Cellular spheroid from subperiosteal regenerative blastema cells of periosteum for bone restoration

Abstract

FIELD: biotechnology.SUBSTANCE: transplant for restoring the bones of a subject. The invention includes cellular spheroids based on autologous periosteal cells of a subject, the size of which ranges from 250 to 600 μm.EFFECT: development and production of a transplant for effective bone restoration based on cellular spheroids possessing pronounced regenerative potential and characterized by the presence of a heterogeneous population of subperiosteal blastema cells in their composition.3 cl, 2 ex

Description

[0001] Technical field

[0002] The invention relates to the field of biomedicine, namely to tissue engineering, regenerative medicine, and is potentially applicable in transplantology, dentistry, plastic surgery, traumatology and orthopedics.

[0003] Technology Level

[0004] The development of scaffold-free tissue engineering is driven by the improvement of bioartificial devices—individual standardized organ building blocks, including cellular spheroids, organoids, and assemblies. These organ building blocks possess the ability to self-assemble and self-organize, enabling the rapid creation of large tissue constructs using the principles of a modular or bottom-up approach known in tissue engineering, which involves the assembly of small cell-filled modules to form larger structures at tissue or organ levels of organization during biofabrication (similar to the levels of structural organization of an individual).Another name for this approach to producing bioartificial organs from organ building blocks is the "embryo" model. In this model, the building blocks are small at the initial stage of organ biofabrication, but over time, their complexity, quantity, and diversity of building blocks increase. Organ building blocks allow for prevascularization of their clusters and enable the creation of a high-density arrangement of cells within their structure, allowing tissue-engineered constructs to approximate the density of their own cellular mass, for example, to limb rudiments formed during individual development or to regenerative blastemas during epimorphic regeneration.

[0005] It is known that periosteal contusions in areas of the skeletal tissue where the bones of the skeleton are palpable (the tibial crest, the posterior surface of the ulna, etc.) are adjacent to the skin are followed by damage to the periosteum and sharp pain. The periosteum is well vascularized, with vessels penetrating the bone through special nutrient openings on its surface and directed into perforating (Volkmann's) canals. Therefore, subperiosteal hemorrhages are often observed with bone contusions, forming a space between the periosteum and the bone that fills with the spilled blood. In this space, the spilled blood is replaced by the proliferation of cells from the trauma-activated periosteum, which form bone substance and bone regenerate. Traces of the contusion can persist for a long time in the form of small nodules—subperiosteal bone growths—which are easily palpated under the skin in these areas.

[0006] The formation of subperiosteal blastema and complete bone regeneration after complete subperiosteal enucleation of the tibia bone diaphyses were observed in experiments on different species of birds and mammals, which were conducted under the supervision of Professor A. N. Studitsky. (Runikhin, Yu. A. Sources of restoration of whole tubular bones after complete subperiosteal enucleation [Text]: (Experimental - morphological study): Abstract of dissertation for the degree of candidate of biological sciences / USSR Academy of Sciences. A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals. - Moscow: [b. i.], 1968. - 24 p.

[0007] The study of skeletal fragment restoration after subperiosteal and subperichondral bone and cartilage enucleation has found a continuation in modern bone regeneration research. A clinical observation using a 3D reconstructed CT image obtained approximately 6 months after surgery demonstrates incomplete bone restoration, cartilage restoration, and costochondral joint restoration in a 42-year-old man. This man required craniofacial reconstruction in the Department of Maxillofacial Surgery due to trauma. For free grafting, a portion of the right 6th rib (8 cm) and adjacent costal cartilage (1 cm) were harvested, preserving the integrity of their periosteum and perichondrium (Srour MK, Fogel JL, Yamaguchi KT, Montgomery AR, Izuhara AK, Misakian AL, Lam S, Lakeland DL, Urata MM, Lee JS, Mariani FV. Natural large-scale regeneration of rib cartilage in a mouse model. J Bone Miner Res. 2015 Feb;30(2):297-308. doi: 10.1002 / jbmr.2326.PMID: 25142306; PMCID: PMC8253918).

