p, hsa-let-7c-5p, hsa-miR-423-3p, hsa-mi R-409-3p, hsa-miR-196b-5p, hsa-miR-221-3p, hsa-miR-382-5p, hsa-miR-619-5p, hsa-miR miR-3613-5p, hsa-miR-3653-5p, hsa-miR-19b-3p, hsa-miR-99b-5p, hsa-miR-376c-3p, hsa-miR-99b-3p, hsa-miR-663b, hsa-miR-495-3p, hsa-miR-454-3p, and combinations thereof.

[0263] In certain embodiments, the cellular miRNAs include hsa-miR-210-3p, hsa-miR-409-3p, hsa-miR-361-3p, hsa-miR-130a-3p, hsa-miR-660-5p, hsa-miR-199b-5p, hsa-miR-3074-5p, hsa-let-7i-5p, hsa-miR-24-3p, hsa-miR-342-3p, hsa-miR-351-3p, hsa-miR-352-3p, hsa-miR-353-3p, hsa-miR-354-3p, hsa-miR-355-3p, hsa-miR-356-3p, hsa-miR-357-3p, hsa-miR-358-3p, hsa-miR-359 ... hsa-miR-214-3p, hsa-miR-199a-5p, hsa-miR-3607-5p, hsa-miR-221-3p, hsa-miR-4449, hsa-miR-382-5p, hsa-miR-196b-5p, hsa-miR-663a, hsa-miR-4485-3p, hsa-miR-6723-5p, and combinations thereof.

[0264] In one embodiment, the cellular miRNAs are hsa-let-7a-5p, hsa-let-7b-5p, hsa-miR-24-3p, hsa-let-7f-5p, hsa-miR-199a-5p, hsa-miR-214-3p, hsa-miR-3607-5p, hsa-miR-125a-5p, hsa-miR-199b-3p, hsa-miR-125b-5p, hsa-miR-21-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-let-7g-5p, hsa-miR-574-3p, hsa-miR-574-5p, hsa-miR-191-5p, hsa-miR-196a-5p, hsa-miR-221-3p, hsa-miR-25-3p, hsa-miR-423-5p, hsa-miR-210-3p, hsa-miR-1273g-3p, hsa-let-7d-5p, hsa-miR-199b-5p, hsa-miR-199a-3p, hsa-miR-193a-5p, hsa-miR-3184-3p, hsa-miR-3653-5p, hsa-miR-342-3p, hsa-miR-28-3p, hsa-miR-23b-3p, hsa-let-7c-5p, hsa-miR-222-3p, hsa-miR-29a-3p, hsa-miR-92a-3p, hsa-miR-30a-3p, hsa-miR-424-3p, hsa-miR-423-3p, hsa-miR-34a-5p, hsa-miR-424-5p, hsa-miR-145-5p, hsa-miR-328-3p, hsa-miR-3074-5p, hsa-let-7d-3p, hsa-miR-93-5p, hsa-miR-23a-3p, hsa-miR-19b-3p, hsa-miR-146b-5p, hsa-miR-320b, hsa-miR-337-3p, hsa-miR-17-5p, hsa-miR-130a-3p, hsa-miR-193b-5p, hsa-miR-382-5p, hsa-miR-30c-5p, hsa-miR-98-5p, hsa-miR-664a-3p, hsa-miR-92b-3p, hsa-miR-4449, hsa-miR-320a, hsa-miR-181a-5p, hsa-miR-3651, hsa-miR-185-5p, hsa-miR-664b-5p,hsa-miR-196b-5p, hsa-miR-27a-3p, hsa-miR-29b-3p, hsa-miR-664b-3p, hsa-miR-99b-5p, hsa-miR-103 a-3p, hsa-miR-6516-3p, hsa-miR-22-3p, hsa-miR-26a-5p, hsa-miR-103b, hsa-miR-1291, hsa-miR-425-5p, hsa-miR-22-5p, hsa-miR-374c-3p, hsa-let-7i-3p , hsa-miR-374b-5p, hsa-miR-455-3p, hsa-miR-532-3p, hsa-miR-619-5p, hsa-m iR-28-5p, hsa-miR-10a-5p, hsa-miR-151a-3p, hsa-miR-30e-3p, hsa-miR-324- 5p, hsa-miR-495-3p, hsa-miR-576-5p, hsa-miR-625-3p, hsa-miR-671-5p, hsa- miR-1271-5p, hsa-miR-186-5p, hsa-miR-23a-5p, hsa-miR-3613-5p, hsa-miR-3 76c-3p, hsa-miR-409-3p, hsa-miR-4461, hsa-miR-454-3p, hsa-miR-6724-5p, hsa-let-7b-3p, hsa-miR-190a-5p, hsa-miR-26b-3p, hsa-miR-3609, hsa-miR-41 l-5p, hsa-miR-425-3p, hsa-miR-4485-3p, and mixtures thereof.

[0265] In certain embodiments, the cellular miRNA is selected from the group including hsa-miR210-3p, hsa-miR-409-3p, hsa-let-7i-5p, hsa-miR-24-3p, hsa-miR-93-5p, hsa-miR-382-5p, hsa-miR-4485-3p, and combinations thereof.

[0266] In some embodiments, at least a portion of the factor content is secreted by the cells, preferably in the form of exosomes or exosome-like vesicles. In such embodiments, at least a portion of the factor content is contained in exosomes or exosome-like vesicles. In some embodiments, at least a portion of the factor content is exosomal miRNA.

[0267] The term "exosomes" as used herein refers to endocytosis-derived nanovesicles secreted by almost all cell types in the body. Exosomes contain proteins, nucleic acids, particularly miRNA, and lipids. In practice, exosomes can be isolated and / or purified by any suitable method known in the art or adapted therefrom. Illustratively, exosome fractions can be isolated by differential centrifugation from the culture medium, by polymer precipitation, or by high-performance liquid chromatography (HPLC). Non-limiting examples of differential centrifugation methods from the medium may include the following steps: - Centrifuge at a speed of approximately 300 x g to approximately 500 x g for 10 to 20 minutes to remove cells. - Centrifuge at a speed of approximately 1,500 x g to approximately 3,000 x g for 10 to 20 minutes to remove dead cells. - Centrifuge at approximately 7,500 x g to approximately 15,000 x g for 20 to 45 minutes to remove cell debris. -To pellet the exosomes, perform one or more rounds of ultracentrifugation at a speed of approximately 100,000 x g to approximately 200,000 x g for 30 to 120 minutes.

[0268] As an alternative method for isolating exosomes, commercially available kits such as the exoEasy Maxi Kit (Qiagen®) or the Total Exosome Isolation Kit (ThermoFisherScientific®) can be used.

[0269] In some embodiments, exosomes or exosome-like vesicles have an average diameter of about 25 nm to about 150 nm, preferably about 30 nm to 120 nm. Within the scope of the present invention, the expression "about 25 nm to about 150 nm" includes 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, and 150 nm.

[0270] In one embodiment, exosomal miRNAs are hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-24-2-5p, hsa-let-7b-5p, hsa-miR-125b-5p, hsa-miR-335-5p, hsa-miR-26a-2-3p, hsa-let-7f-5p, hsa-miR-337-3p, hsa-let-7f-l-3p, hsa-miR-301a-3p, hsa-miR-24-3p, hsa-miR-93-5p, hsa-miR-196b-5p, hsa-miR-98-3p, hsa-miR-21-5p, hsa-miR-409-3p, hsa-miR-3613-3p, hsa-miR-1273 a, hsa-miR-23b-3p, hsa-miR-199a-3p, hsa-miR-23a-5p, hsa-miR-28-5p, hsa-miR-1273g-3p, hsa-miR-145-5p, hsa-miR-374b-5p, hsa-miR-34a-3p, hsa-miR-574-3p, hsa-miR-30a-3p, hsa-miR-660-5p, hsa-miR-425-3p, hsa-miR-25-3p, hsa-miR-382-5p, hsa-miR-186-5p, hsa-miR-505-3p, hsa-let-7e-5p, hsa-miR-19b-3p, hsa-miR-454-3p, hsa-miR-34b-3p, hsa-miR-2l4-3p, hsa-miR-210-3p, hsa-miR-10a-5p, hsa-miR-361-3p, hsa-miR-199a-5p, hsa-miR-619-5p, hsa-miR-495-3p, hsa-miR-10b-5p, hsa-miR-196a-5p, hsa-miR-17-5p, hsa-miR-425-5p, hsa-miR-1306-5p, hsa-miR-199b-5p, hsa-miR-193a-5p, hsa-miR-2053, hsa-miR-22-5p, hsa-miR-221-3p, hsa-miR-320b, hsa-miR-509, hsa-miR-378a-3p, hsa-miR-424-5p, hsa-miR-193b-5p, hsa-miR-494-3p, hsa-miR-41l-5p, hsa-miR-23a-3p,hsa-miR-320a、hsa-miR-27a-3p、hsa-miR-505-5p、hsa-let-7c-5p、hsa-miR-151a-3p、hsa-miR-4449、hsa-miR-664a-3p、hsa-miR-199b-3p、hsa-let-7a-3p、hsa-miR-532-3p、hsa-miR-26a-5p、hsa-miR-191-5p、hsa-miR-30e-3p、hsa-miR-532-5p、hsa-miR-377-3p、hsa-miR-574-5p、hsa-miR-22-3p、hsa-miR-126-5p、hsa-miR-485-3p、hsa-miR-424-3p、hsa-miR-99b-5p、hsa-miR-30c-5p、hsa-miR-590-3p、hsa-miR-423-5p、hsa-miR-625-3p、hsa-miR-130b-3p、hsa-miR-99a-3p、hsa-miR-342-3p、hsa-miR-4668-5p、hsa-miR-136-3p、hsa-miR-143-3p、hsa-let-7d-3p、hsa-miR-29b-3p、hsa-miR-15b-3p、hsa-miR-26b-3p、hsa-miR-130a-3p、hsa-miR-423-3p、hsa-miR-29b-1-5p、hsa-miR-3607-5p、hsa-miR-3184-3p、hsa-miR-376c-3p、hsa-miR-99b-3p、hsa-miR-3651、hsa-miR-222-3p、hsa-let-7b-3p、hsa-miR-127-3p、hsa-miR-374a-3p、hsa-let-7g-5p、hsa-miR-3074-5p、hsa-miR-134-5p、hsa-miR-376a-3p、hsa-miR-125a-5p、hsa-miR-98-5p、hsa-miR-324-5p、hsa-miR-485-5p、hsa-let-7d-5p、hsa-miR-185-5p、hsa-miR-3605-3p、hsa-miR-103b、hsa-miR-29a-3p、hsa-miR-19a-3p、hsa-miR-101-3p、hsa-miR-126-3p、hsa-let-7i-5p、hsa-miR-34a-5p、hsa-miR-103a-3p、hsa-miR-149-5p, hsa-miR-146b-5p, hsa-miR-374c-3p, hsa-miR-1246, hsa-miR-193b-3p, hsa-miR-4454, hsa-miR-181a-5p, hsa-miR-138-5p, hsa-miR-223-3p, hsa-miR-28-3p, hsa-miR-328-3p, hsa-miR-190a-5p, hsa-miR-340-3p, hsa-miR-874-3p, hsa-miR-7847-3p, hsa-miR-6724-5p, hsa-miR-369-5p, and combinations thereof.

[0271] In some embodiments, the exosomal miRNA is selected from the group consisting of hsa-miR-210-3p, hsa-miR-409-3p, hsa-let-7i-5p, hsa-miR-3607-5p, hsa-let-7a-3p, hsa-miR-1246, hsa-miR-335-5p, hsa-miR-4454, hsa-miR-181a-5p, hsa-miR-374c-3p, hsa-miR-619-5p, hsa-miR-29b-3p, hsa-let7e-5p, hsa-miR-23b-3p, and hsa-miR- The miR-664a-3p is selected from the group consisting of iR-4449, hsa-miR-663a, hsa-miR-25-3p, hsa-let-7b-3p, hsa-miR-138-5p, hsa-miR-3613-3p, hsa-miR-6516-3p, hsa-miR-664a-3p, hsa-miR-3648, hsa-miR-3653-5p, hsa-miR-6516-5p, hsa-miR-3651, hsa-miR-3687, hsa-miR-664-5p, hsa-miR-664-3p, and combinations thereof.

[0272] In one embodiment, exosomal miRNAs are hsa-let-7a-5p, hsa-let-7b-5p, hsa-miR-24-3p, hsa-miR-21-5p, hsa-let-7f-5p, hsa-miR-574-3p, hsa-miR-23b-3p, hsa-miR-1273g-3p, hsa-miR-25-3p, hsa-miR-199a-5p, hsa-miR-196a-5p, hsa-miR-214-3p, hsa-miR-125a-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-let-7e-5p, hsa-miR-191-5p, hsa-miR--199b-3p, hsa-miR-342-3p, hsa-miR-23a-3p, hsa-miR-424-3p, hsa-miR-28-3p, hsa-let-7g-5p, hsa-miR-92a-3p, hsa-miR-424-5p, hsa-let-7d-3p, hsa-miR-4454, hsa-miR-146b-5p, hsa-miR-423-5p, hsa-miR-29a-3p, hsa-miR-574-5p, hsa-miR-199b-5p, hsa-miR-125b-5p, hsa-miR-3184-3p, hsa-let-7c-5p, hsa-miR-337-3p, hsa-let-7d-5p, hsa-miR-145-5p, hsa-miR-93-5p, hsa-miR-619-5p, hsa-miR-130a-3p, hsa-let-7i-5p, hsa-miR-409-3p, hsa-miR-210-3p, hsa-miR-199a-3 p, hsa-miR-30a-3p, hsa-miR-320b, hsa-miR-193 a-5p, hsa-miR-382-5p, hsa-miR-423-3p, hsa-miR-17-5p, hsa-miR-19b-3p, hsa-miR-92b-3p, hsa-miR-320a, hsa-miR-3074-5p, hsa-miR-376c-3p, hsa-let-7b-3p, hsa-miR-625-3p, hsa-miR-99b-5p, hsa-miR-34a-5p, hsa-miR-5096, hsa-miR-30e-3p, hsa-miR-22-3p, hsa-miR-151a-3p, hsa-miR-186-5phsa-miR-193b-5p、hsa-miR-328-3p、hsa-miR-4449、hsa-miR-27a-3p、hsa-miR-30c-5p、hsa-miR-494-3p、hsa-miR-98-5p、hsa-miR-10a-5 p、hsa-miR-29b-3p、hsa-miR-374b-5p、hsa-miR-335-5p、hsa-miR-37 4c-3p、hsa-miR-425-5p、hsa-miR-181a-5p、hsa-miR-196b-5p、hsa-l et-7f-l-3p, hsa-miR-4668-5p, hsa-miR-660-5p, hsa-miR-664a-3p, hsa-miR-185-5p, hsa-miR-3651, hsa-miR-495-3p, hsa-let-7a-3p, hsa-miR-28-5p, hsa-miR-99b-3p, hsa-miR-103a-3p, hsa-miR-19a-3p, hsa-miR-126-5p, hsa-miR-2053, hsa-miR-29b-l-5p, hsa-miR-3648 hsa-miR-374a-3p, hsa-miR-454-3p, hsa-miR-532-3p, hsa-miR-136-3p, hsa-miR-361-3p, hsa-miR-1246, hsa-miR-130b-3p, hsa-miR-134-5p, hsa-miR-154-5p, hsa-miR-34a-3p, hsa-miR-576-5p, hsa-miR-874-3p, hsa-miR-100-5p, hsa-miR-103b, hsa-miR-1273a, hsa-miR-1 306-5p, hsa-miR-138-5p, hsa-miR-15b-3p, hsa-miR-26b-3p, hsa-miR-10b-5p, hsa-miR-22-5p, hsa-miR-3613-3p, hsa-miR-655-3p, hsa-miR-7-l-3p, hsa-miR-23a-5p, hsa-miR-24-2-5p, hsa-miR-3605-3p, hsa-miR-6832-3p, hsa-miR-146a-5p, hsa-miR-16-2-3p, hsa-miR-18 lb-5p、hsa-miR-26a-2-3p、hsa-miR-376a-3p、hsa-miR-539-5p、hsa-miR-708-5p、hsa-miR-98-3p, hsa-miR-1237-5p, hsa-miR-223-3p, hsa-miR-532-5p, hsa-miR-542-3p, hsa-miR-663a, hsa-miR-101-3p, hsa-miR-143-3 p, hsa-miR-21-3p, hsa-miR-224-5p, hsa-miR-26a-5p, hsa-miR-27a-5p, hsa-miR-324-5p, hsa-miR-34 0-3p, hsa-miR-379-5p, hsa-miR-409-5p, hsa-miR-543, hsa-miR-5787, hsa-miR-6089, hsa-miR-127-3p , hsa-miR-149-5p, hsa-miR-181c-5p, hsa-miR-193b-3p, hsa-miR-222-5p, hsa-miR-3613-5p, hsa-miR-365b-3p, hsa-miR-3960, hsa-miR-485-3p, hsa-miR-6087, hsa-miR-92a-l-5p and mixtures thereof.

[0273] In some embodiments, the exosomal miRNA is selected from the group including hsa-miR210-3p, hsa-miR-409-3p, hsa-miR-4454, hsa-miR-619-5p, hsa-miR-3607-5p, hsa-miR-3613-3p, hsa-miR-664b-5p, hsa-miR-3687, hsa-miR-3653-5p, hsa-miR-664b-3p, and combinations thereof.

[0274] In one embodiment, the biomaterial comprises altered factor and / or RNA content compared to the factor and RNA content obtained from corresponding viable differentiated cells, including osteo- and / or chondrogenic differentiated cells.

[0275] In certain embodiments, the biomaterial according to the present invention is a multidimensional biomaterial, particularly in the form of a particulate composition, powder, beads, etc. In some embodiments, the multidimensional biomaterial comprises or consists of particles, preferably gelatin, DBM, or ceramic particles, which are coated with cells and extracellular matrix.