[0008] In vivo bioreactor technology is known, which is based on manipulating an intentionally created space inside the body so that it serves as an “in vivo bioreactor,” in which the construction of new growing tissue is achieved by inducing a therapeutic response within the bioreactor space (Stevens MM, Marini RP, Schaefer D, Aronson J, Langer R, Shastri VP. In vivo engineering of organs: the bone bioreactor. Proc Natl Acad Sci USA. 2005 Aug 9; 102(32): 11450-5. doi: 10.1073 / pnas.0504705102. Epub 2005 Jul 29. PMID: 16055556; PMCID: PMC1183576). The authors achieved controlled manipulation of the periosteal space using a procedure they termed hydraulic periosteal elevation. The periosteum was punctured with a syringe needle, bevel side up, and separated from the bone by simultaneously injecting fluid (Ringer's solution) between the cambium and bone and slowly advancing the needle with lateral sweeping movements.After creating a space of the desired geometry, it was filled with calcium alginate gel. This procedure allowed for the creation of a space over the tibia of skeletally mature New Zealand White rabbits, into which 200 mm³ of gel was then reproducibly placed. Filling the space created by hydraulic lifting of the periosteum with gel and Ringer's solution was successively also filled using a syringe with a gel that was obtained by mixing 2% (w / v) sodium alginate (FMC BioPolymer) in 30 mM Hepes containing 150 mM NaCl and 10 mM KCl with an equal volume of a solution containing 75 mM CaCl2 in 10 mM Hepes containing 150 mM NaCl and 10 mM KCl using a sterile Y-mixer. The gel hardened within 1 min and had a Young's modulus of 0.17 MPa. The gel contained growth factors TGF-β1 and FGF-2 in an amount of 10 ng / ml each.The authors found bone regeneration occurring primarily through intramembranous ossification. The fibrous bone matrix scaffold subsequently underwent regenerative remodeling and matured into fully mineralized, compact, lamellar bone exhibiting all the histological markers and mechanical properties of native bone. The bone was resected in the in vivo bioreactor after 6 weeks and transplanted into the post-surgical experimental defects of the contralateral tibia, resulting in complete integration of the free bone graft and fusion of the bone fragments within 6 weeks, without visible complications at the donor site.

[0009] With the observed intramembranous ossification, it is crucial to trigger important, yet poorly understood, mechanisms that underlie the known spatiotemporal associations between angiogenesis and intramembranous osteogenesis, enabling the regeneration of complete and well-vascularized lamellar bone tissue. The role of heterogeneous cell populations formed within the in vivo bioreactor space at the initial stage of in vivo bioreactor-induced bone regeneration is clearly significant. The possibility of isolating the subperiosteal blastema before full-blown intramembranous osteogenesis undoubtedly opens up new opportunities for harvesting large volumes of activated blastema cells, which possess pronounced regenerative osteogenic potential.Subperiosteal blastema allows for the harvesting of tens of times more osteogenic cells from comparable areas of excised and surgically harvested intact periosteum. Furthermore, blastema cells are activated and clearly contain diverse cell populations. In our invention, we use in vivo bioreactor technology not for bone augmentation, but to produce a subperiosteal regenerative blastema, which, as we discovered, forms at the interface between the exfoliated periosteum and the gel of a known in vivo bioreactor.

[0010] The closest analogue of the present invention is the spheroid described in patent No. 2744664 C1 Russian Federation, IPC C12N 5 / 077, A61F 2 / 30. for the group of inventions "Method for producing spheroids from cultured periosteum cells to ensure reparative osteogenesis": No. 2020109215: declared. 02.03.2020: published. 12.03.2021 / A. V. Kovalev, M. M. Smorchkov; applicant Limited Liability Company "International Center for Medical Research and Development". The inventions provide for obtaining a transplant in the form of cellular spheroids from autologous periosteum cells of a subject, which have pronounced regenerative potential for the effective restoration of skeletal connective tissues, in particular articular cartilage and bone. Histologically, two layers are distinguished in the periosteum: the outer or adventitial (fibrous) and the inner bone-forming (osteogenic, or cambial).According to the known invention, the spheroid consists of cultured cells from the inner layer of the periosteum, which serves as the cambial zone for skeletal connective tissues, supplying progenitor and stem cells for physiological and reparative regeneration. The inner layer of the periosteum contains osteogenic cells.