[0276] In some embodiments, the multidimensional biomaterial according to the present invention is added to or contained within a predetermined 3D shape or scaffold, such as, for example, a portion of freeze-dried human bone tissue, using techniques available to those skilled in the art. In some embodiments, the multidimensional biomaterial according to the present invention is structured to form a predetermined 3D shape or scaffold, such as, for example, a cube, using techniques available to those skilled in the art.

[0277] In some embodiments, the multidimensional biomaterial is in the form of particles having an average diameter of about 100 μm to about 1.5 mm, preferably about 500 μm to about 1 mm. Within the scope of the present invention, the expression "about 100 μm to about 1.5 mm" includes 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, and 1.5 mm.

[0278] In practice, the average particle size can be measured by any suitable method known in the art or adapted therefrom, non-limiting examples of which include atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS).

[0279] In some embodiments, the dry biomaterial is obtained by freeze-drying.

[0280] Freeze-drying, or lyophilization as it is called, can be carried out accordingly by any one of the protocols disclosed in the art, or a protocol adapted therefrom.

[0281] In some embodiments, freeze-drying of the biomaterial is carried out under vacuum at a temperature of about -80°C.

[0282] In practice, sterilization can be carried out by any suitable method known in the art, or a method adapted therefrom, non-limiting examples of suitable methods include irradiation, such as electron beam irradiation, X-ray irradiation, gamma irradiation, or ultraviolet irradiation.

[0283] In one embodiment, the sterilized biomaterial is obtained by gamma irradiation, preferably at a dose of about 7 kGy to about 45 kGy, more preferably at room temperature. Within the scope of the present invention, the expression "about 7 kGy to about 45 kGy" is understood to mean 7 kGy, 8 kGy, 9 kGy, 10 kGy, 11 kGy, 12 kGy, 13 kGy, 14 kGy, 15 kGy, 16 kGy, 17 kGy, 18 kGy, 19 kGy, 20 kGy, 21 kGy, 22 kGy, 23 kGy, 24 kGy, , 25kGy, 26kGy, 27kGy, 28kGy, 29kGy, 30kGy, 31kGy, 32kGy, 33kGy, 34kGy, 35kGy, 36kGy, 37kGy, 38kGy, 39kGy, 40kGy, 41kGy, 42kGy, 43kGy, 44kGy, and 45kGy.

[0284] In some embodiments, the biomaterial is obtained by gamma irradiation at a dose of about 10 kGy to about 40 kGy.

[0285] Within the scope of the present invention, the term "room temperature" refers to a temperature of about 18°C ​​to about 22°C, including 18°C, 19°C, 20°C, 21°C, and 22°C. In some embodiments, room temperature is about 20°C.

[0286] The inventors have observed that gamma irradiation of the biomaterials of the present invention can be carried out at room temperature with little effect from overheating, despite the fact that samples subjected to gamma irradiation generally tend to overheat and destroy potentially valuable components.

[0287] In some embodiments, gamma irradiation may be carried out at or below about 10° C., preferably on ice (about 0° C.). Within the scope of the present invention, temperatures below about 10° C. include 9.5° C., 8.5° C., 8° C., 7.5° C., 7° C., 6.5° C., 6° C., 5° C., 4° C., 3° ​​C., 2° C., 1° C., 0° C., −1° C., −2° C., −3° C., −4° C., −5° C., −10° C., −20° C., −30° C., −40° C., −50° C., −60° C., −70° C., and −80° C.

[0288] In practice, gamma irradiation is applied to the size (e.g., mm) of the biomaterial to be sterilized. 3 or cm 3 The administration may be over a period of time that depends on the amount (expressed, for example, in mg or g) and / or the dose (expressed, for example, in mg or g).

[0289] In one embodiment, gamma ray irradiation may be carried out for about 10 seconds to about 24 hours, preferably about 5 minutes (300 seconds) to about 12 hours, and more preferably about 10 minutes (600 seconds) to about 3 hours (10,800 seconds). Within the scope of the present invention, the expression "about 10 seconds to about 24 hours" includes 10 seconds, 12 seconds, 14 seconds, 16 seconds, 18 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 1 minute 30 seconds, 2 minutes, 2 minutes 30 seconds, 3 minutes, 3 minutes 30 seconds, 4 minutes, 4 minutes 30 seconds, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, and the like. , 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1 hour 30 minutes, 2 hours, 2 hours 30 minutes, 3 hours, 3 hours 30 minutes, 4 hours, 4 hours 30 minutes, 5 hours, 5 hours 30 minutes, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, and 24 hours.

[0290] In one embodiment, the dry, sterile biomaterial is obtained by freeze-drying and gamma irradiation.

[0291] In some embodiments, the biomaterial is autologous (self-derived). In some embodiments, the biomaterial is allogeneic.

[0292] In some embodiments, the biomaterial is xenogenic. In one embodiment, the biomaterial is derived from an animal, such as a vertebrate, a non-human mammal, or a human.

[0293] The present invention relates to a method for producing a sterile, dry biomaterial comprising devitalized differentiated cells and particulate material, said cells and said particulate material being embedded in an extracellular matrix, said method comprising the steps of: -(1) contacting (i) differentiable living cells with (ii) particulate material to obtain a first combination; - (2) culturing the first combination obtained in step (1) in a culture medium so that the cells secrete an extracellular matrix and synthesize factor contents to obtain tissue regeneration and / or tissue repair properties, wherein the cells and the particulate material are embedded in the extracellular matrix to form a multidimensional structure; - (3) subjecting the multidimensional structure obtained in step (2) to drying to obtain a dried biomaterial; - (4) subjecting said dried biomaterial obtained in step (3) to sterilization, preferably by gamma irradiation, to obtain a sterile, dried biomaterial.

[0294] In some embodiments, the devitalized differentiated cells have regenerative and / or repair properties.

[0295] The present invention also relates to a method for producing a sterile dry biomaterial comprising devitalized differentiated cells having tissue regenerative and / or tissue repair properties and gelatin, said cells and said gelatin being embedded in an extracellular matrix, said method comprising the steps of: -(1) contacting (i) differentiable living cells with (ii) gelatin to obtain a first combination; - (2) culturing the first combination obtained in step (1) in a culture medium so that the cells secrete an extracellular matrix and synthesize factor contents to obtain tissue regeneration and / or tissue repair properties, wherein the cells and the gelatin are embedded in the extracellular matrix to form a multidimensional structure; - (3) subjecting the multidimensional structure obtained in step (2) to drying to obtain a dried biomaterial; - (4) subjecting said dried biomaterial obtained in step (3) to sterilization, preferably by gamma irradiation, to obtain a sterile, dried biomaterial.

[0296] As used herein, the phrase "viable differentiable cells" refers to a population of cells that are capable of differentiating into cells with tissue regenerative and / or tissue repair properties.

[0297] The present invention also relates to a method for producing a sterile, dry biomaterial comprising devitalized osteo- and / or chondrocyte-differentiated cells and particulate material, said cells and said particulate material being embedded in an extracellular matrix, said method comprising the steps of: -(1) contacting (i) viable cells capable of osteogenic and / or chondrogenic differentiation with (ii) particulate material to obtain a first combination; - (2) culturing the first combination obtained in step (1) in a culture medium so that the cells secrete an extracellular matrix and synthesize factor contents to obtain osteogenic and / or chondrogenic properties, wherein the cells and the particulate material are embedded in the extracellular matrix to form a multidimensional structure; - (3) subjecting the multidimensional structure obtained in step (2) to drying to obtain a dried biomaterial; - (4) subjecting said dried biomaterial obtained in step (3) to sterilization, preferably by gamma irradiation, to obtain a sterile, dried biomaterial.

[0298] As used herein, the phrase "viable cells capable of osteogenic and / or chondrogenic differentiation" refers to a population of cells capable of differentiating into cells having osteogenic and / or chondrogenic properties.

[0299] As used herein, the term "embedded in" means "enclosed closely in" or "being an integral part of." In other words, "cells and particulate material embedded in the extracellular matrix" means that the cells, particulate material, and extracellular matrix are intimately associated with one another, with the three components forming a unique structure.

[0300] In some embodiments, the differentiable live cells are selected from the group consisting of primary cells, stem cells, particularly stem cells from adipose tissue, bone marrow, or umbilical cord blood, genetically modified cells, and mixtures thereof. In some embodiments, the primary cells may be cultured in a suitable culture medium to allow for cell proliferation or maintenance.

[0301] In certain embodiments, the stem cells and / or genetically modified cells may be cultured in a culture medium that allows for cellular differentiation into cell populations with tissue regenerative and / or tissue repair properties.

[0302] In certain embodiments, the stem cells and / or genetically modified cells may be cultured in a culture medium that allows for cellular differentiation into cell populations with osteogenic and / or chondrogenic properties.

[0303] In one embodiment, the biomaterial comprises about 10 2 ~about 10 16 cells, preferably about 10 per gram of biomaterial 6 ~about 10 12 Within the scope of the present invention, the expression "about 10 2 ~about 10 16 "10 cells" 2 cells, 5 x l02 cells, 10 3 cells, 5 x l0 3 cells, 10 4 cells, 5 x l0 4 cells, 10 5 cells, 5 x 10 5 cells, 10 6 cells, 5 x l0 6 cells, 10 7 cells, 5 x l0 7 cells, 10 8 cells, 5 x l0 8 cells, 10 9 cells, 5 x l0 9 cells, 10 10 cells, 5 x l0 10 cells, 10 11 cells, 5 x l0 11 cells, 10 12 cells, 5 x l0 12 cells, 10 13 cells, 5 x l0 13 cells, 10 14 cells, 5 x l0 14 cells, 10 15 cells, 5 x l0 15 cells, and 10 16 Contains cells.

[0304] As used herein, "culture medium" refers to the definition generally accepted in the field of cell biology, i.e., any medium suitable for promoting the growth of cells of interest.

[0305] In some embodiments, a suitable culture medium may comprise a chemically defined medium, i.e., a nutrient medium that contains only specific components, preferably components of known chemical structure.

[0306] In some embodiments, the chemically defined medium can be blood-free and / or feeder-free. As used herein, "blood-free" medium refers to a culture medium without added serum. As used herein, "feeder-free" medium refers to a culture medium without added feeder cells.

[0307] Culture media for use in accordance with the present invention may be aqueous media that may contain combinations of substances such as one or more salts, carbon sources, amino acids, vitamins, minerals, reducing agents, buffers, lipids, nucleosides, antibiotics, cytokines, and growth factors.

[0308] Examples of suitable media include, but are not limited to, RPMI medium, Williams' Medium E, Basal Medium Eagle (BME), Eagle's Minimum Essential Medium (EMEM), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-10 medium, Ham's F-12 medium, Kaighn's modified Ham's F-12 medium, DMEM / F-12 medium, and McCoy's 5A medium, which may further contain any of the substances listed above.

[0309] In some embodiments, the media of the present invention may be synthetic media such as Roswell Park Memorial Institute (RPMI) medium or Connaught Medical Research Laboratories (CMRL)-1066 medium.

[0310] In practice, both media may be supplemented with additional additives commonly used in the field. In some embodiments, the additional additives may be intended to promote osteogenesis, chondrogenesis, myogenesis, angiogenesis, neurogenesis, epitheliogenesis, endotheliogenesis, or adipogenesis. In some embodiments, the additional additives may be intended to promote osteogenesis and / or chondrogenesis. Non-limiting examples of suitable additional additives include growth factors, transcription factors, bone cell activating factors, osteoblast activating factors, osteoclast inhibitors, chondrocyte activating factors, etc., and mixtures thereof.

[0311] In fact, temperature, pH, salinity, O 2 and CO 2 Culture parameters, such as the level of ATP, are adjusted based on standards established in the art. Illustratively, the culture temperature for cells according to the present invention may be about 30°C to about 42°C, preferably about 35°C to about 40°C, and more preferably about 36°C to about 38°C. Within the scope of the present invention, the expression "about 30°C to about 42°C" includes 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C.

[0312] In some embodiments, the CO2 level is maintained constant during the course of the culture and ranges from about 1% to about 10%, preferably from about 2.5% to about 7.5%. Within the scope of the present invention, "about 1% to about 10%" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%. In some embodiments, CO 2 during the culture process 2 The level is maintained constant and is in the range of about 1% to about 10%, preferably about 2.5% to about 7.5%. Within the scope of the present invention, the expression "about 1% to about 10%" includes 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10%.

[0313] In one embodiment, step (2) is carried out in the presence of one or more exogenous factors selected from the group comprising growth factors, transcription factors, osteogenic and / or chondrogenic factors, and mixtures thereof.

[0314] Indeed, under appropriate culture conditions, the cells secrete extracellular matrix and synthesize polypeptides and nucleic acids that promote osteogenesis and / or chondrogenesis, which may be considered osteogenic and / or chondrogenic biomarkers and can be monitored at the polypeptide and / or nucleic acid level by the methods described above.

[0315] In some embodiments, the biomaterial further comprises one or more exogenous factors having osteogenic and / or chondrogenic properties selected from the group including growth factors, transcription factors, osteogenic and / or chondrogenic factors, osteoinductive or chondrogenic nucleic acids, and mixtures thereof.

[0316] Here, the biomaterial according to the present invention has osteogenic and / or chondrogenic properties. In practice, the osteogenic and / or chondrogenic properties of a biomaterial can be evaluated by any suitable method available in the art after administration to an individual. Illustratively, biomarkers of osteoinduction can be measured when the biomaterial according to the present invention is administered to an individual. Non-limiting examples of such biomarkers include BMPR-1A, BMPR-2, CSF-1, IGF-1R, RUNX2, SMAD-2, SMAD-3, SMAD-4, SMAD-5, and TWIST-1.

[0317] One aspect of the present invention relates to the sterile dry biomaterial obtained by the method of the present invention.

[0318] Another aspect of the present invention relates to a pharmaceutical composition comprising a biomaterial according to the present invention, and a pharmaceutically acceptable vehicle.

[0319] As used herein, "pharmaceutically acceptable vehicle" refers to any solvents, dispersion media, coatings, antibacterial and / or antifungal agents, isotonic and absorption delaying agents, and the like.

[0320] In practice, the pharmaceutically acceptable vehicle may contain one or more components selected from the group consisting of additives such as polypeptides, amino acids, lipids, and carbohydrates, including sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, and esterified sugars; and polysaccharides or sugar polymers.

[0321] Examples of suitable pharmaceutically acceptable vehicles include polypeptides such as gelatin and casein.

[0322] In some embodiments, the pharmaceutical composition is in the form of a paste or film. In certain embodiments, the paste is a moldable paste. In some embodiments, the moldable paste or film can be easily handled, manipulated, and grafted.

[0323] A further aspect of the present invention relates to a medicament comprising a biomaterial according to the present invention.

[0324] Another aspect of the invention relates to a medical device comprising a biomaterial or pharmaceutical composition according to the invention.

[0325] In some embodiments, the medical device is a dressing for topical application. In some embodiments, the dressing may comprise a woven or nonwoven fabric. In some embodiments, the medical device is coated with / by a composition according to the present invention. In some embodiments, the medical device according to the present invention is configured to allow for controlled release of the pharmaceutical composition. In some embodiments, the medical device is in the form of a patch.

[0326] In some embodiments, the medical device is an implant. In some embodiments, the implant can be in the form of an organic or inorganic scaffold. In certain embodiments, the implant is resorbable.

[0327] The present invention further relates to an implant comprising a multidimensional biomaterial according to the present disclosure. In some embodiments, the implant is allogeneic. In some embodiments, the implant is autologous (self-derived). In some embodiments, the implant is xenogeneic. In some embodiments, the implant is freeze-dried and sterilized, preferably by gamma irradiation.

[0328] The uses and methods may be carried out in vivo or ex vivo.

[0329] In one aspect, the present invention relates to a biomaterial or a pharmaceutical composition according to the present invention for use as a medicament.

[0330] In some embodiments, the present invention relates to a biomaterial or pharmaceutical composition according to the present invention for use in preventing and / or treating tissue damage.

[0331] In some embodiments, the biomaterial or pharmaceutical composition for use in accordance with the present invention is for preventing and / or treating bone and / or cartilage disorders.

[0332] The present invention further relates to the use of a biomaterial or a pharmaceutical composition according to the invention for the manufacture or preparation of a medicament, in particular for the prevention and / or treatment of tissue disorders.

[0333] The present invention further relates to the use of a biomaterial or a pharmaceutical composition according to the invention for the manufacture or preparation of a medicament, and to the use of a biomaterial or a pharmaceutical composition according to the invention for the prevention and / or treatment of bone and / or cartilage disorders.

[0334] The present invention also relates to a method for preventing and / or treating tissue damage in an individual in need thereof, comprising administering a therapeutically effective amount of a biomaterial or pharmaceutical composition according to the present invention.

[0335] The present invention also relates to a method for preventing and / or treating bone and / or cartilage disorders in an individual in need thereof, comprising administering a therapeutically effective amount of a biomaterial or pharmaceutical composition according to the present invention.

[0336] In certain embodiments, the tissue is selected from the group comprising bone tissue, cartilage tissue, skin tissue, muscle tissue, epithelial tissue, endothelial tissue, nervous tissue, connective tissue, and adipose tissue.

[0337] In one embodiment, the term "tissue" includes or consists of defects of bone, cartilage, skin, muscle, endothelium, epithelium, nerve, connective tissue, and adipose tissue.

[0338] In certain embodiments, the tissue disorder is selected from the group comprising: aplasia cutis congenita; burns; cancer, including breast cancer, skin cancer, and bone cancer; compartment syndrome (CS); epidermolysis bullosa; giant congenital nevi; ischemic muscle injuries of the lower extremities; muscle contusions, ruptures, or strains; post-radiation lesions; diabetic ulcers, particularly ulcers including diabetic foot ulcers; arthritis; fractures; bone fragility; Caffey's disease; congenital nonunions; cranial deformities; cranial malformations; delayed union; bone infiltration disorders; hyperostosis; decreased bone mineral density; metabolic bone loss; osteogenesis imperfecta; osteomalacia; osteonecrosis; osteopenia; osteoporosis; Paget's disease; pseudoarthrosis; sclerotic lesions; spina bifida; spondylolisthesis; spondylolysis; chondrodysplasia; costochondritis; enchondroma; hallux rigidus; labral tears of the hip; osteochondrosis dissecans; osteochondrodysplasia; polychondritis; and the like.

[0339] In some embodiments, the tissue disorder is selected from the group comprising: aplasia cutis congenita; burns; cancer, including breast cancer, skin cancer; compartment syndrome (CS); epidermolysis bullosa; giant congenital nevi; ischemic muscle injuries of the lower limbs; muscle contusions, ruptures or strains; post-irradiation lesions; ulcers, including diabetic ulcers, particularly diabetic foot ulcers.

[0340] The term "cancer" as used herein includes solid cancers, particularly cancers including or selected from the group consisting of bone cancer, brain cancer, skin cancer, breast cancer, central nervous system cancer, cervical cancer, upper aerodigestive tract cancer, colorectal cancer (colon cancer), endometrial cancer, germ cell cancer, bladder cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, neuroblastoma, esophageal cancer, ovarian cancer, pancreatic cancer, pleural cancer, prostate cancer, retinoblastoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, and thyroid cancer.

[0341] In some embodiments, the bone disorder is selected from the group consisting of arthritis, bone cancer, bone fracture, bone fragility, Caffey's disease, congenital pseudarthrosis, cranial deformity, cranial malformation, delayed union, bone infiltrative disorders, hyperostosis, decreased bone mineral density, metabolic bone loss, osteogenesis imperfecta, osteomalacia, osteonecrosis, osteopenia, osteoporosis, Paget's disease, pseudoarthrosis, sclerotic lesions, spina bifida, spondylolisthesis, and spondylolysis.

[0342] In certain embodiments, the cartilage disorder is selected from the group comprising arthritis, chondrodysplasia, costochondritis, enchondroma, hallux rigidus, labral tears, osteochondrosis dissecans, osteochondrodysplasia, and polychondritis.

[0343] In one embodiment, the biomaterial or pharmaceutical composition is for use in tissue reconstruction.

[0344] In one embodiment, the tissue reconstruction includes or is selected from the group consisting of bone reconstruction, cartilage reconstruction, dermal reconstruction, muscle or myogenic reconstruction, endothelial reconstruction, epithelial reconstruction, connective tissue reconstruction, nerve reconstruction, and adipogenic reconstruction. Examples of bone and skin reconstruction include, but are not limited to, dermal and / or epidermal reconstruction, wound healing, diabetic ulcer treatment such as diabetic foot ulcers, post-burn lesion reconstruction, post-radiation lesion reconstruction, and reconstruction after breast cancer or breast deformity.

[0345] Examples of cartilage reconstruction include, but are not limited to, knee chondroplasty, nose or ear reconstruction, rib or sternum reconstruction.

[0346] Examples of myogenic reconstruction include, but are not limited to, skeletal muscle reconstruction, reconstruction after abdominal wall injury, reconstruction after ischemic muscle injury of the lower extremities, and reconstruction associated with compartment syndrome (CS).

[0347] Examples of endothelial reconstruction include, but are not limited to, recellularization of vascular patches for vascular anastomoses such as venous arteriosclerosis shunts.

[0348] Examples of lipoplastic reconstruction include, but are not limited to, cosmetic surgery, rejuvenation, and lipofilling reconstruction. Examples of adipogenic reconstruction include, but are not limited to, cosmetic surgery, rejuvenation, and lipofilling reconstruction.

[0349] In some aspects, the present invention relates to biomaterials or pharmaceutical compositions for use according to the present invention for skin reconstruction, preferably for the treatment of skin wounds.

[0350] The present invention also relates to a method for skin reconstruction, preferably for treating a skin wound, in an individual in need thereof, comprising the administration of a therapeutically effective amount of a biomaterial or pharmaceutical composition according to the present invention.

[0351] In one embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention is for use in treating skin tissue disorders. In one embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention is for use in skin reconstruction, including reconstruction of the dermis and / or epidermis. In one embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention is for dermal and / or epidermal reconstruction, wound healing, treatment of diabetic ulcers such as diabetic foot ulcers, reconstruction of post-burn lesions, reconstruction after radiation lesions, reconstruction after breast cancer or breast deformity. In a specific embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention is for use in treating or in treating skin wounds, preferably diabetic skin wounds. In one embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention is for promoting wound closure. In one embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention is for reducing wound thickness, particularly during wound healing.

[0352] In certain embodiments, the biomaterial, pharmaceutical composition, or medical device of the invention is for use in the treatment of or in the treatment of epidermolysis bullosa, giant congenital nevus, and / or congenital aplasia cutis.

[0353] In yet another aspect, the present invention relates to a biomaterial, a pharmaceutical composition, or a medical device of the present invention for use in reconstructive and / or aesthetic surgery.

[0354] In one embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention may be used as an allogeneic or autologous implant, hi one embodiment, the biomaterial, pharmaceutical composition, or medical device of the present invention may be used for tissue grafting.

[0355] In one embodiment, the subject has already been treated for the tissue defect. In another embodiment, the subject has not been treated for the tissue defect.

[0356] In one embodiment, the subject has failed to respond to at least one other treatment for the tissue defect.

[0357] In one embodiment, the subject has diabetes. In one embodiment, the subject suffers from a diabetic wound.

[0358] In another aspect, the invention relates to a biomaterial or pharmaceutical composition for use in accordance with the invention to compensate for side effects of and / or enhance primary treatment of tissue damage.

[0359] The present invention further relates to a method for compensating for side effects of a primary treatment for tissue damage and / or enhancing a primary treatment for tissue damage in an individual in need thereof, comprising the administration of a therapeutically effective amount of a biomaterial or pharmaceutical composition according to the present invention.

[0360] In certain embodiments, the first line treatment may be selected from the group including anti-inflammatory treatments, cancer treatments, ulcer treatments, burn treatments, and the like, and combinations thereof.

[0361] In practice, the biomaterial or pharmaceutical composition according to the invention may be administered before, during or after the primary treatment.

[0362] In one aspect, the present invention relates to a biomaterial or pharmaceutical composition for use in accordance with the present invention that compensates for the side effects of therapeutic treatments known to have deleterious effects on tissue, particularly bone, cartilage, skin, muscle, epithelial, endothelial, nervous, connective, and adipose tissue.

[0363] In certain embodiments, the therapeutic treatment may be selected from the group including anti-inflammatory treatment, cancer treatment, antibiotic treatment, immunotherapy, chemotherapy, etc., and combinations thereof.

[0364] In another aspect, the present invention also relates to a biomaterial or pharmaceutical composition for use according to the present invention for promoting osteogenesis and / or reducing osteoclastogenesis and / or promoting chondrogenesis and / or reducing chondroclastogenesis.

[0365] In some embodiments, the biomaterial may be for further use in promoting angiogenesis. The present invention further relates to a method for promoting osteogenesis and / or reducing osteoclast formation and / or promoting chondrogenesis and / or reducing chondroclast formation in an individual in need thereof, comprising administering a therapeutically effective amount of a biomaterial or pharmaceutical composition according to the present invention.

[0366] Another aspect of the present invention also relates to biomaterials or pharmaceutical compositions for use in accordance with the present invention to inhibit abnormal or dysfunctional bone and / or cartilage formation. In some embodiments, the biomaterials or pharmaceutical compositions of the present invention are for use in restoring abnormal or dysfunctional bone and / or cartilage formation.

[0367] In some embodiments, the individual in need thereof has or is susceptible to developing a bone disorder selected from the group comprising arthritis, bone cancer, bone fracture, bone fragility, Caffey's disease, congenital pseudoarthrosis, cranial deformity, cranial malformation, delayed union, bone infiltrative disorders, hyperostosis, reduced bone mineral density, metabolic bone loss, osteogenesis imperfecta, osteomalacia, osteonecrosis, osteopenia, osteoporosis, Paget's disease, pseudoarthrosis, sclerotic lesions, spina bifida, spondylolisthesis, and spondylolysis.

[0368] In certain embodiments, the individual in need thereof has or is susceptible to developing a cartilage disorder selected from the group including arthritis, chondrodysplasia, costochondritis, enchondroma, hallux rigidus, labral tears, osteochondrosis dissecans, osteochondrodysplasia, and polychondritis.

[0369] Another aspect of the present invention also relates to a biomaterial or pharmaceutical composition for use according to the present invention to compensate for side effects of and / or enhance the first-line treatment of bone and / or cartilage disorders.

[0370] The present invention further relates to a method for compensating for side effects of and / or enhancing primary treatment for bone and / or cartilage disorders in an individual in need thereof, comprising the administration of a therapeutically effective amount of a biomaterial or pharmaceutical composition according to the present invention.

[0371] In some embodiments, the primary treatment may be selected from the group comprising anti-inflammatory treatment, bone cancer treatment, etc., and combinations thereof. In practice, the biomaterial or pharmaceutical composition according to the present invention may be administered before, during, or after the primary treatment.

[0372] Another aspect of the present invention also relates to a biomaterial or pharmaceutical composition for use according to the present invention to compensate for the side effects of therapeutic treatments known to have deleterious effects on bone and / or cartilage.

[0373] In certain embodiments, the therapeutic treatment may be selected from the group including anti-inflammatory treatment, cancer treatment, antibiotic treatment, immunotherapy, chemotherapy, etc., and combinations thereof.

[0374] In one embodiment, the biomaterial or pharmaceutical composition according to the present invention is combined with any one of an isotonic aqueous solution, a scaffold material, another pharmaceutical composition, a medical device, a material of biological origin, and combinations thereof prior to use.

[0375] In some embodiments, the biomaterials or pharmaceutical compositions according to the present invention may be formulated in any suitable form known in the art, such as injectable solutions or suspensions, tablets, coated tablets, capsules, syrups, suppositories, creams, ointments, lotions, gels, and the like.

[0376] In some embodiments, the biomaterials of the present invention may be rehydrated prior to administration. Illustratively, the biomaterials of the present invention may be rehydrated with a sterile saline composition, particularly a sterile saline composition containing about 0.75% to about 1.25% NaCl, more preferably a sterile saline composition containing about 0.90% NaCl.

[0377] In some embodiments, the biomaterial or pharmaceutical composition according to the present invention may be formulated as a putty, emollient, cream, ointment, lotion, gel, salve, controlled release matrix, liposome or lipid particle formulation, microcapsules or nanocapsules, suppository, transdermal delivery system, or combinations thereof.

[0378] In some embodiments, the biomaterial or pharmaceutical composition is in a semi-solid form. In some embodiments, the pharmaceutical composition is in the form of a paste, ointment, cream, plaster, or gel. In some embodiments, the pharmaceutical composition may be in the form of a moldable paste or a manipulable and graftable film.

[0379] In some embodiments, the biomaterial or pharmaceutical composition of the present invention may be processed into a semi-solid form, preferably a paste, together with a suitable excipient. Suitable excipients are, in particular, excipients commonly used to prepare paste bases. Particularly suitable according to the present invention are excipients commonly used to produce gel-like paste bases, such as gel-forming agents. Gel-forming agents are substances that form gels with dispersing agents such as water. Examples of gel-forming agents of the present invention include layered silicates, carrageenans, xanthan gum, acacia gum, alginates, alginic acid, pectins, modified celluloses, or poloxamers.

[0380] In some embodiments, the biomaterial or pharmaceutical composition in semi-solid form, preferably in paste form, is ready for use, hi other embodiments, the pharmaceutical composition in semi-solid form, preferably in paste form, must be prepared extemporaneously.

[0381] In some embodiments, the content of factors, including miRNA, contained in the biomaterials of the present invention is encapsulated, i.e., immobilized in a vesicle system. In one embodiment, the encapsulation is bilayer encapsulation. In another embodiment, the encapsulation is monolayer encapsulation. In yet another embodiment, the encapsulation is matrix encapsulation.

[0382] In one embodiment, the vesicles encapsulating the miRNA-containing factor content are composed of biopolymers. In another embodiment, the vesicles encapsulating the miRNA-containing factor content are extracellular vesicles. In a specific embodiment, the vesicles encapsulating the miRNA-containing factor content are exosomes. In some embodiments, the exosomes are cell-derived exosomes, preferably exosomes from which the miRNA-containing factor content is derived. In another specific embodiment, the exosomes are engineered exosomes.

[0383] Exosome engineering may be performed by any suitable method known in the art or adapted therefrom, see, for example, "Exosome engineering: Current progress in cargo loading and targeted delivery" (Fu et al., Nanoimplant, 2020, Volume 20, 100261).

[0384] According to one embodiment, the biomaterial, pharmaceutical composition, medicament or medical device of the present invention is administered by any suitable route, including: enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual; intratracheal instillation, bronchial instillation, and / or inhalation; and / or oral spray, nasal spray, and / or aerosol.

[0385] According to one embodiment, the biomaterial, pharmaceutical composition, medicament, or medical device of the present invention is administered locally or by surgical implantation. In one embodiment, the biomaterial, pharmaceutical composition, medicament, or medical device is administered at the site of tissue damage.

[0386] In one embodiment, the biomaterial, pharmaceutical composition, agent or medical device is administered to the site of the bone and / or cartilage disorder.

[0387] In some embodiments, biomaterials or medical devices according to the present invention are combined with one or more components for bioprinting the biomaterial or medical device. As used herein, the term "bioprinting" refers to a technique that allows for the creation of three-dimensional structures that mimic natural tissues and / or organs. In some embodiments, the one or more components include natural polymers such as cellulose, gelatin, alginate, and chitosan, and synthetic polymers such as polyvinyl polymers, polyethylene glycol polymers, etc.

[0388] For examples of suitable bioprinting processes, reference may be made to Aljohani et al. (Internat. J. Biol. Macromol., Volume 107, Part A, 2018, p261-275), Daly et al. (Adv Healthc Mater. 2017 Nov;6(22)); Gu et al. (Adv Exp Med Biol. 2018;1078:15-28), Mataiet al. 30 (Biomaterials. 2020 Jan;226:119536).

[0389] The present invention further relates to a multidimensional biomaterial comprising differentiated human mesenchymal stem cells (MSCs), a particulate material, an extracellular matrix, vascular endothelial growth factor (VEGF), and insulin-like growth factor 1 (IGF-1), wherein the differentiated MSCs are devitalized, the devitalized MSCs and the particulate material are embedded in the extracellular matrix, and the multidimensional biomaterial is enriched with exosomes or exosome-like vesicles containing one or more nucleic acids that promote normal tissue differentiation and / or inhibit abnormal tissue differentiation.

[0390] In some embodiments, the biomaterial is three-dimensional. In some embodiments, the mesenchymal stem cells are adipose tissue-derived stem cells (ASCs). In some embodiments, the ASCs are late-passage ASCs. In some embodiments, the MSCs are devitalized by lyophilization. In some embodiments, the biomaterial is sterilized, optionally by gamma irradiation. In some embodiments, the lyophilized and sterilized biomaterial retains its three-dimensional structure. In some embodiments, the VEGF and IGF-1 are biologically active. In some embodiments, the biomaterial comprises at least 10 ng of VEGF per gram of biomaterial. In some embodiments, the biomaterial comprises at least 60 ng of VEGF per gram of biomaterial. In some embodiments, the biomaterial comprises at least 20 ng of IGF-1 per gram of biomaterial. In some embodiments, the biomaterial comprises at least 40 ng of IGF-1 per gram of biomaterial. In some embodiments, the extracellular matrix comprises one or more matrisome proteins secreted by differentiated MSCs and specific to soft tissue or mineralized tissue. In some embodiments, the soft tissue is skin tissue. In some embodiments, the mineralized tissue is bone. In some embodiments, the extracellular matrix is ​​secreted by differentiated MSCs and comprises collagen. In some embodiments, the particulate material is selected from the group consisting of demineralized bone particles, gelatin particles, and ceramic particles. In some embodiments, the particulate material is demineralized bone particles. In some embodiments, the particulate material is gelatin particles. In some embodiments, the particulate material is ceramic particles. In some embodiments, the ceramic particles are calcium phosphate particles. In some embodiments, the calcium phosphate particles are hydroxyapatite (HA) and / or β-tricalcium phosphate (β-TCP) particles. In some embodiments, the one or more nucleic acids are one or more microRNAs (miRs). In some embodiments, the one or more miRs are selected from the group consisting of miR-210-3p and hsa-miR-361-3p. In some embodiments, the one or more miRs are miR-210-3p.In certain embodiments, miR-210-3p promotes bone formation. In some embodiments, the one or more miRs is hsa-miR-361-3p.

[0391] Another aspect of the present invention also relates to a method for producing a multidimensional biomaterial according to the present disclosure, said method comprising: (a) culturing the isolated human MSCs using a proliferation medium; (b) differentiating the MSCs using a differentiation medium; (c) adding the particulate material to the differentiating MSCs and culturing the MSCs with the differentiation medium for an additional period to generate a multidimensional tissue; and (d) freeze-drying the multidimensional tissue to produce a multidimensional biomaterial.

[0392] In some embodiments, the method further comprises the step of (e) sterilizing the multidimensional biomaterial, optionally by gamma irradiation. In some embodiments, the MSCs are adipose-derived stem cells (ASCs). In some embodiments, step (c) differentiates the MSCs into a cell type selected from the group consisting of osteoblasts, chondrocytes, keratinocytes, myofibroblasts, endothelial cells, and adipocytes. In some embodiments, the particulate material is selected from the group consisting of demineralized bone particles, gelatin particles, and ceramic particles.

[0393] The present invention further relates to a multidimensional biomaterial obtainable by the method according to the present disclosure.

[0394] Another aspect of the present invention also relates to a medical device comprising a multidimensional biomaterial according to the present disclosure or an implant according to the present disclosure. Another aspect of the present invention relates to a medical device comprising a multidimensional biomaterial according to the present disclosure or an implant according to the present disclosure.

[0395] The present invention also relates to a kit comprising a multidimensional biomaterial according to the present disclosure or an implant according to the present disclosure and suitable fixation means.