[0011] The well-known spheroid possesses high regenerative potential. However, its main drawback is the difficulty of obtaining it, as the periosteum is quite thin, and the cambial layer of periosteal cells may be poorly developed or completely absent in older specimens. It is well known that the inner layer of the periosteum in adults is poorly visible and normally consists of loose fibrous connective tissue containing flat, spindle-shaped cells—resting osteoblasts and their precursors (preosteoblasts). The periosteum is firmly attached to the bone by thick bundles of collagenous perforating (Sharpey's) fibers, which penetrate the extracellular bone matrix and intertwine with the layer of the outer common lamellae of the bone, the part of the bone to which the periosteum is adjacent. Therefore, when attempting to separate the periosteum and harvest it during surgery, the cambial layer is partially destroyed.Under normal conditions, periosteal cambium cells ensure bone thickening during growth and physiological bone tissue regeneration. Blastema cells, by definition and intrinsically, possess a higher regenerative potential, and the blastema itself is a source of a heterogeneous cell population, already activated by mechanical trauma, actively participating in reparative regeneration and regenerative bone remodeling.

[0012] Disclosure of invention

[0013] The objective of the present invention is to develop a new transplant based on cell spheroids from cells of the subperiosteal blastema of the periosteum formed at the border of the periosteum exfoliated from the bone and the alginate gel of an in vivo bioreactor installed in the subperiosteal space, for effective bone restoration, as well as for the development of new regenerative technologies for bone organ restoration, including new bioprinting methods.

[0014] The present invention relates to a transplant for restoring skeletal connective tissues, comprising cellular spheroids based on cells of the subperiosteal regenerative blastema of a subject, formed at the border of the periosteum exfoliated from the bone and the gel of an in vivo bioreactor installed in the subperiosteal space.

[0015] In particular embodiments of the invention, cellular spheroids are cellular spheroids based on cultured cells of the subperiosteal regenerative blastema of a subject.

[0016] In particular embodiments of the invention, cellular spheroids based on autologous cells of the subperiosteal regenerative blastema of a subject.

[0017] In particular embodiments of the invention, the subject is a human being.

[0018] In particular embodiments of the invention, the size of the cell spheroids is 250-650 μm.

[0019] The technical result of this invention is the development and production of a transplant for effective bone restoration based on cellular spheroids that have a pronounced regenerative potential and are characterized by the presence of a heterogeneous population of subperiosteal blastema cells in their composition.

[0020] The technical result of the present invention also consists in the fact that the in vivo use of a bioreactor makes it possible to obtain a large amount of cellular biomass of osteogenic cells of the subperiosteal blastema, compared to the cellular mass of the cambial intact layer of the periosteum.

[0021] The technical result of the present invention is also achieved by using autologous cells of the subperiosteal blastema of the periosteum of a subject to obtain an autologous personalized transplant in the form of cellular spheroids with high regenerative potential.

[0022] The technical result of the present invention is also achieved by using an in vivo bioreactor in the form of an alginate gel introduced under the periosteum, which was obtained in a known manner by mixing 2% (weight / volume) sodium alginate (FMC BioPolymer) in 30 mM Hepes containing 150 mM NaCl and 10 mM KCl, with an equal volume of a solution containing 75 mM CaCl2 in 10 mM Hepes containing 150 mM NaCl and 10 mM KCl, using a sterile Y-shaped mixer, the gel hardens within 1 min and has a Young's modulus of 0.17 MPa, the gel contains growth factors TGF-β1 and FGF-2 in an amount of 10 ng / ml each.

[0023] Definitions and Terms. Various terms related to the objects of the present invention are used above and also in the description and claims. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement with equivalent techniques known to those skilled in the art.

[0024] In the description of this invention, the terms "includes" and "including" are interpreted to mean "includes, among other things." These terms are not intended to be construed as meaning "consists only of."

[0025] The term "adherent culture" in this document means a monolayer culture of cells that are capable of attaching to a culture plastic or glass through their receptors; under certain conditions, these cells will attach to the substrate and will normally divide and spread in this manner.

[0026] The cells of the cambial layer of the periosteum are located in the internal bone-forming (osteogenic, or cambial) layer of the periosteum, containing skeletogenic cells - osteoblasts, preosteoblasts and skeletogenic stem cells, which belong to the external cambium.