[0396] Another aspect of the present invention relates to a pharmaceutical composition comprising a multidimensional biomaterial according to the present disclosure and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0397] [Figure 1A] Figures 1A and 1B are a set of graphs showing cell viability (expressed as luminescence (RLU)) in NVDX2 (Figure 1A) and NVDX3 (Figure 1B) compared with ASC viability at various cell concentrations of 1%, 10%, 50%, and 100% (Tukey's test, n=3-6). Average: RLU 3.5 for NVDX2, RLU -91.9 for NVDX3, RLU 6820.3 for 10% ASC, and RLU 2235 for 1% ASC. **: p<0.01, ***: p<0.001, ns=not significant. [Figure 1B] Figures 1A and 1B are a set of graphs showing cell viability (expressed as luminescence (RLU)) in NVDX2 (Figure 1A) and NVDX3 (Figure 1B) compared with ASC viability at various cell concentrations of 1%, 10%, 50%, and 100% (Tukey's test, n=3-6). Average: RLU 3.5 for NVDX2, RLU -91.9 for NVDX3, RLU 6820.3 for 10% ASC, and RLU 2235 for 1% ASC. **: p<0.01, ***: p<0.001, ns=not significant. [Figure 2A] Figures 2A and 2B are a set of graphs showing glucose consumption (in mmol) by NVDX2 (Figure 2A) and NVDX3 (Figure 2B) compared with glucose consumption by ASC at various cell concentrations of 1%, 10%, 50%, and 100% (Fisher's LSD test, n=3-6). Mean: -0.0031 mmol for NVDX2, 0.0020 mmol for 1% ASC. Mean: -0.0011 mmol for NVDX3, 0.0018 mmol for 10% ASC, and 0.0020 mmol for 1% ASC. *: p<0.05, **: p<0.01, ***: p<0.001. [Figure 2B]Figures 2A and 2B are a set of graphs showing glucose consumption (in mmol) by NVDX2 (Figure 2A) and NVDX3 (Figure 2B) compared with glucose consumption by ASC at various cell concentrations of 1%, 10%, 50%, and 100% (Fisher's LSD test, n=3-6). Mean: -0.0031 mmol for NVDX2, 0.0020 mmol for 1% ASC. Mean: -0.0011 mmol for NVDX3, 0.0018 mmol for 10% ASC, and 0.0020 mmol for 1% ASC. *: p<0.05, **: p<0.01, ***: p<0.001. [Figure 3A] Figures 3A and 3B are a series of graphs showing lactate production (in mmol) by NVDX2 (Figure 3A) and NVDX3 (Figure 3B) compared with lactate production by ASC at various cell concentrations of 1%, 10%, 50%, and 100% (Fisher's LSD test, n = 3-6). ***: p < 0.001, ****: p < 0.0001, ns = not significant. [Figure 3B] Figures 3A and 3B are a series of graphs showing lactate production (in mmol) by NVDX2 (Figure 3A) and NVDX3 (Figure 3B) compared with lactate production by ASC at various cell concentrations of 1%, 10%, 50%, and 100% (Fisher's LSD test, n = 3-6). ***: p < 0.001, ****: p < 0.0001, ns = not significant. [Figure 4A] 4A-4D are a series of plots showing the water content (in %) of NVD002 (FIG. 4A), NVDX2 (FIGS. 4A and 4B), NVD003 (FIG. 4C), and NVDX3 (FIGS. 4C and 4D). [Figure 4B] 4A-4D are a series of plots showing the water content (in %) of NVD002 (FIG. 4A), NVDX2 (FIGS. 4A and 4B), NVD003 (FIG. 4C), and NVDX3 (FIGS. 4C and 4D). [Figure 4C]4A-4D are a series of plots showing the water content (in %) of NVD002 (FIG. 4A), NVDX2 (FIGS. 4A and 4B), NVD003 (FIG. 4C), and NVDX3 (FIGS. 4C and 4D). [Figure 4D] 4A-4D are a series of plots showing the water content (in %) of NVD002 (FIG. 4A), NVDX2 (FIGS. 4A and 4B), NVD003 (FIG. 4C), and NVDX3 (FIGS. 4C and 4D). [Figure 5] Graph showing the amount of VEFG (expressed in ng / g of biomaterial) in the biomaterials NVD002 (fresh), NVD002 lyo (lyophilized), and NVDX2 (lyophilized and sterilized) obtained from 3D induction in the presence of gelatin. [Figure 6] Graph showing IGF-1 amounts (expressed in ng / g of biomaterial) in biomaterials NVD002 (fresh), NVD002 lyo (lyophilized), and NVDX2 (lyophilized and sterilized) obtained from 3D induction in the presence of gelatin. [Figure 7] FIG. 1 is a graph showing the amount of SDF-1α (expressed in ng / g of biomaterial) in the biomaterials NVD002 (fresh), NVD002 lyo (lyophilized), and NVDX2 (lyophilized and sterilized) obtained from 3D induction in the presence of gelatin. [Figure 8] Graph showing the total protein content (expressed in arbitrary units) in the biomaterials NVD002 (fresh), NVD002 lyo (lyophilized), and NVDX2 (lyophilized and sterilized) obtained from 3D induction in the presence of gelatin. [Figure 9A] Figures 9A and 9B are a series of graphs showing the relative expression of miR-199-5p (Figure 9A) and miR-361-3p (Figure 9B) in NVD00X2 (lyophilized NVD002) and NVDX2 (lyophilized and gamma-irradiated NVD002). [Figure 9B] Figures 9A and 9B are a series of graphs showing the relative expression of miR-199-5p (Figure 9A) and miR-361-3p (Figure 9B) in NVD00X2 (lyophilized NVD002) and NVDX2 (lyophilized and gamma-irradiated NVD002). [Figure 10A] Figures 10A and 10B are a series of graphs showing CD3 mobilization in the ischemic and non-ischemic hindlimb. Mean values ​​± SD include rats treated once and twice overall (no difference between the two "treatment" groups). Figure 10A: CD3 mobilization in the ischemic hindlimb. Figure 10B: CD3 mobilization in the non-ischemic hindlimb. These means were obtained by several counts performed on HE-stained histological slides in the periphery and center of the wound area and mixed together (CD3+ cells / mm2). [Figure 10B] Figures 10A and 10B are a series of graphs showing CD3 mobilization in the ischemic and non-ischemic hindlimb. Mean values ​​± SD include rats treated once and twice overall (no difference between the two "treatment" groups). Figure 10A: CD3 mobilization in the ischemic hindlimb. Figure 10B: CD3 mobilization in the non-ischemic hindlimb. These means were obtained by several counts performed on HE-stained histological slides in the periphery and center of the wound area and mixed together (CD3+ cells / mm2). [Figure 11A] Figures 11A and 11B are a series of graphs showing CD68 mobilization in the non-ischemic hindlimb. Mean values ​​± SD (no difference between the two "treatment" groups) include rats treated once and twice globally with NVDX2. Figure 11A: CD68 mobilization in the ischemic hindlimb. Figure 11B: CD68 mobilization in the non-ischemic hindlimb. These mean values ​​were obtained by multiple counts performed on HE-stained histological slides in the periphery and center of the wound area and mixed together (CD68+ cells / mm2). [Figure 11B] Figures 11A and 11B are a series of graphs showing CD68 mobilization in the non-ischemic hindlimb. Mean values ​​± SD (no difference between the two "treatment" groups) include rats treated once and twice globally with NVDX2. Figure 11A: CD68 mobilization in the ischemic hindlimb. Figure 11B: CD68 mobilization in the non-ischemic hindlimb. These mean values ​​were obtained by multiple counts performed on HE-stained histological slides in the periphery and center of the wound area and mixed together (CD68+ cells / mm2). [Figure 12A] Figures 12A and 12B are a series of photographs obtained by scanning electron microscopy showing the microstructure of fresh (NVD003) (Figure 12A) and freeze-dried non-irradiated (NVDX3) (Figure 12B) biomaterials obtained according to Example 1 after 3D induction in the presence of HA / β-TOR. The upper panels represent microscopic views obtained at 25x zoom. The lower panels represent microscopic views obtained at 1,200-1,300x zoom. [Figure 12B] Figures 12A and 12B are a series of photographs obtained by scanning electron microscopy showing the microstructure of fresh (NVD003) (Figure 12A) and freeze-dried non-irradiated (NVDX3) (Figure 12B) biomaterials obtained according to Example 1 after 3D induction in the presence of HA / β-TOR. The upper panels represent microscopic views obtained at 25x zoom. The lower panels represent microscopic views obtained at 1,200-1,300x zoom. [Figure 13A] Figures 13A and 13B are a series of graphs showing the expression profiles of the genes VEGFA (Figure 13A) and VEGFB (Figure 13B) in the biomaterials NVD003 and NVD003l1yo compared to HA / β-TCP. *: p<0.05. [Figure 13B] Figures 13A and 13B are a series of graphs showing the expression profiles of the genes VEGFA (Figure 13A) and VEGFB (Figure 13B) in the biomaterials NVD003 and NVD003l1yo compared to HA / β-TCP. *: p<0.05. [Figure 14A] Figures 14A-14D are a series of graphs showing the expression profiles of genes SMAD2 (Figure 14A), SMAD3 (Figure 14B), SMAD4 (Figure 14C), and SMAD5 (Figure 14D) in biomaterials NVD003 and NVD003 lyo compared to HA / β-TCP. *: p<0.05, **: p<0.01. [Figure 14B]Figures 14A-14D are a series of graphs showing the expression profiles of genes SMAD2 (Figure 14A), SMAD3 (Figure 14B), SMAD4 (Figure 14C), and SMAD5 (Figure 14D) in biomaterials NVD003 and NVD003 lyo compared to HA / β-TCP. *: p<0.05, **: p<0.01. [Figure 14C] Figures 14A-14D are a series of graphs showing the expression profiles of genes SMAD2 (Figure 14A), SMAD3 (Figure 14B), SMAD4 (Figure 14C), and SMAD5 (Figure 14D) in biomaterials NVD003 and NVD003 lyo compared to HA / β-TCP. *: p<0.05, **: p<0.01. [Figure 14D] Figures 14A-14D are a series of graphs showing the expression profiles of genes SMAD2 (Figure 14A), SMAD3 (Figure 14B), SMAD4 (Figure 14C), and SMAD5 (Figure 14D) in biomaterials NVD003 and NVD003 lyo compared to HA / β-TCP. *: p<0.05, **: p<0.01. [Figure 15A] Figures 15A-15C are a series of graphs showing the expression profiles of the genes ITGAV (Figure 15A), ITGB1 (Figure 15B), and VCAM1 (Figure 15C) in the biomaterials NVD003 and NCD003 lyo compared to HA / β-TCP. *: p<0.05. [Figure 15B] Figures 15A-15C are a series of graphs showing the expression profiles of the genes ITGAV (Figure 15A), ITGB1 (Figure 15B), and VCAM1 (Figure 15C) in the biomaterials NVD003 and NCD003 lyo compared to HA / β-TCP. *: p<0.05. [Figure 15C] Figures 15A-15C are a series of graphs showing the expression profiles of the genes ITGAV (Figure 15A), ITGB1 (Figure 15B), and VCAM1 (Figure 15C) in the biomaterials NVD003 and NCD003 lyo compared to HA / β-TCP. *: p<0.05. [Figure 16A]Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16B] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16C] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16D] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16E]Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16F] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16G] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16H] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16I]Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16J] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 16K] Figures 16A to 16K are a series of graphs showing the expression profiles of the genes ACVR1 (Figure 16A), BMPRIA (Figure 16B), BMPR1B (Figure 16C), BMPR2 (Figure 16D), CSF1 (Figure 16E), EGFR (Figure 16F), FGFR1 (Figure 16G), IGF1R (Figure 16H), RUNX2 (Figure 16I), TGFBR1 (Figure 16J), and TWIST1 (Figure 16K) in the biomaterials NVD003 and NVD0031yo obtained from 3D induction in the presence of HA / / β-TOR. [Figure 17A] Figures 17A and 17B are a series of graphs showing the relative miRNA expression profiles of a subset of miRNAs (Figure 17A: hsa-miR-4485-3p; Let-7i-5p; hsa-miR-24-3p; hsa-miR-210-3p; Figure 17B: miR-4454; miR-619-5p; miR-3607-5p; miR-3653-5p) in fresh NVD003 (a) and freeze-dried NVD003 (b) biomaterials. [Figure 17B]Figures 17A and 17B are a series of graphs showing the relative miRNA expression profiles of a subset of miRNAs (Figure 17A: hsa-miR-4485-3p; Let-7i-5p; hsa-miR-24-3p; hsa-miR-210-3p; Figure 17B: miR-4454; miR-619-5p; miR-3607-5p; miR-3653-5p) in fresh NVD003 (a) and freeze-dried NVD003 (b) biomaterials. [Figure 18A] Figures 18A and 18B are a series of graphs showing the relative expression of miR-210-3p (Figure 18A) and miR-24-3p (Figure 18B) in fresh NVD003 and lyophilized and gamma-irradiated NVD003 (NVDX3). [Figure 18B] Figures 18A and 18B are a series of graphs showing the relative expression of miR-210-3p (Figure 18A) and miR-24-3p (Figure 18B) in fresh NVD003 and lyophilized and gamma-irradiated NVD003 (NVDX3). [Figure 19] 1 is a graph showing the content of OPG, IGF1 and VEGF (expressed in ng / g of biomaterial) in the freeze-dried biomaterial NVD003 upon irradiation with 12 kGy or 25 kGy at room temperature (RT) or −80° C. (−80). [Figure 20] Graph showing the relative expression of hsa-miR-210-3p in biomaterials NVD003 lyo and NVDX3 (gamma-irradiated freeze-dried biomaterial NVD003). [Figure 21A] Figures 21A and 21B are a series of graphs showing the level of inhibition of osteoclast formation (Figure 21A) or mature osteoclasts (Figure 21B) obtained versus the dose (mg) of biomaterial NVD003 obtained from 3D induction in the presence of HA / / β-TOR. [Figure 21B] Figures 21A and 21B are a series of graphs showing the level of inhibition of osteoclast formation (Figure 21A) or mature osteoclasts (Figure 21B) obtained versus the dose (mg) of biomaterial NVD003 obtained from 3D induction in the presence of HA / / β-TOR. [Figure 22A]Figures 22A and 22B are a series of graphs showing the level of inhibition of osteoclast formation (Figure 22A) or mature osteoclasts (Figure 22B) obtained versus the dose (mg) of HA / β-TCP. [Figure 22B] Figures 22A and 22B are a series of graphs showing the level of inhibition of osteoclast formation (Figure 22A) or mature osteoclasts (Figure 22B) obtained versus the dose (mg) of HA / β-TCP. [Figure 23A] Figures 23A and 23B are a series of graphs showing the level of inhibition of osteoclast formation (Figure 23A) or mature osteoclasts (Figure 23B) obtained versus the dose (mg) of biomaterial NVDX3 obtained from 3D induction in the presence of HA / / β-TCP. [Figure 23B] Figures 23A and 23B are a series of graphs showing the level of inhibition of osteoclast formation (Figure 23A) or mature osteoclasts (Figure 23B) obtained versus the dose (mg) of biomaterial NVDX3 obtained from 3D induction in the presence of HA / / β-TCP. [Figure 24A] Figures 24A and 24B are a series of graphs showing the relative induction of BGLAP (osteocalcin) (Figure 24A) or SPP-1 (osteopontin) (Figure 24B) obtained for adipose stem cells in osteogenic differentiation medium ("MD"; ​​control group) in the presence of sclerotin (SCL) for 10 days with or without the biomaterial NVDX3. [Figure 24B] Figures 24A and 24B are a series of graphs showing the relative induction of BGLAP (osteocalcin) (Figure 24A) or SPP-1 (osteopontin) (Figure 24B) obtained for adipose stem cells in osteogenic differentiation medium ("MD"; ​​control group) in the presence of sclerotin (SCL) for 10 days with or without the biomaterial NVDX3. [Figure 25]1 is a graph showing the viability of adipose stem cells in osteogenic differentiation medium in the presence or absence of 10 ng / ml (SCL10) or 100 ng / ml (SCL100) of sclerotin and in the presence or absence of the biomaterial NVDX3. Viability is expressed as a percentage compared to the viability of adipose stem cells in osteogenic differentiation medium supplemented with 0.5% human platelet lysate (% vs. MD0.5%). [Figure 26A] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26B]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26C] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26D]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26E] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26F]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26G] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26H]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26I] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26J]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26K] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26L]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26M] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26N]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26O] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26P]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26Q] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26R]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26S] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26T]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26U] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26V]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26W] 26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 26X]26A-26X show the effects of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), TWIST (FIG. 26J), and BMPR1A (FIG. 26C) on the expression of RUNX-2 (FIG. 26A), BGLAP (FIG. 26B), BMPR1A (FIG. 26C), SMAD5 (FIG. 26D), SMAD2 (FIG. 26E), SPP-11 (FIG. 26F), CSF-1 (FIG. 26G), EGFR (FIG. 26H), and TWIST (FIG. 26J) as a function of time (7 days (7J) or 14 days (14J)) and dose of the biomaterial NVDX3 (5 mg, 20 mg, or 100 mg). 26A-26C are a series of graphs showing the relative levels (expressed as fold induction) of 1 (FIG. 26I), TGFB-1 (FIG. 26J), TGFB2 (FIG. 26K), SMAD4 (FIG. 26L), ITGA1 (FIG. 26M), ITGA3 (FIG. 26N), ICAM1 (FIG. 26O), HIF1a (FIG. 26P), THBS1 (FIG. 26Q), leptin (FIG. 26R), MMP-2 (FIG. 26S), EDN1 (FIG. 26T), ENG (FIG. 26U), EFNA1 (FIG. 26V), VEGFA (FIG. 26W), and EFNB2 (FIG. 26X). The control condition (C or CTL) was performed using differentiation medium without the biomaterial NVDX3. [Figure 27A] Figures 27A-27D are a series of plots showing cytotoxicity (Figure 27A), viability (Figure 27B), LDH content (Figure 27C), and DNA content (Figure 27D) as a percentage of 10 mg, 20 mg, 40 mg, 100 mg, 200 mg of NVDX3 or 200 mg of HA / βTOR, Triton, or differentiation medium (MD). [Figure 27B] Figures 27A-27D are a series of plots showing cytotoxicity (Figure 27A), viability (Figure 27B), LDH content (Figure 27C), and DNA content (Figure 27D) as a percentage of 10 mg, 20 mg, 40 mg, 100 mg, 200 mg of NVDX3 or 200 mg of HA / βTOR, Triton, or differentiation medium (MD). [Figure 27C] Figures 27A-27D are a series of plots showing cytotoxicity (Figure 27A), viability (Figure 27B), LDH content (Figure 27C), and DNA content (Figure 27D) as a percentage of 10 mg, 20 mg, 40 mg, 100 mg, 200 mg of NVDX3 or 200 mg of HA / βTOR, Triton, or differentiation medium (MD). [Figure 27D]Figures 27A-27D are a series of plots showing cytotoxicity (Figure 27A), viability (Figure 27B), LDH content (Figure 27C), and DNA content (Figure 27D) as a percentage of 10 mg, 20 mg, 40 mg, 100 mg, 200 mg of NVDX3 or 200 mg of HA / βTOR, Triton, or differentiation medium (MD). [Figure 28A] Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 28B] Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 28C] Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 28D]Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 28E] Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 28F] Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 28G] Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 28H]Figures 28A-28H are a series of graphs showing the relative levels (expressed as fold induction) of BMPR1A (Figure 28A), CSF-1 (Figure 28B), IGF1R (Figure 28C), TWIST1 (Figure 28D), SMAD2 (Figure 28E), SMAD3 (Figure 28F), SMAD4 (Figure 28G), and SMAD5 (Figure 28H) one month after implantation of HA / TCP, NVD003, or NVDX3 biomaterials obtained from 3D induction in the presence of HA / / β-TCP. [Figure 29A] Figures 29A and 29B are a series of graphs showing the time course of median anti-HLA IgM (Figure 29A) or anti-HLA IgG (Figure 29B) antibody levels in the serum of transplanted female Wistar rats over time (1, 3, 7, 15, and 30 days after transplantation) with the biomaterials NVD003 or NVDX3 obtained from 3D induction in the presence of HA / / β-TCP. [Figure 29B] Figures 29A and 29B are a series of graphs showing the time course of median anti-HLA IgM (Figure 29A) or anti-HLA IgG (Figure 29B) antibody levels in the serum of transplanted female Wistar rats over time (1, 3, 7, 15, and 30 days after transplantation) with the biomaterials NVD003 or NVDX3 obtained from 3D induction in the presence of HA / / β-TCP. [Figure 30A] Figures 30A and 30B are a series of plots showing the cell viability (expressed as percentages) of HDFa (Figure 30A) and ASC (Figure 30B) incubated for 48 hours in the presence of 10 μM dexamethasone (GC: glucocorticoid) with or without three different doses (20 mg or 50 mg) of NVDX2 and measured by metabolic activity (CCK-8 assay) (n=2, t-test). [Figure 30B]Figures 30A and 30B are a series of plots showing the cell viability (expressed as percentages) of HDFa (Figure 30A) and ASC (Figure 30B) incubated for 48 hours in the presence of 10 μM dexamethasone (GC: glucocorticoid) with or without three different doses (20 mg or 50 mg) of NVDX2 and measured by metabolic activity (CCK-8 assay) (n=2, t-test). [Figure 31A] Figures 31A and 31B are a series of plots showing DNA quantification (expressed as percentages) of HDFa (Figure 31A) and ASC (Figure 31B) incubated for 48 hours in the presence of 10 μM dexamethasone (GC: glucocorticoid) with or without two different doses (20 or 50 mg) of NVDX2 (n=2, t-test). [Figure 31B] Figures 31A and 31B are a series of plots showing DNA quantification (expressed as percentages) of HDFa (Figure 31A) and ASC (Figure 31B) incubated for 48 hours in the presence of 10 μM dexamethasone (GC: glucocorticoid) with or without two different doses (20 or 50 mg) of NVDX2 (n=2, t-test). [Figure 32A] Figures 32A and 32B are a series of plots showing the proliferation (Figure 32A) and linear regression (Figure 32B) of human osteosarcoma cell H143B in the absence (black curve) or presence of NVD002-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 32A: Proliferation is expressed as percent viability (DO) versus the time of co-culture of cells with exosomes. Figure 32B: Results are expressed as the percentage of live cells vs. the negative control (no exosomes) at each time point. **: p<0.01, ***: p<0.005, ****: p<0.0001, no statistical difference. [Figure 32B]Figures 32A and 32B are a series of plots showing the proliferation (Figure 32A) and linear regression (Figure 32B) of human osteosarcoma cell H143B in the absence (black curve) or presence of NVD002-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 32A: Proliferation is expressed as percent viability (DO) versus the time of co-culture of cells with exosomes. Figure 32B: Results are expressed as the percentage of live cells vs. the negative control (no exosomes) at each time point. **: p<0.01, ***: p<0.005, ****: p<0.0001, no statistical difference. [Figure 33A] Figures 33A and 33B are a series of plots showing the proliferation (Figure 33A) and linear regression of proliferation loss (Figure 32B) of human osteosarcoma cells H143B in the absence (black curve) or presence of NVD003-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 33A: Proliferation is expressed as percent viability (DO) versus the time of co-culture of cells with exosomes. Figure 33B: Results are expressed as the ratio (%) of live cells to the negative control group (no exosomes) at each time point. *: p<0.05, **: p<0.01, -: no statistical difference. [Figure 33B] Figures 33A and 33B are a series of plots showing the proliferation (Figure 33A) and linear regression of proliferation loss (Figure 32B) of human osteosarcoma cells H143B in the absence (black curve) or presence of NVD003-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 33A: Proliferation is expressed as percent viability (DO) versus the time of co-culture of cells with exosomes. Figure 33B: Results are expressed as the ratio (%) of live cells to the negative control group (no exosomes) at each time point. *: p<0.05, **: p<0.01, -: no statistical difference. [Figure 34A]Figures 34A and 34B are a series of plots showing linear regression of proliferation (Figure 34A) and loss of proliferation (Figure 34B) of human melanoma cells A375 in the absence (black curve) or presence of NVD002-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 34A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 34B: Results are expressed as the ratio (%) of live cells to the negative control (no exosomes) at each time point. *: p<0.05, **: p<0.01, ****: p<0.0001, -, 00: p<0.01, 0000: p<0.0001, -, no statistical difference. [Figure 34B] Figures 34A and 34B are a series of plots showing linear regression of proliferation (Figure 34A) and loss of proliferation (Figure 34B) of human melanoma cells A375 in the absence (black curve) or presence of NVD002-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 34A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 34B: Results are expressed as the ratio (%) of live cells to the negative control (no exosomes) at each time point. *: p<0.05, **: p<0.01, ****: p<0.0001, -, 00: p<0.01, 0000: p<0.0001, -, no statistical difference. [Figure 35A] Figures 35A and 35B are a series of plots showing linear regression of proliferation (Figure 35A) and proliferation loss (Figure 35B) of human melanoma cells A375 in the absence (black curve) or presence of NVD003-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 35A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 35B: Results are expressed as the ratio (%) of live cells to the negative control group (no exosomes) at each time point. ***: p<0.005, ****: p<0.0001, 0000: p<0.0001, -: no statistical difference. [Figure 35B]Figures 35A and 35B are a series of plots showing linear regression of proliferation (Figure 35A) and proliferation loss (Figure 35B) of human melanoma cells A375 in the absence (black curve) or presence of NVD003-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 35A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 35B: Results are expressed as the ratio (%) of live cells to the negative control group (no exosomes) at each time point. ***: p<0.005, ****: p<0.0001, 0000: p<0.0001, -: no statistical difference. [Figure 36A] Figures 36A and 36B are a series of plots showing linear regression of proliferation (Figure 36A) and proliferation loss (Figure 36B) of human glioblastoma cells E187 in the absence (black curve) or presence of NVD002-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 36A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 36B: Results are expressed as the ratio (%) of live cells to the negative control (no exosomes) at each time point. *: p<0.05, ****: p<0.0001, 0000: p<0.0001, no statistical difference. [Figure 36B] Figures 36A and 36B are a series of plots showing linear regression of proliferation (Figure 36A) and proliferation loss (Figure 36B) of human glioblastoma cells E187 in the absence (black curve) or presence of NVD002-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 36A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 36B: Results are expressed as the ratio (%) of live cells to the negative control (no exosomes) at each time point. *: p<0.05, ****: p<0.0001, 0000: p<0.0001, no statistical difference. [Figure 37A]Figures 37A and 37B are a series of plots showing linear regression of proliferation (Figure 37A) and loss of proliferation (Figure 37B) of human glioblastoma cells U87 in the absence (black curve) or presence (light gray curve) of NVD003-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 37A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 37B: Results are expressed as the ratio (%) of live cells to the negative control (no exosomes) at each time point. ***: p<0.005, ****: p<0.0001, 0000: p<0.0001, -: no statistical difference. [Figure 37B] Figures 37A and 37B are a series of plots showing linear regression of proliferation (Figure 37A) and loss of proliferation (Figure 37B) of human glioblastoma cells U87 in the absence (black curve) or presence (light gray curve) of NVD003-Exosomes at 2.5 μg / ml (dark gray curve) and 25 μg / ml (light gray curve). Figure 37A: Proliferation is expressed as percent viability (DO) versus time of co-culture of cells with exosomes. Figure 37B: Results are expressed as the ratio (%) of live cells to the negative control (no exosomes) at each time point. ***: p<0.005, ****: p<0.0001, 0000: p<0.0001, -: no statistical difference. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0398] The present invention is further illustrated by the following examples.