[0027] Prevascularization is a tissue engineering concept aimed at creating a pre-formed microvascular network in tissue constructs prior to their implantation. This approach aims to reduce the period during which the constructs are avascular and the living cells within them are exposed to hypoxic conditions.

[0028] The term "spheroids" as used in this application refers to densely packed, spherical cell aggregates formed by three-dimensional cell culture in agarose or special plastic wells. Their important properties include their ability to adhere to each other and subsequently undergo tissue fusion, as well as their ability to adhere to elements of the extracellular matrix.

[0029] The term "blastema" in this document refers to a heterogeneous cell mass that, through migration and proliferation, is temporarily formed at the site of injury and undergoes morphogenesis to form the missing organ.

[0030] Subperiosteal regenerative blastema is a subperiosteal heterogeneous cellular mass containing skeletogenic cells, originating from the hypertrophied periosteum, activated by trauma and deprived of direct contact with bone. Detached periosteum can form a subperiosteal regenerative blastema (blastoma subperiosteal regenerate) between 3 and 8 days after injury.

[0031] Sub-, pod- (Sub-) - a prefix denoting located below, under something.

[0032] In vivo bioreactors are regenerative medicine devices that are implanted into the body and utilize the body's natural regenerative ability to generate new tissue. The space occupied by the in vivo bioreactor contacts the body's living tissues, creating local conditions and internal space between living tissues within the body, in which the formation of regenerates occurs.

[0033] An assembly is an in vitro model that combines two or more organoids, spheroids, or cultured cell types to recreate the structural and functional properties of an organ. It represents a three-dimensional structure formed by the fusion and functional integration of multiple cell types. Assemblies mimic the complex cellular interactions that give rise to organs in the body.

[0034] Intramembranous ossification is a characteristic mechanism of formation of the flat bones of the skull and shell of the turtle. During intramembranous ossification of the skull, mesenchymal cells of the neural crest proliferate and condense into compact nodules (ossification centers). The ossification centers grow deeply and superficially, forming radially directed bony crossbars connected by bony trabeculae. Most dermal bones have multiple ossification centers that fuse during ossification. The non-ossified superficial layer of mesenchyme forms the periosteum.

[0035] Enucleation is the surgical removal of bone (from a joint capsule, a tumor, or a cyst from a capsule, etc.). It is a minimally invasive surgical procedure that is one way to remove tumors or organs. This surgical technique is used in neurosurgery to remove neuromas and other conditions.

[0036] Subperiosteal bone enucleation is the surgical removal of a large segment of the diaphysis of a long tubular bone, while maintaining the integrity of the periosteum - the connective tissue membrane covering the bone.

[0037] The term “regenerative potential” in this document represents the ability of tissue-engineered constructs to initiate reparative or transplantative regeneration, to be a source of cellular regeneration, to support reparative regeneration with the achievement of a positive result - complete or partial restoration of a damaged organ.

[0038] Detailed disclosure of the invention

[0039] Spheroids, which are dense three-dimensional cellular aggregates, have the ability to stimulate regeneration, tissue fusion, and can act as a source of regeneration. However, the regenerative potential and optimal application of cellular spheroids for regenerative medicine purposes largely depend on the spheroid's cellular composition, its biofabrication conditions, and its size.

[0040] Epimorphosis, in which bones of regrowing limbs are regenerated, and an example of regeneration in mice, where amputation of the distal region of the terminal phalanx of mice induces an initial wound healing response followed by blastema formation and complete regeneration of the fingertip along with the distal phalanx (Fernando WA, Leininger E, Simkin J, Li N, Malcom CA, Sathy-amoorthi S, Han M, Muneoka K. Wound healing and blastema formation in regenerating digit tips of adult mice. Dev Biol. 2011 Feb 15;350(2):301-10. doi: 10.1016 / j.ydbio.2010.11.035. Epub 2010 Dec 8. PMID: 21145316; PMCID: PMC3031655) indicate the critical role of the blastema in bone regeneration.

[0041] According to the present invention, subperiosteal regenerative blastema cells are used as a source of blastema cells for cell spheroid transplants. Such blastema can be produced in the recipient's own body. This requires the creation of an in vivo bone bioreactor using a known method.

[0042] In an experiment on skeletally mature Californian rabbits, a hydraulic elevation technique was used to manipulate the periosteum covering the tibia (Marini RP, Stevens MM, Langer R, Shastri VP. Hydraulic elevation of the periosteum: a novel technique for periosteal harvest. J Invest Surg. 2004 Jul-Aug;17(4):229-33. doi: 10.1080 / 08941930490472073. PMID: 15371165.). The muscle on the anteromedial side of the metaphyseal and diaphyseal parts of the tibia was cut and retracted caudally to expose the periosteum. A 5 / 8-inch, 25-gauge needle was bent at a 45° angle at half its length and attached to a 3-ml syringe filled with Ringer's solution. The periosteum was punctured with the needle, with the beveled side facing upward. The periosteum was separated from the bone by simultaneously injecting fluid between the inner cambium layer of the periosteum and the adjacent bone.Slowly advancing the needle with sweeping movements of the needle tip to the right and left along the insertion path, gently detaches the periosteum. After creating a subperiosteal space of the planned size, gel was injected into the space between the periosteum and bone through an entry hole the size of a pinhole using a thicker needle until fully distended (bioreactor dimensions: length 3 cm, width 0.7 cm, height above the tibial plane 1 mm; volume ~200 mm³). The periosteal opening was sutured with absorbable sutures, and the incision itself was sutured.

[0043] Alginate gel was prepared according to a known method (Stevens MM, Marini RP, Martin I, Langer R, Prasad Shastri V. FGF-2 enhances TGF-betal-induced periosteal chondrogenesis. J Orthop Res. 2004 Sep;22(5):1 114-9. doi: 10.1016 / j.orthres.2003.12.021. PMID: 15304287), and gelation was initiated by mixing 2% (w / v) sodium alginate (FMC BioPolymer) in 30 mM Hepes containing 150 mM NaCl and 10 mM KCl with an equal volume of a solution containing 75 mM CaCl2 in 10 mM Hepes. containing 150 mM NaCl and 10 mM KCl, using a sterile Y-mixer. The gel cured for 1 min and had a Young's modulus of 0.17 MPa. Growth factors (TGF-β1 and FGF-2, R&D Systems) were included in the gel at a concentration of 10 ng / ml.

[0044] Unlike the known methods of using a bone in vivo bioreactor, we have found that at the border of the periosteum exfoliated from the bone and the gel of a known in vivo bioreactor, a subperiosteal regenerative blastema develops in rabbits within 4-7 days, the cells of which we use after isolation in laboratory conditions or during isolation and subsequent cultivation for aggregation into a cellular spheroid.

[0045] To extract cells from the subperiosteal regenerative blastema of the periosteum, the periosteum above the gel of the in vivo bioreactor is completely excised during the second surgical procedure (stage 1 involved creating a bone in vivo bioreactor). The periosteum is laid out on a sterile preparation table, and with constant wetting, the cell mass is scraped from the inner surface of the periosteum. The periosteum completely regenerates on the donor site after some time.

[0046] The isolated portion of periosteum containing the blastema was subjected to further mechanical and enzymatic disaggregation (collagenase type 1). After centrifugation and resuspension, the sample was seeded into culture flasks with a treated surface. To maintain the growth of the primary cell culture and at all stages of passaging, culture flasks with a non-ventilated lid and a treated surface were used, and a culture nutrient medium of the following composition was used: DMEM / P12 450 ml, L-glutamine 292 mg, 50 ml of thrombolysate, penicillin 100 U / ml, streptomycin 100 μg / ml (at 100% humidity above the layer of nutrient medium inside the culture flask and a gas mixture for filling the culture flask consisting of 5% CO2, 10% oxygen and 85% nitrogen), the nutrient medium in the flask is replaced at least once every 3 days. Subcultivation of a monolayer culture of subperiosteal regenerative blastema cells is carried out until the third - fifth passage.After the final passage, cells are removed from the plastic vials with trypsin / EDTA. The resulting cell suspension is washed to remove trypsin / EDTA, including with DMEM medium supplemented with 10% autologous serum, followed by centrifugation (10 minutes at 200g). The cells are then transferred to 81-well agarose plates with a well diameter of 800 µm at a concentration of up to 1.4 million cells per plate. Cell spheroids are formed in the wells from the cultured cells. Cell aggregation and spheroid maturation occur in the agarose wells for up to 7 days.