[0399] Example 1: Production of a biomaterial according to the present invention a) Isolation of hASCs Human subcutaneous adipose tissue was harvested by liposuction according to the Coleman method in the abdominal region after informed consent and serological screening.

[0400] Human adipose tissue-derived stem cells (hASCs) were rapidly isolated from the incoming adipose tissue. Lipoaspirate can be stored at +4°C for 24 hours or at -80°C for longer periods.

[0401] First, a fraction of the lipoaspirate was separated for quality control purposes, and the remaining volume of the lipoaspirate was measured. The lipoaspirate was then digested with collagenase solution (NB1, Serva Electrophoresis® GmbH, Heidelberg, Germany) prepared with HBSS (final concentration: 8 μl / mL). The volume of enzyme solution used for digestion was twice the volume of the adipose tissue. This digestion was carried out at 37°C ± 1°C for 50–70 min. The first round of intermittent shaking was performed after 15–25 min, and the second round of intermittent shaking was performed after 35–45 min. The digestion was stopped by the addition of MP medium (growth medium or growth medium). MP medium consisted of DMEM medium (4.5 g / L glucose and 4 mM AlA-Gin; Sartorius Stedim Biotech®, Göttingen, Germany) supplemented with 5% human platelet lysate (hPL) (v / v). DMEM is a standard medium containing salts, amino acids, vitamins, pyruvate, and glucose, buffered with a carbonate buffer, and has a physiological pH (7.2-7.4). The DMEM used contained Ala-Gin. Human platelet lysate (hPL) is a rich source of growth factors used to stimulate the in vitro growth of mesenchymal stem cells (e.g., hASCs).

[0402] The digested adipose tissue was centrifuged (500 x g, 10 min, 20°C) and the supernatant was removed. The pelleted stromal vascular fraction (SVF) was resuspended in MP medium and passed through a 200-500 μm mesh filter. The filtered cell suspension was centrifuged again (500 x g, 10 min, 20°C). The pellet containing hASCs was resuspended in MP medium. A small portion of the cell suspension was kept for cell counting, and the entire remaining cell suspension was used to make one 75 cm 2 T-flasks (referred to as passage P0) were seeded with 1000 ng / ml of PBS containing ...

[0403] On the day after the separation process (day 1), 75 cm 2The growth medium was removed from the T-flasks. The cells were rinsed three times with phosphate buffer, and then freshly prepared MP medium was added to the flasks.

[0404] b) Growth and expansion of human adipose tissue-derived stem cells During the expansion phase, hASCs were passaged four times (P1, P2, P3 and P4) to obtain sufficient amounts of cells for subsequent steps in the process.

[0405] Between P0 and the fourth passage (P4), cells were cultured in T-flasks and fed with fresh MP medium. Cells were passaged when they reached 70%–100% confluence (target confluence: 80–90%). All recipient cells from one batch were passaged simultaneously. At each passage, TrypLE (Select 1×; 75 cm 2 9 mL for flask, 150 cm 2 Cells were detached from the culture vessel using recombinant animal-free cell-dissociation enzyme (12 mL for flasks). TrypLe digestion was carried out at 37°C ± 2°C for 5–15 min and terminated by the addition of MP medium.

[0406] The cells were then centrifuged (500xg, 5 min, 20°C) and resuspended in MP medium. To ensure a homogenous cell suspension, the harvested cells were pooled. After resuspension, the cells were counted.

[0407] At passages P1, P2, and P3, the remaining cell suspension was then diluted to the appropriate cell density with MP medium and plated onto larger tissue culture surfaces. 2 The flask was seeded with 15 mL of cell suspension and 150 cm 2 Each flask was seeded with 30 mL of cell suspension. At each passage, 0.5 × 10 cells were 4 ~0.8×10 4 cells / cm 2The cells were seeded at 100°C. Between different passages, the culture medium was changed every 3–4 days. Cell behavior and growth rate may vary slightly from donor to donor. Therefore, the duration between two passages and the number of medium changes between passages may vary from donor to donor.

[0408] c) osteogenic differentiation At passage P4 (i.e., the fourth passage), cells were centrifuged twice and resuspended in MD medium (differentiation medium). After resuspension, cells were counted a second time before diluting to the appropriate cell density with MD medium, and 70 mL of cell suspension was collected at 150 cm. 2 The cells were seeded in flasks and fed with osteogenic MD medium. According to this method, the cells were directly cultured in osteogenic MD medium after the fourth passage. Therefore, osteogenic MD medium was added before the cells reached confluence.

[0409] Osteogenic MD medium consisted of growth medium (DMEM, Ala-Gln, hPL 5%) supplemented with dexamethasone (1 μM), ascorbic acid (0.25 mM), and sodium phosphate (2.93 mM).

[0410] Cell behavior and growth rate may vary slightly from donor to donor, therefore the duration of the osteogenic differentiation process and the number of medium changes between passages may vary from donor to donor.

[0411] d) Multidimensional cell guidance Multidimensional induction of ASCs was initiated when cells reached confluence, morphological changes appeared, and at least one osteoid nodule (i.e., the unmineralized organic portion of the bone matrix that forms before the maturation of bone tissue) was observed in the flask.

[0412] -3D induction with gelatin particles After exposure to osteogenic MD medium, culture vessels containing confluent monolayers of adherent osteogenic cells were inoculated with gelatin particles (Cultispher-S, Percell Biolytica, Åstrup, Sweden) at 150 cm 2 1.5cm for a container 3The solution was dispersed slowly and evenly at a concentration of .

[0413] Cells were maintained in MD medium. Periodic medium changes were performed every 3–4 days during multidimensional induction. These medium changes were performed carefully to prevent the removal of gelatin particles and allow the structures to develop.

[0414] After approximately 15 days, scaffold-free 3D cultures (NVD-002 biomaterial) were developed and separated from the T-flask. Cultures were maintained for 5-8 weeks after particle addition with medium changes every 3-4 days.

[0415] -3D induction by HA / Zβ-TCP particles (ceramic particles) After exposure to osteogenic MD medium, HA / b-TCP particles (60 / 40 ratio) were added to the culture vessel containing a confluent monolayer of adherent osteogenic cells at a density of 150 cm. 2 3 cc / cm for flasks (Biomatlante®, France) 2 Spread it slowly and evenly.

[0416] Cells were maintained in MD medium. Periodic medium changes were performed every 3–4 days during multidimensional induction. These medium changes were performed carefully to prevent the removal of ceramic material particles and allow the structure to develop.

[0417] After approximately 15 days, scaffold-free 3D cultures (biomaterial NVD-003) were developed and detached from the T-flasks. The cultures were maintained for 5-8 weeks after particle addition with medium changes every 3-4 days.

[0418] e) Freeze-drying and gamma irradiation The resulting biomaterials (hereafter referred to as NVD002 (gelatin) and NVD003 (HA / TCP)) were further freeze-dried (NVD002 lyo and NVD003 lyo) or freeze-dried and further sterilized to obtain dry sterile biomaterials, hereafter referred to as NVDX2 and NVDX3, respectively.

[0419] Freeze-drying was performed by sublimation of fresh biomaterials at −80°C under vacuum for at least 24 hours (<0.05 mBar, −50°C, 24–36 hours).

[0420] Sterilization was carried out by subjecting the freeze-dried biomaterial to a dose of about 12 kGy to about 25 kGy (for 730 seconds) at about 20°C to about -80°C.

[0421] Example 2: Viability of cells in biomaterials NVDX2 and NVDX3 1. Materials and Methods The biopsy tissues of NVDX 2 or NVDX 3 were compared with the concentration of ASC, and the biopsy rate of the biopsy tissues was determined. To determine the viable cell content in the material, NVDX2 or NVDX3 biopsies were compared with known concentrations of ASC corresponding to 100%, 50%, 10%, and 1% viable cells.

[0422] NVDX2 and NVDX3 were placed in 2 ml of differentiation medium (MD) for 24 hours. The cell numbers of the NVD003 and NVD002 biopsies were counted, yielding 2.1 x 10 cells per 300 mg of tissue. 6 The doubling time of ASCs was estimated at approximately 30 hours, which represented 100% of the viable cells.

[0423] Viable cells were measured in two different ways: 1) To estimate viable cells in ASCs and in NVDX2 and NVDX3 biopsies, a cell viability assay (CellTiter-Glocell® viability assay) was performed after 24 hours of culture. The CellTiter-Glocell® luminescent cell viability assay is a uniform method for measuring the number of viable cells in culture based on the quantification of intracellular ATP, which indicates the presence of metabolically active cells. 2) Quantitative measurements of glucose or lactate in the differentiation medium after 24 hours of culture were performed using a CedexBio® analyzer. Glucose measurement was based on the rate of NADPH formation and was directly proportional to the glucose concentration in the medium. Lactate measurement was based on the production of a dye and was directly proportional to the L-lactate concentration in the medium.