[0047] For use, cell spheroids are collected from the plates and transferred to a test tube, where they settle to the bottom without further centrifugation. The spheroids are then transferred using an applicator with a needle opening of at least 1 mm. 2 .

[0048] Implementation of the invention.

[0049] Example 1.

[0050] In an experiment on 8-month-old male Californian rabbits, a surgical approach to the anteromedial aspect of the metaphyseal and diaphyseal portions of the tibia was performed under operating conditions. The periosteum was exposed. Using a syringe and needle and Ringer's solution, the periosteum was hydraulically elevated. The subperiosteal space was then filled with alginate gel, forming a bone bioreactor measuring 3 cm in length, 0.7 cm in width, and 4 mm in height above the tibial plane. The periosteal opening was further sutured with absorbable sutures, and the wound was closed layer by layer. Alginate gel preparation was carried out in a known manner, gelation was initiated by mixing 2% (w / v) sodium alginate (FMC BioPolymer) in 30 mM Hepes containing 150 mM NaCl and 10 mM KCl with an equal volume of a solution containing 75 mM CaCl2 in 10 mM Hepes.containing 150 mM NaCl and 10 mM KCl, using a sterile Y-mixer. The gel cured for 1 min and had a Young's modulus of 0.17 MPa. Growth factors (TGF-β1 and FGF-2) were included in the gel at a concentration of 10 ng / ml.

[0051] Five days after the in vivo bone bioreactor was installed, the periosteum containing the developed subperiosteal regenerative blastema was removed from the rabbits. The periosteum was laid out on a sterile preparation table, and the cellular mass was scraped from the inner surface of the periosteum under constant moistening. The isolated and partially crushed portion of the periosteum containing the blastema was then subjected to mechanical and enzymatic disaggregation (using collagenase type I). After centrifugation and resuspension, the isolated cells were seeded into culture flasks with a treated surface.To maintain the growth of the primary cell culture and at all stages of passaging, we used culture flasks with a non-ventilated lid and a treated surface; we used a culture nutrient medium of the following composition: DMEM / P12 450 ml, L-glutamine 292 mg, 50 ml of rabbit thrombolysate, penicillin 100 U / ml, streptomycin 100 μg / ml (at 100% humidity above the nutrient medium layer inside the culture flask, we used a ready-made gas mixture to fill the culture flask consisting of 5% CO2, 10% oxygen and 85% nitrogen), the nutrient medium and gas mixture in the culture flask were replaced at least once every 3 days. Subcultivation of the monolayer culture of subperiosteal regenerative blastema cells was carried out until the fifth passage. After the last passage, cells were removed from the surface of plastic flasks with trypsin / EDTA.The resulting cell suspension was washed from trypsin / EDTA using DMEM medium with 10% autologous serum, followed by centrifugation and transferred to 81-well agarose plates with a well diameter of 800 µm at a concentration of up to 1.4 million cells per plate. 3D cell cultivation was performed in the wells, during the initial stages of which cell aggregation occurred with the formation of cell spheroids. The production of cell spheroids using 3D agarose Petri Dish®, cell aggregation, and maturation of cell spheroids in agarose wells were carried out taking into account the recommendations of the silicone mold manufacturer (MicroTissues Inc.®).

[0052] 3D cell culture growth, cell aggregation and spheroid maturation occurred in agarose wells for 5 days.

[0053] For transplantation, cell spheroids were collected from agarose wells and transferred to a test tube, where they settled to the bottom without further centrifugation. The spheroids were then transferred using an applicator with a needle lumen of at least 1 mm2.

[0054] A 1-cm-diameter through-the-bone defect in the cranial vault of rabbits was surgically created in the operating room using a burr and rongeurs. This defect was filled with cellular spheroids. After hemostasis, the cellular spheroids were placed in a single layer on the exposed dura mater using an applicator. Morphological and histological examination of the regenerated bone was performed 6 months after cellular spheroid transplantation. Prior to histological examination of the bone regenerated bone, microCT scanning of the rabbit skull with digital 3D reconstruction of the image was performed.

[0055] A 1 cm diameter cranial vault bone defect is considered critical and never spontaneously closes with bone regeneration. It was found that cellular spheroids from blastema cells provide complete and robust regeneration of the cranial vault bones in such bone defects, forming organotypic bone regeneration in the form of spongy tissue located between the inner and outer layers of the parietal bone regeneration with well-developed red bone marrow in its intertrabecular spaces. However, the total bone mass of the regenerated bone is less than the mass of the removed parietal bone fragment that was located at the defect site prior to the experiment.

[0056] Example 2.

[0057] In another experiment, surgical access to the tibia was performed in an operating room on 12 8-month-old male Californian rabbits. The periosteum was exposed, hydraulically elevated, and the subperiosteal space was filled with alginate gel, the composition and preparation method of which are described above. A bone bioreactor was formed, measuring 3 cm in length, 0.7 cm in width, and 4 mm above the plane of the tibia. The integrity of the periosteum was restored, and the wound was sutured layer by layer. Alginate gel was prepared using a known method. Gel formation was initiated by mixing 2% (w / v) sodium alginate (FMC BioPolymer) in 30 mM Hepes containing 150 mM NaCl and 10 mM KCl with an equal volume of a solution containing 75 mM CaCl2 in 10 mM Hepes containing 150 mM NaCl and 10 mM KCl, using a sterile Y-mixer. The gel cured within 1 min and had a Young's modulus of 0.17 MPa.Growth factors (TGF-β1 and FGF-2) were included in the gel at a concentration of 10 ng / ml.

[0058] Also, 5 days after the installation of the in vivo bone bioreactor, the periosteum containing the developed subperiosteal regenerative blastema was removed from the rabbits. The scraped blastema cell mass from the inner surface of the periosteum was then partially minced and enzymatically disaggregated using collagenase type I. After centrifugation and resuspension, the isolated cells were 3D cultured in agarose 3D Petri Dishes®, bypassing the 2D culture step in culture flasks. Self-assembly of 3D microtissue—cellular spheroids from subperiosteal regenerative blastema cells—was carried out in an agarose well for 4 days. In a culture nutrient medium of the following composition: DMEM / P12 450 ml, L-glutamine 292 mg, 50 ml rabbit thrombolysate, penicillin 100 U / ml, streptomycin 100 μg / ml, the nutrient medium in agarose 3D Petri Dish® was replaced at least once every 2 days.The cultivation of cell spheroids was carried out in multi-gas incubators maintaining the composition of the gas environment in the inner chamber: 5% CO2, 10% oxygen and 85% nitrogen.

[0059] Then the spheroids were transferred to culture flasks with a non-adhesive surface for further cultivation; the so-called osteogenic cocktail was added to the culture nutrient medium of the above-described composition, creating concentrations of 100 nM dexamethasone, 0.05 nM L-ascorbic acid and 10 mM β-glycerophosphate in the culture nutrient medium, in which further cultivation of the cell spheroids was carried out for 12 days.

[0060] Cellular spheroids were subjected to histochemical studies, determining alkaline phosphatase expression levels and the degree of mineralization of the intercellular matrix of the cellular spheroids, with the detection of insoluble phosphate deposits within the spheroids (von Kossa staining). Signs of bone differentiation were detected.

Claims

1. A transplant for restoring the bones of a subject, comprising cellular spheroids based on autologous cells of the periosteum of the subject, the size of which is from 250 to 600 μm, characterized in that the spheroids consist of cells of the subperiosteal regenerative blastema of the subject, formed at the border of the periosteum exfoliated from the bone and the gel in vitro bioreactor, formed under the action of the bone in vivo bioreactor, in the form of an alginate gel introduced under the periosteum, which was obtained by mixing 2 vol.% sodium alginate in 30 mM Hepes, with an equal volume of a solution containing 75 mM CaCl2 in 10 mM Hepes, while the number of cells in the spheroid is 8000 pieces.

2. The graft of claim 1, wherein the cellular spheroid consists of uncultured subperiosteal regeneration blastema cells.

3. The graft of claim 1, wherein the cellular spheroid consists of cultured adherent cells of the subperiosteal regeneration blastema.