[0424] Statistical analysis was performed with PrismGraphPad2 using Tukey's test (all pairwise comparisons) and Fisher's LSD test (standalone comparisons). *: p-value < 0.05, **: p-value < 0.01, ***: p-value 0.001, ns = not significant.

[0425] 2.Results 2.1 Survival rate After lyophilization and gamma irradiation, the presence of viable cells was measured using the Cell Titer-GloCell® Viability Assay. This method is based on estimating the number of viable cells in culture based on the quantification of intracellular ATP, which indicates the presence of metabolically active cells (viable cells). Cells served as a source of ATP, and the resulting luminescence was proportional to the number of viable cells. As shown in Figures 1A and 1B, the percentage of viable cells in NVDX2 and NVDX3 was significantly lower, less than 10%.

[0426] 2.2 Glucose consumption After freeze-drying and gamma irradiation, glucose consumption in the culture medium, which reflects viable cell consumption, was measured using a CedexBio analyzer. This technique is based on measuring glucose in the medium consumed by viable cells.

[0427] In the presence of hexokinase (HK), glucose was phosphorylated by ATP to glucose 6-phosphate (G-6-P), which was then oxidized by NADPH in the presence of glucose-6-phosphate dehydrogenase (G-6-PDH). The rate of NADPH formation was measured by UV photometry and was directly proportional to the glucose concentration.

[0428] Glucose + ATP → glucose-6P + ADP Glucose-6P+NADP + → Gluconate-6P + NADPH + H +

[0429] Glucose consumption by NVDX2 was less than that of 1% of viable cells (Figure 2A), and glucose consumption by NVDX3 was less than that of 10% of viable cells (Figure 2B).

[0430] 2.3 Lactic acid production After freeze-drying and gamma irradiation, lactate production in the culture medium, which reflects part of the viable cell metabolism, was measured using a CedexBio analyzer. The principle of this technique is based on the measurement of lactate production in the medium by viable cells. L-lactate was oxidized by lactate oxidase (LaOD) to pyruvate and H2O2, which in the presence of peroxidase (POD) produced a dye. The photometric absorbance of the dye was directly proportional to the L-lactate concentration in the medium.

[0431] L-Lactic acid + O2 → Pyruvate + H2O2 H2O2 + H donor + 4-AAP → H2O + chromogen

[0432] The percentage of viable cells in NVDX2 has been shown to be close to 10% ASC (Figure 3A).

[0433] However, this value is relative to the volume loaded into the well and may not reflect the actual amount of lactate produced. NVDX2, which contains gelatin beads as a component, is highly porous and absorbs the liquid present in the medium, resulting in less than 2 mL of residual liquid. This reduction in effective liquid volume affected the estimation of the presence of lactate in the medium compared to the presence of lactate in the medium + ASC. Lactate was concentrated, which led to an overestimation of the remaining viable cells. Therefore, we estimated that the percentage of remaining viable cells was less than 10% in NVDX2, yielding 0.0037 ± 0.0010 mmol of lactate with NVDX2 and 0.0030 ± 0.0008 mmol of lactate with 10% ASC (Figure 3A).

[0434] Lactate production from 10% of ASCs was estimated to be 0.0030 mmol (±0.0008 mmol), but was only 0.0014 mmol (±0.001 mmol) (Figure 3B). However, due to the small sample size, the difference was not significant.

[0435] 2.4 Conclusion NVD002 and NVD003 were large, moldable structural compounds of ASCs entrapped in an extracellular matrix and Cultispher-S or HA / β-TOR particles, respectively. After freeze-drying and gamma irradiation, the structure of both 3D grafts (NVDX2 and NVDX3, respectively) was significantly altered.

[0436] After viability testing and considering the balance of major cellular metabolites, the viable cell content in the lyophilizates was less than 10%, and probably up to 1% for both NVDX2 and NVDX3 materials.

[0437] Example 3: Effect of freeze-drying on the water content of biomaterials NVD002 and NVD003 The objective of this study was to evaluate the effect of freeze-drying on the moisture content of NVD002 and NVD003.

[0438] 1. Materials and Methods a) Biopsy Residual moisture content was assessed using a moisture meter by comparing biopsies from NVD002 and NVD003 with biopsies from freeze-dried NVD002 (NVD00X2) and freeze-dried NVD003 (NVD00X3), respectively. Analyses were performed on NVD002 and NVD00X2 from one donor, NVD003 from one donor, and NVD00X3 from two donors.

[0439] b) Moisture analysis A moisture analyzer (Ohaus® moisture analyzer MB120) was used to measure the moisture content of the samples. The principle of this technique is to first measure the weight of the sample. The sample is then rapidly heated in a dryer unit to evaporate the moisture. During drying, the instrument continuously measures the weight of the sample. Once drying is complete, the results are expressed as % moisture content.

[0440] NVD002, NVD003, NVD00X2, and NVD00X3 were weighed, then the products were heated with infrared light until the samples no longer lost weight, and the moisture content was calculated. The total weight loss was used to calculate the moisture content.

[0441] 2.Results NVD002 was a malleable, translucent 3D sheet-like structure without a scaffold. NVD003 was a moldable, whitish-yellow 3D structure without a scaffold. After freeze-drying, NVD00X2 was pink-brown like skin and dried like flakes. NVD00X3 was a whitish-yellow dry powder. After freeze-drying, the moisture content was measured using a moisture meter MB120OHAUS®. The principle of this technique is to first measure the weight of the sample. The sample was rapidly heated in a dryer unit to evaporate the moisture. The weight of the sample was continuously measured during drying. Once drying was complete, the results were expressed as moisture content (%).

[0442] The water content (%) was approximately 90% for fresh NVD002 biomaterial (Figure 4A) and approximately 55% for fresh biomaterial NVD003 (Figure 4C). After lyophilization, the water content was less than 5%, i.e., less than 2.5% for NVD00X2 (Figures 4A and 4B) and less than 0.25% for NVD00X3 (Figures 4C-4D). Good lyophilizates were observed.

[0443] Example 4: Characterization of the biomaterials NVD002, NVD002 lyo and NVDX2 obtained after 3D induction in the presence of gelatin 1. Materials and Methods a) mRNA was isolated from biopsies. mRNA was extracted using the miRNeasy Kit Master Mix (Qiagen®, Hilden, Germany) according to the manufacturer's protocol. RNA concentration was measured using a Nanodrop (Therm oFisher®, Waltham, MA, USA).

[0444] b) To quantify miRNA expression, 50 ng of RNA was reverse transcribed into cDNA using the qScript miRNA cDNA Synthesis Kit (Quanta Biosciences®), and qRT-PCR was performed in triplicate using Perfecta SYBR Green Super Mix (Quanta Biosciences®). Thermal cycling was performed on an Applied Biosystems 7900HT Detection System (Applied Biosystems®). Data were normalized to miR-16-5p and U6 small nuclear RNA using the ΔΔCt method.

[0445] c) Exosomes were isolated from the culture medium by differential centrifugation, whereby larger "contaminants" were first filtered out by increasing the centrifugation speed to pellet exosomes, small extracellular vesicles, and even protein aggregates, before being pelleted at very high speeds (~100,000xg).

[0446] 2.Results

[0447] [Table 13-1]

[0448] [Table 13-2]

[0449] [Table 14]

[0450] [Table 15]

[0451] [Table 16]

[0452] Compared to 2D culture, the miRNA content of the biomaterials obtained from the present invention was altered. For example, exosomal and cellular hsa-miR-210-3p and hsa-let-7i-5p were both upregulated, while exosomal hsa-miR-664b-3p and hsa-miR-664b-5p, and cellular hsa-miR-4485-3p and hsa-miR-6723-5p were downregulated. This suggests that freeze-drying and sterilization affect the properties of the biomaterial NVDX2 compared to fresh, non-dried, non-frozen biomaterials.

[0453] Example 5: Characterization of the compositions NVD002, NVD002 lyo and NVDX2 obtained from 3D induction in the presence of gelatin 1. Materials and Methods Samples of "fresh" biomaterial (NVD002), dried biomaterial (NVD002 lyo), and dried and sterilized biomaterial (NVDX2) in the presence of gelatin obtained in Example 1 were processed for protein and miRNA extraction.

[0454] a) Protein extraction Proteins were extracted from non-irradiated and irradiated samples with guanidine HCl (4 M), benzamidine (5 mM), N-ethylmaleimide (10 mM), and PMSF (1 mM) for 24 hours at 4°C, followed by Tris-HCl (50 mM) at pH 7.4 for 5 hours (all from Sigma-Aldrich®, St. Louis, MO, USA) and purified on a desalting column (PD10 de GE Healthcare®, Chicago, MO, USA). Quantification of growth factor VEGF, IGF-1, and SDF-1α content was performed by ELISA (Human VEGF quantitation ELISA Kit, Human SDF-1 quantitation ELISA Kit, Human IGF-1 quantitation ELISA Kit; R&D Systems®, Minneapolis, MN, USA).

[0455] Protein levels of VEGF and SDF-1 were upregulated in biomaterial NVDX2 compared to both fresh biomaterial NVD002 and dried biomaterial NVD002 lyo (see Figures 5 and 7, respectively). Meanwhile, protein levels of IGF-1 in biomaterials NVD002 and NVDX2 were comparable and upregulated compared to biomaterial NVD002 lyo (see Figure 6). Finally, total protein levels in these three biomaterials were globally similar (see Figure 8).

[0456] b) miRNA extraction and RT-PCR miRNA was extracted using the miRNeasy Kit Master Mix (Qiagen®, Hilden, Germany) according to the manufacturer's protocol. RNA concentration was measured by Nanodrop (ThermoFisher®, Waltham, MA, USA).

[0457] For quantification of miRNA expression, 50 ng RNA was reverse transcribed into cDNA using the qScript miRNA cDNA Synthesis Kit (Quanta Biosciences®), and qRT-PCR was performed in triplicate using PerfectaSYBR® Green Supermix (Quanta Biosciences®).

[0458] Thermal cycling was performed on an Applied Biosystems® 7900HT Detection System (Applied Biosystems®). Data were normalized to miR-16-5p and U6 small nuclear RNA using the ΔΔCt method.

[0459] 2.Results As shown in Figures 9A and 9B, no significant differences were observed in the expression of miR-199-5p and miR-361-3p between NVD00X2 (lyophilized NVD002) and NVDX2 (lyophilized and gamma-irradiated NVD002), respectively.

[0460] Example 6: Efficacy of biomaterial NVDX2 in hyperglycemia and ischemia models The purpose of this study was to evaluate the efficacy of the biological powder NVDX2 (human-derived) used as a wound dressing in the treatment of ischemic / hyperglycemic wounds in a Wistar rat model. NVDX2 is a lyophilized, gamma-irradiated version of NVD002, a scaffold-free 3D graft composed of a mixture of human adipose tissue-derived stem cells (ASCs) and porcine gelatin beads (Cultispher® S, Percell) embedded in an extracellular matrix produced by the ASCs.

[0461] 1. Materials and Methods a) Experimental design The efficacy of NVDX2 was evaluated in a xenogeneic (human-to-rat) model of ischemic (vs. non-ischemic) wound in hyperglycemic Wistar rats (n=13). The 13 hyperglycemic rats were divided into two subgroups according to the number of NVDX2 applications to the wound: one application on day 1 (×1), and two applications on days 1 and 7 (×2).

[0462] Surgical procedure This in vivo study was approved by the Ethics Committee of the CER-Groupe, Department of Biotechnology, B6900 AYE, Belgium. The protocol was developed by the Department of Plastic, Reconstructive and Cosmetic Surgery, "University of Geneva", University of Geneva, Geneva Medical School, Geneva, Switzerland (Andre-Levigne et al., Wound Repair and Regeneration. 2016 - Alizadeh et al., Wound Repair and Regeneration. 2007). b) Surgical procedures This in vivo experiment was approved by the Ethics Committee of the CER-Groupe (Biotechnology Department, B6900 AYE, Belgium). This protocol was inspired by a model developed by the Department of Plastic, Reconstructive, and Cosmetic Surgery at the "Hopitaux Universitaires de Genève" Faculty of Medicine, University of Geneva, Geneva, Switzerland (André-Levigne et al., Wound Repair and Regeneration. 2016; Alizadeh et al., Wound Repair and Regeneration. 2007).

[0463] Thirteen male Wistar rats weighing approximately 250-300 g were used in this study. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ) [50 mg / kg] to rats with a minimum body weight of 250 g. Animals exhibiting blood glucose levels above 9 mM were considered hyperglycemic and enrolled in the study. Of the 13 rats of origin, all were enrolled. Seven to 10 days after STZ injection, the hyperglycemic rats were surgically operated on to induce a unilateral ischemia model. This model was obtained by resecting a portion of the femoral artery in the rat's left hind limb (from the groin to the knee). This model allowed for the presence of an ischemic (left hind) and a non-ischemic (right hind) limb in the context of hyperglycemia in each individual rat. Once the artery was resected, the incision was sutured, and an ALZET 2ML2 pump delivering buprenorphine [0.3 mg / ml] at 5 μl / h was administered subcutaneously to the rat's back. Then, the skin on the sole of the foot is removed down to the tendon, and 1cm 2 The wound was made and photographed using the measuring device as a reference. The test item was then applied to the wound. One rat died during surgery.

[0464] For non-ischemic limbs, the wound was covered by pouring NVDX2 directly onto the wound. For ischemic limbs, several drops of sterile physiological solution were applied (2-3 drops) to allow NVDX2 (powder) to "glue" onto the "dry wound." Once the test item was placed on the affected area, a bandage was created by applying one layer of Tegaderm® followed by another layer of adhesive tape, and a specific collar was placed around the rat.

[0465] c) Follow-up Animals were subjected to daily clinical follow-up, and following the course of wound healing, rats were euthanized, both hind paws were photographed, and these paws were amputated and placed in 10% formol for histological analysis. For most rats, interim photographs were taken three times a week between postoperative days 13 and 37.

[0466] d) Final Steps Animals were sacrificed by a lethal intraperitoneal injection of pentobarbital.

[0467] e) Macroscopic assessment of wound healing Wound healing kinetics was assessed by measuring wound area on photographs of the paw taken from day 0 to day 37.

[0468] To quantify wound closure, wound area was measured by image analysis using Image J software by two independent operators. The remaining wound area was calculated based on the wound area measured at each time point between D0 and D37 and expressed relative to the wound area at a fixed 100% time of implantation (D0).

[0469] To account for wound contraction and epithelialization, wound area was divided into different components of the wound: 1) initial wound (blue area), 2) hairless closed wound (white; epithelialized wound). Wounds closed by contraction were calculated by subtracting the areas of the hairless closed and non-closed wounds from the total wound area.

[0470] f) Histopathological and 2D histomorphometric analysis The lower limb was dissected to remove wound tissue, and the latest section was oriented laterally to provide full-thickness histological slides. Histological slides were prepared at 5 PM and stained with HE, TM, CD3, CD68, KU80, and a-SMA.

[0471] To assess immune and inflammatory responses, CD3 (T lymphocytes) and CD68 (macrophages) immunostaining was performed. The number of CD3- and CD68-positive cells was manually counted using NDPview2 software. A region of interest was manually delineated to define the area of ​​the "implant site."

[0472] To evaluate the pro-angiogenic properties of the implanted tissue, quantification of the area occupied by blood vessels (Masson's Trichrome staining) was performed: Regions of interest were manually delineated based on tissue characteristics to define the area of ​​the "implant site." Each blood vessel was manually delineated, and the area occupied by the vessel in the region of interest was quantified. The number of blood vessels and their corresponding surface were reported relative to the total area of ​​the "implant site." KU80 staining was performed to highlight the presence of human cells at the "implant site." aSMA immunostaining was performed to quantify the presence of smooth muscle fibers responsible for wound contraction. Smooth muscle fibers were identified within approximately 2.9 mm. 2 The wound thickness was measured on histological slides to assess the formation of hypertrophic scars.

[0473] g) Image analysis Histological slides were examined using NDP.view.2 (Hamamatsu Photonics). Image analysis was performed using ImageJ2 (NIH).

[0474] 2.Results Of the 13 rats that received streptozotocin injections, all were hyperglycemic (blood glucose levels >9 mM) and selected for the study, but only one developed surgical complications and died during surgery and was therefore excluded from the study.

[0475] To confirm the maintenance of hyperglycemic state and to assess the possibility of weight loss due to this hyperglycemic state, both blood glucose and body weight changes were closely followed. Clinical blood glucose follow-up showed that all rats had blood glucose levels above 9 mM during all in vivo phases of the study (up until euthanasia).

[0476] It was observed that after STZ injection (first or second), all rats lost some weight, reaching a critical weight loss of 20% compared with the weight measured before the first STZ injection.

[0477] a) Macroscopic assessment of wound healing Macroscopic photographs of the wounds were taken during follow-up and at the end of the study.

[0478] In the non-ischemic limbs treated with NVDX2, better wound healing was observed from post-surgery day 15 (D15), with total wound closure estimated around post-surgery day 22 and 23 (average of all surviving rats). A delay in the wound healing process was observed in the ischemic limbs treated with NVDX2 from post-surgery day 15 (D15), with complete wound closure estimated at post-surgery day 31 (average of all surviving rats).

[0479] The kinetics of wound closure was measured by photographing the wound. The wound surface remained constant for the first 7–8 days after NVDX2 treatment, but wound healing was observed on days 13–16, accompanied by a reduction in the wound surface. Complete and irreversible wound closure in the non-ischemic limb was estimated on days 22–23 after NVDX2 treatment (one or two NVDX2 applications). Complete and irreversible wound closure in the ischemic limb was estimated on day 31 after NVDX2 treatment (one or two NVDX2 applications) (Table 17).

[0480] [Table 17]

[0481] b) Microscopic assessment of wound healing Histological slides stained with hematoxylin and eosin were examined for each animal on the day of sacrifice. In the two rats sacrificed early, full-thickness wounds were visible on day 2, and granulation tissue development was observed on day 15. Test items were clearly visible up to day 15, but only some particles were visible at later time points.

[0482] Each wound was found to be fully healed with a complete epithelial layer by up to 36 / 37 days (with the exception of the ischemic limb of one rat (2x treatment); no data available for one rat (1x treatment) that was sacrificed on day 49).

[0483] In both the ischemic and nonischemic limbs, a complete epithelial layer was observed 28 days after surgery in one rat (2× treatment) and 29 days after surgery in one rat (1× treatment). Compared with the subepithelial layer in the ischemic limb of one rat (1× treatment) at 29 days, the subepithelial layer was not completely reconstituted at 28 days in the ischemic limb of one rat (2× treatment).

[0484] c) Lymphocyte CD3 recruitment and macrophage CD68 recruitment CD3 and CD68 mobilization in nonischemic and ischemic wounds at each sacrifice time point are shown in Figures 10A and 10B, and 11A and 11B, respectively. Note that the number of rats at each "time point" varies. One rat was analyzed on day 2, one on day 15, two on days 28 / 29, and seven on days 36 / 37.

[0485] For both treated limbs, mild and transient CD3+ mobilization was observed in the non-ischemic limb beginning on day 15 (Figure 10A), peaking on days 28 / 29, and decreasing significantly on days 36 / 37. In the ischemic limb (Figure 10B), peaking on days 28 / 29 and decreasing slightly on days 36 / 37.

[0486] The different treatment groups were differentiated and the results are shown in Tables 18 and 19. The mean ± SD indicated the differentiation between rats treated with NVDX2 once and twice. On HE-stained histological slides, the peripheral and central areas of the wound area were analyzed and mixed together (CD3+ cells / mm 2 ) The average value was obtained by counting several times.

[0487] [Table 18]

[0488] [Table 19]

[0489] From Tables 19 and 20, differences in CD3+ cell mobilization at day 28 / 29 were observed between rats treated once with NVDX2 and rats treated twice with NVDX2 in both treated limbs: 29.4 CD3+ cells / mm for the ischemic and non-ischemic limbs, respectively. 2 vs224,0CD3+ cells / mm 2 , and 59,7 CD3+ cells / mm 2 vs284,2CD3+ cells / mm 2 .

[0490] This difference could be explained by an immune response to NVDX2 during the second application, which occurred one week after the first application. This second application of NVDX2 triggered an immune response that led to typical graft rejection.

[0491] This difference observed on days 28 / 29 appeared to resolve on days 36 / 37, with similar numbers of CD3+ cells / mm3 in both treatment groups, with 98.8 vs. 69.9 (1xNVDX2) vs. 86.3 vs. 48.9 (2xNVDX2) and 51.8 vs. 25.3 (1xNVDX2) vs. 47.6 vs. 18.1 (2xNVDX2) for the ischemic and non-ischemic limbs, respectively. 2 Data on days 36 / 37 were obtained from 5 and 3 rats for 1xNVDX2 and 2xNVDX2 applications, respectively.

[0492] In both treated limbs, an increase in CD68+ mobilization was observed from day 15 onwards for both treated limbs (Figures 11A and 11B). This increase continued or was maintained afterwards, reaching 546.9 ± 376.9 CD68+ cells / mm for the ischemic and non-ischemic limbs, respectively. 2 , and 437.2 ± 174.6 CD68+ cells / mm 2 was thought to reach

[0493] Tables 20 and 21 summarize the results obtained after differentiation of the different treatment groups. Mean values ​​± SD represent the differentiation of rats treated once with NVDX2 from rats treated twice with NVDX2. These measures were performed on HE-stained histological slides in the periphery and center of the wound area and mixed together (CD68+ cells / mm 2 ) The average value was obtained by counting several times.

[0494] [Table 20]

[0495] [Table 21]

[0496] From Tables 20 and 21, similarities in CD68+ cell mobilization at day 28 / 29 were observed between rats treated once with NVDX2 and rats treated twice with NVDX2 in both treated limbs: 477.1 CD68+ cells / mm for the ischemic and non-ischemic limbs, respectively. 2 vs413,2CD68+ cells / mm 2 , and 404,0 CD68+ cells / mm 2 vs375,9CD68+ cells / mm 2 . CD68+ cells / mm 2 This similarity in terms of β was also observed on days 36 / 37, with 582.3 ± 430.1 (1 × NVDX2) vs 546.6 ± 203.9 (2 × NVDX2) and 582.3 ± 430.1 (1 × NVDX2) vs 546.6 ± 203.9 (2 × NVDX2) for the ischemic and non-ischemic limbs, respectively.

[0497] This CD68 recruitment did not differ between the two treatment groups (1×NVDX2 vs. 2×NVDX2), which could be explained by a typical response to a foreign substance.

[0498] 3. Conclusions and Discussion The objective of this study was to evaluate the efficacy of a biological freeze-dried and gamma-irradiated dressing, NVDX2 (human origin), in the treatment of ischemic / hyperglycemic wounds in a Wistar rat model.

[0499] This recognized deep ischemic / hyperglycemic wound model was chosen because it is a highly rigorous model of hypoxic wounds with impaired angiogenesis, such as those seen in diabetic patients. Indeed, in most cases, chronic wounds are the result of severe tissue ischemia, particularly in diabetic patients or smokers. Ischemia has been shown to decrease fibroblast replication, collagen production, increase collagen degradation, and reduce wound contraction (Hunt et al., Surg Gynecol Obstet 1972; Steinbrech et al., J Surg Res 1999; Yamanaka et al., J Dermatol Sci 2000; Alizadeh et al., Wound Repair and Regeneration 2007).

[0500] Furthermore, hyperglycemia exponentially worsens the adverse effects of ischemia on wound repair, particularly wound contraction and myofibroblast differentiation (Tobalem et al., Plast Reconstr Surg Glob Open 2015). Similarly, in vitro hypoxia inhibits myofibroblast differentiation (Modarressi et al., J Invest Dermatol 2010). In this study using NVDX2, 13 male Wistar rats were intraperitoneally injected with 50 mg / kg STZ. The animals' hyperglycemic state was confirmed by blood glucose levels above 9 mM, and ischemia was induced by ligation of the femoral artery of one hind limb before NVDX2 implantation.

[0501] Wound closure kinetics, based on macroscopic assessment of the wound, were measured using photographs of the wound. The wound surface remained constant for the first 7–8 days after NVDX2 treatment, but wound healing was observed on days 13–16, accompanied by a reduction in the wound surface. Complete and irreversible wound closure in the non-ischemic limb was estimated on days 22–23 after NVDX2 treatment (one or two NVDX2 applications). Complete and irreversible wound closure in the ischemic limb was estimated on day 31 after NVDX2 treatment (one or two NVDX2 applications).

[0502] Each wound was found to be fully healed with complete epithelialization in up to 36 / 37 days. Complete epithelialization was observed on both the ischemic and non-ischemic limbs at 28 and 29 days post-surgery.

[0503] Macrophage infiltration was observed in the NVDX2 groups (1xNVDX2 and 2xNVDX2) from day 15 after implantation until the total wound closure time, shown at days 36 / 37. This macrophage recruitment, known as a typical response to foreign bodies, was associated with transient T lymphocyte recruitment, which in some cases was observed in the dermis until day 37, indicating an immune response to the implantation product. The peak of T lymphocyte recruitment was observed on days 28 / 29, and showed completely different profiles following one or two NVDX2 applications. This difference could be explained by an immune response to NVDX2 during the second application, which occurred one week after the first application. This second application of NVDX2 triggered an immune response, leading to classic graft rejection. These data demonstrated the efficacy of NVDX2 in a rigorous xenogeneic model of hyperglycemic and ischemic deep wounds.

[0504] Example 7: Comparative analysis by scanning electron microscope between fresh biomaterial (NVD003) obtained in Example 1 using HA / / β-TCP particles and freeze-dried non-irradiated biomaterial (NVD0031yo) Samples of the biomaterial NVD003 and freeze-dried NVD003 (NVD0031yo) biomaterial were fixed in 2% glutaraldehyde for 2 hours and then washed three times with PBS (3 x 10 min). The samples were dehydrated in baths of increasing concentrations of ethanol (10%, 30%, 50%, 60%, 70%, 80%, and 100%) for 15 minutes in each bath. The samples were dried using a CPD critical drying device and mineralized with gold. Finally, the samples were observed using a JEOL7600F SEM (JEOL®, Japan).

[0505] Figure 12A shows the structure of biomaterial NVD003, and Figure 12B shows the structure of biomaterial NVD0031yo. NVD0031yo has a similar microscopic structure to NVD003; however, when viewed at 25x magnification, the average particle size was significantly smaller. Furthermore, both materials appeared to be aggregated particles, with aggregation being achieved by the extracellular matrix and ceramic material. In biomaterial NVD003, all particles appeared to be linked by the ECM. In contrast, NVD0031yo biomaterial appeared to be composed of multiple particle clusters linked by the ECM.

[0506] Example 8: Effects of NVD003 and NVD0031yo on gene expression of bone marrow MSCs in vitro The objective of this study was to evaluate the ability of the NVD003 and NVD0031yo biomaterials to induce osteoblast differentiation.

[0507] 1. Materials and Methods Bone marrow mesenchymal stem cells (BMSCs) were cultured in basal medium (DMEM supplemented with 5% FBS) with or without 100 or 500 mg of HA / bTCP, 100 or 500 mg of NVD003, or 100 or 500 mg of NVD0031yo. A positive control for osteoblast differentiation (MD) was performed by culturing MSCs in osteoblast differentiation medium (DMEM supplemented with 5% FBS, BMP-2 (100 ng / mL), ascorbic acid (50 μg / mL), and β-glycerophosphate (10 mM).

[0508] Treatments were performed in 6-well plates and applied three times over 7 or 14 days. Cells were detached and frozen in 10% FBS / DMSO on days 7 and 14. Supernatants were also collected during medium refreshment and at the end of treatment and stored at -20°C.

[0509] One of the three replicates collected for each treatment was subjected to RNA extraction for further analysis.

[0510] First, gene expression profiling of 92 bone differentiation markers (using Taqman® osteopanel array) was performed on the positive and negative control samples. This screening showed that in the presence of bone differentiation medium, 13 genes were induced by more than two-fold in BMSCs on day 7, and less than that on day 14 (see Tables 22, 23, and 24 below). The mRNA expression levels of each sample were normalized to the respective expression levels measured in the negative control group.

[0511] 2.Results More globally, three gene expression patterns were identified. The first pattern included genes induced by both NVD003 and NVD0031yo biomaterial treatment (Table 22). The second pattern included genes induced by biomaterial NVD003 treatment but not by biomaterial NVD0031yo treatment (Table 23). Finally, the third pattern included genes induced by biomaterial NVD0031yo treatment but not by biomaterial NVD003 treatment (Table 24).

[0512] [Table 22]

[0513] [Table 23]

[0514] [Table 24]

[0515] These results confirmed that the gene expression profiles induced by the biomaterials NVD003 and NVD0031yo were not equivalent.

[0516] Example 9: Effect of freeze-drying the biomaterial NVD003 on its in vivo bioactivity 1. Materials and Methods Implantation of (NVD003) into critical-sized femoral bone defects was performed in 56 male nude rats, of which only 42 were enrolled in the study. Analyses performed: histology, pCT scans, and q-RT-PCR (rat primers).

[0517] One month after transplantation, total RNA was extracted from explants using Qiazol lysis reagent (Qiagen®, Hilden, Germany) and a Precellys homogenizer (Bertin instruments®, Montigny-le-Bretonneux, France). RNA was purified using the Rneasy Mini Kit (Qiagen®, Hilden, Germany) with on-column DNase digestion according to the manufacturer's instructions. RNA quality and quantity were measured using a spectrophotometer (Spectramax190, MolecularDevices®, California, USA). A commercially available PCR array (Human RT) was used. 2 Profiler Assay-Angiogenesis; Human RT 2 RT for osteogenesis and angiogenesis gene expression profiles in the Profiler Assay-Osteogenesis (Qiagen®). 2 cDNA was synthesized from 0.5 pg of total RNA using an RNA First Strand Kit (Qiagen®, Hilden, Germany). The ABI Quantstudio 5 system (Applied Biosystems®) and SYBR Green ROX Mastermix (Qiagen®, Hilden, Germany) were used to detect the amplified products. Quantification was performed using the DDOT method, and the final results for each sample were normalized to the mean expression levels of three housekeeping genes (ACTB, B2M, and GAPDH).

[0518] The expression of osteogenic genes was compared between explants obtained from the biomaterial of the present invention at one month after implantation. Next, 84 osteogenic genes were tested in the explants.

[0519] For quantification of miRNA expression, 50 ng of RNA was reverse transcribed into cDNA using the qScript miRNA cDNA Synthesis Kit (Quanta Biosciences®), and qRT-PCR was performed in triplicate using Perfecta SYBR Green Super Mix (Quanta Biosciences®). Thermal cycling was performed on an Applied Biosystems 7900HT Detection System (Applied Biosystems®). Data were normalized to miR-16-5p and U6 small nuclear RNA using the ΔΔCt method.

[0520] One month after implantation, the grafts were explanted and osteoinduction was assessed at the molecular level using biomarkers including VEGFA, VEGFB, and IGF-1; SMAD2, SMAD3, SMAD4, and SMAD5; ITGAV, ITGB1, and VCAM1.

[0521] 2.Results Assays for VEGF-A (Figure 13A), SMAD-2-5 (Figures 14A-14D), ITGAV, ITGB-1, and VCAM-1 (Figures 15A-15C) demonstrated similar overall osteoinductive profiles at the molecular level for the novel NVD003 and NVD0031yo compared to HA / bTOR particles alone. Meanwhile, the relative amount of VEGF-B in biomaterial NVD0031yo was significantly increased compared to biomaterial NVD003 (Figure 13B), while the relative amount of IGF-1 in biomaterial NVD0031yo was significantly decreased compared to biomaterial NVD003.

[0522] Bone induction was confirmed histologically by Alcian blue staining for endochondral ossification (*).

[0523] Example 10: Effect of freeze-drying fresh biomaterial NVD003 on osteogenic gene stability and miR cellular content compared to fresh biomaterial NVD003 1. Stability of bone formation genes Total RNA was extracted as described in Example 3.

[0524] When biomarkers of skeletal development were evaluated in biomaterials NVD003 and NVD0031yo, the levels of these biomarkers were generally altered (Figures 16A-16K). For example, the mean relative amounts of factors ACVR-1, CSF-1, EGFR, FGFR-1, and IGF-IR were generally increased in biomaterial NVD0031yo compared to biomaterial NVD003. Meanwhile, the mean relative amounts of factors BMPR-1A, BMPR-1B, BMPR-2, RUNX2, TGFBR-2, and TWIST-1 were generally decreased in biomaterial NVD0031yo compared to biomaterial NVD003. These data suggest that lyophilization of fresh biomaterial NVD003 had an overall effect on the content of factors expressed in ASCs.

[0525] 2.miRNA content 2.1.RNA Extraction mRNA isolation was performed from biopsies of NVD003, lyophilized NVD003 (NVD0031yo), and lyophilized / irradiated NVD003 (NVDX3). mRNA was extracted using the miRNeasy Mastermix Kit (Qiagen®, Hilden, Germany) according to the manufacturer's protocol. RNA concentration was measured using a Nanodrop™ (ThermoFisher®, Waltham, MA, USA).

[0526] 2.2. Quantitative RT-PCR (QRT-PCR) Quantification of miRNA expression was performed as described in Example 3.

[0527] 2.3.Exosome purification Exosomes were isolated from the culture medium by differential centrifugation, whereby larger “contaminants” were first eliminated by pelleting at increased centrifugation speeds before exosomes, small extracellular vesicles, and even protein aggregates were pelleted at very high speeds (~100,000xg).

[0528] 2.4.Results

[0529] [Table 25-1]

[0530] [Table 25-2]

[0531] [Table 26]

[0532] [Table 27]

[0533] [Table 28]

[0534] Comparing biomaterials NVD003 and NVDX3, we found that the overall miRNA content in cells was altered with either an increase in the average abundance of individual miRNAs or a decrease in the average abundance of individual miRNAs (Figures 17A and 17B and Figures 18A and 18B). These data suggest that lyophilization of fresh biomaterial NVD003 had an overall effect on the cellular and exosomal content of miRNAs synthesized and secreted by ASCs, respectively.

[0535] Example 11: Effect of freeze-drying and gamma irradiation of NVD003 on osteogenic gene stability and cellular miR profile between fresh biomaterial NVD003 and freeze-dried NVD003 (NVD0031yo) Freeze-dried NVD003 biopsies from three donors were gamma-irradiated under four different conditions (dose (12 kGy vs. 25 kGy) and temperature (20°C vs. -80°C)) and compared with non-irradiated tissue in terms of growth factor and mRNA expression.

[0536] Fifteen samples, approximately 350 mg of biomaterial from each donor (NVD0031yo), were weighed and placed in glass vials. Three samples served as negative controls (non-irradiated samples), and 12 samples were sent to Sterigenics for irradiation. The negative control samples were also sent to Sterigenics and stored under the same conditions as the irradiated samples.

[0537] After sterilization, samples were processed for protein extraction and growth factor quantification by ELISA, as well as mRNA expression by q-RT-PCR and cellular miR-210-3p expression.

[0538] To extract proteins from irradiated and non-irradiated samples, these samples were placed in guanidine HCl (4 M), benzamidine (5 mM), N-ethylmaleimide (10 mM), and PMSF (1 mM) for 24 hours at 4°C. Then, they were added to Tris-HCl (50 mM) (all from Sigma-Aldrich®, St. Louis, MO, USA) for 5 hours at pH 7.4. The samples were then purified on a desalting column (PD 10 deGE Healthcare®, Chicago, MO, USA). Quantification of the growth factors VEGF, IGF1, SDF1a, and OPG content was performed by ELISA (Human VEGF quantikine ELISA Kit, Human SDF1a quantikine ELISA Kit, Human IGF1 quantikine ELISA Kit, OPG Duo Set ELISA, R&D Systems®, Minneapolis, MN, USA).

[0539] Total RNA was extracted and quantified as described in Example 3.

[0540] OPG, IGF-1, and VEGF factors were totally preserved by irradiation with two test doses (12 kGy and 25 kGy) at either room temperature or -80°C (Figure 19).

[0541] Similarly, irradiation of the biomaterials had no adverse effect on the content of hsa-miR-210-3p (Figure 20).

[0542] Although gamma irradiation is generally known to have significant adverse effects on the structure of polypeptides and nucleic acids, experimental data suggest that both the factor content (polypeptides) and the cellular content of miRNAs from fresh biomaterial NVD003 are protected from degradation by gamma irradiation.

[0543] Example 12: In vitro evaluation of osteoclastogenesis inhibition 1. Effect of freeze-drying and gamma irradiation of NVD003 on the inhibition of osteoclast maturation and activity 1.1.Effect of NVDX3 on osteoclast precursor differentiation Human CD14+ monocytes were isolated from the peripheral blood of healthy volunteers, obtained with consent from the “Etablissement franc,ais du sang”.

[0544] After isolation of peripheral blood mononuclear cells, freshly isolated precursors were seeded into 24-well culture plates in medium supplemented with 1% FBS, 25 ng / mL human MCSF + / - 100 ng / mL human RANKL, and incubated for 2 hours (minimum cell attachment time). NVD003 (n = 3 donors), HA / β-TCP (n = 4 different batches), and NVDX3 (n = 3 donors) were added to transwells at 5, 20, and 100 mg / well. The medium was changed on day 4.

[0545] TRAP staining was performed after 5 or 6 days (depending on the donor of CD14+ cells) in the presence of multinucleated cells. The number of TRAP-positive cells containing three or more nuclei was determined in each well.

[0546] 1.2. Effect of NVS on mature osteoclasts (cytotoxicity) Osteoclast precursor cells were isolated from peripheral blood. After separating peripheral blood mononuclear cells by Ficoll-Hypaque centrifugation, monocytes (CD14+ cells) were sorted (MACS®, Miltenyi Biotec). Freshly isolated precursors were differentiated into osteoclasts for 5–6 days (depending on the donor of CD14+ cells) in the presence of M-CSF and RANKL ("+RANKL" control group). Cells in medium without RANKL served as a negative control ("no RANKL" control group). When multinucleated cells were observed in the positive control group, NVD003 (n = 3 donors), HA / bTER (n = 4 different batches), and NVDX3 (n = 3 donors) were added to the transwell at 5, 20, and 100 mg / well.

[0547] NVD003, HA / β-TCP , and Forty-eight hours after the addition of NVDX3, TRAP staining was performed, and the number of TRAP-positive cells containing three or more nuclei was counted in each well.

[0548] Compared with HA / β-TCP particles alone (Figures 22A and 22B), the dose-response for inhibition of osteoclast maturation and osteoclast activity was maintained between fresh NVD003 (Figures 21A and 21B) and freeze-dried / irradiated NVD003 (NVDX3; Figures 23A and 23B), which more significantly inhibited mature osteoclasts (functional tests assay the effect of the biomaterial on osteoclast viability). When comparing each product at different doses, no significant differences were found between the inhibition of osteoclast-forming cells and mature osteoclasts.

[0549] 2. Effect of freeze-drying and gamma-irradiation of NVD003 on bone formation Countering the adverse effects of sclerotin Adipose stem cells (ASCs) at passage 4 were plated in 12-well plates for 2 days, then 5 days later, the medium was changed and the cells were incubated in osteogenic differentiation medium (MD = positive control group) [MD + sclerostin (SCL) 100 mg / ml or MD + sclerostin (SCL) 100 mg / ml + NVDX3 100 mg / well] for 10 days. Additionally, cells were placed in proliferation medium (MP) as a negative control.

[0550] After 10 days of culture, cells were placed in Qiazol lysis reagent (Qiagen®, Hilden, Germany) and RNA was isolated, extracted and quantified as described in Example 3.

[0551] In another experiment, adipose stem cells at passage 4 were plated in 12-well plates in osteogenic differentiation medium (0.5% HPL (MD = positive control), 0.5% HPL at MD + sclerostin (SCL) 10 or 100 mg / ml, or 0.5% HPL at MD + sclerostin (SCL) 10 or 100 mg / ml + NVDX3 400 mg / well) for 11 days. Cells were then plated in osteogenic differentiation medium with 5% HPL. Viability was monitored over 9 days (48 hours) using the RealTime Glo MT Cell Viability Assay (Promega® G9711). Experiments were performed in duplicate.

[0552] The miRNA content in NVDX3 (after freeze-drying and gamma irradiation) showed in vitro promotion of mesenchymal stem cell (derived from adipose stem cells) osteogenesis.

[0553] As shown in Figures 24A and 24B, the NVDX3 biomaterial counteracted the adverse effects of sclerotin on the osteogenic properties of osteocalcin (BGLAP) and osteopontin (SPP-1).

[0554] Furthermore, NVDX3 (lyophilized and irradiated) demonstrated the ability to antagonize the effects of sclerostin on adipose stem cells in terms of viability (FIG. 25).

[0555] 2.2.Effect of NVGDXS on osteoblast precursor differentiation The osteoinductive effect of NVDX3 was evaluated. A model of osteoblast formation from human mesenchymal stem cells was used. Mesenchymal stem cells were thawed according to the supplier's recommendations. For cell expansion, cells were seeded in flasks in the supplier's recommended medium (RoosterBio, KT-001) and cultured. Four days after thawing, human MSCs were detached with trypsin-EDTA and counted. Cells were seeded at 2.104 cells per well and cultured in monolayer in 24-well plates in DMEM medium supplemented with 1% FBS for 4 days (the seeding day is designated as day 4). After 4 days of culture in DMEM medium, cells were plated in transwells at 5, 20, and 100 mg / well in basal medium (DMEM 1% FBS + ascorbic acid (50 μg / mL) and β-glycerophosphate (10 mM)), differentiation medium (positive control) (DMEM 1% FBS + ascorbic acid (50 μg / mL) and β-glycerophosphate (10 mM), dexamethasone (10 M), and vitamin D3 (10 M)), or basal medium and NVDX3 (n = 2 donors) (the first day of treatment is "day 0"). Medium and treatments were changed on days 4, 7, and 11. All treatments and controls were performed in duplicate.

[0556] Cells were lysed using Qiazol lysis reagent (Qiagen®, Hilden, Germany) on days 7 and 14. Total RNA was extracted from cell lysates and quantified as described in Example 8.

[0557] The expression of osteogenic and angiogenic genes varied depending on the evaluation time (7 days vs. 14 days) and the dose of the test material applied. However, the majority of the tested genes were at least transiently overexpressed after NVDX3 treatment at each test dose, as seen in the positive control group (differentiation medium) compared to the negative control group (basal medium, set at 1). These results demonstrated that NVDX3 has osteoinductive properties and can promote the osteogenic differentiation of osteoblast precursors (Figures 26A-26X). Furthermore, these results indicated that the osteogenic / angiogenic effects of the biomaterial NVDX3 are contact-independent and mediated by soluble factors.

[0558] 2.3. Effect of NVDX3 on mature osteoblasts (cytotoxicity) SaOS2 cells were grown in MeCoy's medium + 10% FBS, 1% P / S, and 0.1% amphotericin B in 12-well plates for 2–3 days and then added to MeCoy's medium with 1% FBS and NVDX3 (n=3) or HA / β-TCP (12mm Transwell with 0.4μm pore membrane insert) in triplicate. SaOS2 cells were plated in 12-well plates in MeCoy medium + 10% FBS, 1% P / S, and 0.1% amphotericin B for 2–3 days, then placed in MeCoy with 1% FBS and NVDX3 (n=3) or HA / β-TCP and added to transwells (12 mm transwells with 0.4 μm pore polyester membrane inserts). The following conditions were performed in triplicate: - Cells + medium (1% FBS + 2% Triton® "control group") - Cells + medium (1% FBS "control group") -Cells + medium (1% FBS + 10mg NVDX3) -Cells + medium (1% FBS + 20mg NVDX3) -Cells + medium (1% FBS + 40mg NVDX3) -Cells + medium (1% FBS + 100mg NVDX3) -Cells + medium (1% FBS + 200mg NVDX3) - Cells + medium (1% FBS + 200 mg HA / β-TOR) (Biomatlante®)

[0559] Cytotoxicity was assessed at 24 hours by viability assay, cytotoxicity assay, and LDH production was assessed.

[0560] The positive control group (Triton®) showed rapid cytotoxicity (Figure 27A) with no viability (Figure 27B), no cell turnover associated with low DNA content, low levels of "free DNA" staining (cytotoxicity; Figure 27D), and no LDH production (Figure 27C).

[0561] Basal conditions (MD) showed low cytotoxicity (Figure 27A) and therefore low cell turnover, which was associated with low cumulative mitochondrial activity (low viability; Figure 27B), high DNA content (Figure 27D), and moderate LDH activity (Figure 27C) at 24 h.

[0562] The negative control group (HA / bTOR) showed high viability (FIG. 27B) associated with low cytotoxicity (FIG. 27A), high DNA content (FIG. 27D), and moderate LDH activity (FIG. 27C).

[0563] A dose-response cytotoxic effect was observed with increasing doses of NVDX3 at 10 mg / well (12-well plates; Figure 27A). Compared to MD cells, NVDX3 at 100 and 200 mg induced significantly higher cytotoxicity (p<0.01; Figure 27A). In contrast, no cytotoxicity was observed with 200 mg HA / β-TOR (Figure 27A). However, similar viability (FIG. 27B), DNA content (FIG. 27D), and LDH production (FIG. 27C) were measured for doses of NVDX3 and HA / β-TOR tested up to 200 mg / well.

[0564] 3. Conclusions and Discussion The purpose of this study was to describe the anti-resorptive and osteogenic properties of NVDX3. Using an in vitro model of human osteoclastogenesis, we evaluated the effects of these products on human osteoclast formation and viability. Furthermore, we used an in vitro model of human osteoblastogenesis to study the effects of NVDX3 on human osteoblast formation and viability.

[0565] In this study, NVDX3 showed a total inhibitory effect on osteoclast differentiation at the three test doses (5 mg, 20 mg, and 100 mg). This inhibitory effect was more significant at lower doses (5 mg and 20 mg) with NVDX3 treatment compared with NVD00X, NVD003, and HA / β-TCP.

[0566] NVDX3 also exhibited an inhibitory effect on mature osteoclasts, suppressing osteoclast viability by 35% in the presence of 5 mg, 30% in the presence of 20 mg, and 80% in the presence of 100 mg.

[0567] These results indicate that the anti-resorptive properties of NVD003 are maintained after freeze-drying and gamma irradiation.

[0568] Furthermore, NVDX3 was shown to promote osteogenic differentiation of osteoblast precursors and, while exhibiting cytotoxic effects on mature osteoblasts, was associated with rapid cell turnover without affecting cell content or cell viability.

[0569] In conclusion, NVDX3 exhibited antiresorptive and osteogenic properties in vitro, associated with a lack of cytotoxicity in osteoblasts.

[0570] Example 13: NVDX3 promotes bone formation in vivo 1. Bone induction NVDX3 (lyophilized and gamma-irradiated) showed significantly higher osteoinduction at the molecular level one month after implantation compared with HA / β-TCP alone and fresh NVD003 (Figures 28A-H).

[0571] 2. Immune response - Detection of anti-HLA1 (anti-human leukocyte antigen 11) antibodies This significantly higher bone induction (at the molecular level) was associated with a significantly lower immune response in terms of antibody production (anti-HLA), indicating tolerance of freeze-dried irradiated NVD003 (NVDX3).

[0572] To assess the humoral response after NVD003 (or HA / β-TCP or NVDX3) implantation in critical-sized bone defects in Wistar rats, we used the FlowPRA™ Class I Screening Test technique. The protocols varied widely depending on the Ig type being investigated.

[0573] a) Anti-HLAl IsG detection Serum samples were mixed with FlowPRA™ Class I Screening Test beads (OneLambda®, USA-FL1-30) in appropriate tubes according to the manufacturer's instructions. The mixture was incubated for 30 minutes in the dark at 20°C. Two washes with PBS-BSA 0.5% were performed with 2-minute centrifugation steps at 9,000 x g. Biotinylated anti-IgG (BioLegend®, USA-405428) was added, followed by a 30-minute incubation in the dark at 20°C. Two washes with PBS-BSA 0.5% were performed with 2-minute centrifugation steps at 9,000 x g. PE-streptavidin (BD Biosciences®, USA-554061) was added, followed by a 30-minute incubation in the dark at 20°C. Two washes with PBS-BSA 0.5% were performed with 2-minute centrifugation steps at 9,000 x g. 0.5% PFA was added, transferred to a reading plate, and 5,000 to 10,000 beads were acquired using a cytometer (Beckman Coulter).

[0574] b) Anti-HLAl IgM detection Before mixing with specific beads, serum samples needed to be depleted of IgG. Depletion was performed by adding biotinylated anti-IgG (BioLegend®, USA-405428) to the samples and then incubating them in the dark at 4°C for 20 minutes. Magnetic beads combined with streptavidin (Streptavidin Particles...

Claims

1. A sterile and dried biomaterial comprising devitalized cells having tissue regenerative and / or tissue repair properties and particulate material, wherein the cells and the particulate material are embedded in an extracellular matrix, and the cells secrete the extracellular matrix, The sterilized dry biomaterial comprises: (1) contacting differentiable living cells (i) with particulate material (ii) to obtain a first combination; Culturing the first combination obtained in step (1) in a culture medium so that the cells secrete an extracellular matrix and synthesize growth factors and / or transcription factors to obtain tissue regeneration and / or tissue repair properties, wherein step (2) embeds the cells and the particulate material in the extracellular matrix to form a multidimensional structure; Step (3) of subjecting the multidimensional structure obtained in step (2) to drying to obtain a dried biomaterial; and Step (4) of subjecting the dried biomaterial obtained in step (3) to sterilization, for example, by gamma irradiation, to obtain a sterile, dried biomaterial; and a particulate material obtained by a method comprising: up to 10% of the cells are viable, A sterilized, dry biomaterial.

2. 2. The sterilized, dry biomaterial of claim 1, wherein the cells are selected from the group consisting of primary cells, stem cells, genetically modified cells, and combinations thereof.

3. 3. The sterilized, dry biomaterial of claim 1 or 2, wherein at most 1% of the cells are viable.

4. The particulate material is Organic materials, including demineralized bone matrix (DBM), gelatin, agar / agarose, alginate chitosan, chondroitin sulfate, collagen, elastin or elastin-like peptides (ELPs), fibrinogen, fibrin, fibronectin, proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, polysaccharides, laminin, and cellulose derivatives; Calcium phosphate (CaP) particles, calcium carbonate (CaCO ３ ) particles, calcium sulfate (CaSO ４ ) particles, calcium hydroxide (Ca(OH) ２ ) particles, or a combination thereof; polymers, including polyanhydrides, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene oxide / polyethylene glycol (PEO / PEG), poly(vinyl alcohol) (PVA), fumaric acid-based polymers, oligo(poly(ethylene glycol) fumarate) (OPF), poly(n-isopropylacrylamide) (PNIPPAAm), poly(aldehyde guluronate) (PAG), poly(n-vinylpyrrolidone) (PNVP), or combinations thereof; Gels, including self-assembled oligopeptide gels, microgels, nanogels, particulate gels, hydrogels, thixotropic gels, xerogels, responsive gels, or combinations thereof; and combinations thereof; 4. The sterilized, dry biomaterial of claim 1 , comprising or selected from the group consisting of:

5. 5. The sterilized, dried biomaterial of claim 1, wherein the biomaterial comprises an altered factor content of growth factors and / or transcription factors compared to the factor content of a corresponding fresh, non-sterile, non-dried biomaterial.

6. The sterilized dry biomaterial according to claim 5, wherein the growth factors and / or transcription factors comprise IGF-1 and / or VEGF and / or SDF-1α and / or OPG.

7. 6. The sterilized, dry biomaterial of claim 5, wherein the factor content comprises RNA content.

8. 8. The sterilized dry biomaterial according to claim 1, wherein the dry biomaterial is a lyophilisate of the biomaterial.

9. A sterilized dry biomaterial described in any one of claims 1 to 8, wherein in the sterilization by gamma ray irradiation in step (4), the sterilized biomaterial is irradiated with gamma rays at room temperature at a dose of 7 kGy to 45 kGy of the biomaterial.

10. 10. A method for producing the sterile dry biomaterial of claim 1, comprising: (1) contacting differentiable living cells (i) with particulate material (ii) to obtain a first combination; (2) culturing the first combination obtained in step (1) in a culture medium so that the cells secrete an extracellular matrix and synthesize growth factors and / or transcription factors to obtain tissue regeneration and / or tissue repair properties, wherein the cells and the particulate material are embedded in the extracellular matrix to form a multidimensional structure; (3) subjecting the multidimensional structure obtained in step (2) to drying to obtain a dried biomaterial; and (4) sterilizing the dried biomaterial obtained in step (3) by gamma irradiation to obtain a sterilized dried biomaterial; 1. A method for producing a sterile, dry biomaterial comprising devitalized, differentiated cells and particulate material, wherein said cells and said particulate material are embedded in an extracellular matrix, comprising:

11. A pharmaceutical composition comprising the sterilized dry biomaterial of any one of claims 1 to 9 and a pharmaceutically acceptable vehicle.

12. A medical device comprising a sterile dry biomaterial according to any one of claims 1 to 9 and a pharmaceutical composition according to claim 11.

13. A pharmaceutical composition comprising a sterile dry biomaterial according to any one of claims 1 to 9 or a medical device according to claim 12 for use as a medicament.

14. The pharmaceutical composition according to claim 13, which is used for preventing and / or treating tissue damage.

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