RU2386453C1 - Biotransplant based on foamed ceramic carriers of zirconium oxide and aluminium oxide system and multipotent stromal cells of human bone marrow for restoring extended bone tissue defects and method for making thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and represents a biotransplant based on a high-porous ceramic material of zirconium oxide - aluminium oxide system and multipotent stromal cells of human bone marrow for restoring the extended bone tissue defects characterised by that contains autologous or donor multipotent mesenchimal stromal cells (MMSC) of bone marrow and a carrier made of the high-porous ceramic material of zirconium oxide - aluminium oxide system by polyurethane foam base doubling with the carrier being densely seeded with MMSC cultivated within 1 to 3 passages; one carrier contains 200 to 500 thousand cells immobilised of the vitality at least 90%, while the functional targeting is verified by the ability to the directed differentiation of the mesodermal lines and the expression of stromal markers CD90 and CD105 in 60 - 90% of cells and the absence of expression of marker CD34 is shown.

EFFECT: invention provides accelerated repair processes and more effective osteointegration.

5 cl

Description

The invention is intended for use in regenerative medicine in order to repair bone defects resulting from traumatic and degenerative diseases.

The range of currently used osteoplastic matrices is very wide. Their main required characteristics are biocompatibility, osteoconductivity and sufficient mechanical strength. The introduction of the cellular component (osteoblasts, stem cells, etc.) into the composition of these materials makes it possible to impart osteoinductive properties to it. Of greatest interest for tissue engineering are multipotent stromal bone marrow cells (MSCs), capable of differentiation in osteogenic, chondrogenic and adipogenic directions [1]. The introduction of cells leads to the replacement of lost cellular elements, the induction of regeneration due to the allocation of growth factors and differentiation, the formation of an extracellular matrix, which significantly accelerates the restoration of damaged tissue [2]. Immobilization of cultured cells on a carrier matrix provides the highest concentration of cells in the area of damage, differentiation in a given direction and increases cell survival during transplantation.

Demineralized bone matrix (DCM) due to the natural architectonics, osteogenic potential due to the presence of biologically active substances, is one of the most popular materials for osteoplasty. The effectiveness of using carriers based on DCM with immobilized MSCs in the restoration of defects of the parietal bones in rats and dogs was shown [3]. Patented technology for the treatment of false joints using a biograft based on DCM and autologous MSCs [4]. The disadvantages of this xenogenic material are the need for treatment with aggressive fluids (reducing cell adhesion), potential immunogenicity and the possibility of transfer of infectious disease pathogens.

Widely used synthetic biodegradable carriers based on polylactic and polyglycolic acids and their copolymers in combination with MSCs also demonstrate in vitro induction of osteogenesis and bone formation after 6–9 weeks in animal experiments [5]. Membranes made of polylactides and / or polyglycolides impregnated with chondrocytes or bone marrow MSCs are patented as a method of treating bone and cartilage defects in humans and animals [6]. The disadvantages of such carriers is the rapid and not always predictable degradation of the matrices, which is accompanied by a local decrease in pH in the implant region, which reduces the effectiveness of their use [7].

The required mechanical strength of the matrices for bone tissue regeneration is determined by the use of non-resorbable carriers based on ceramics and alloys of various metals. It is known to use osteogenic MSCs of bone marrow deposited on a porous skeleton made of an alloy of titanium nickelide, previously coated with a collagen solution, for modeling the reparative function of the cancellous bone [8].

The present invention is the creation of a biograft (tissue engineering design) to make up for extended defects of bone tissue based on a porous ceramic foam carrier system of zirconium oxide-alumina and multipotent stromal cells of the human bone marrow.

The carrier proposed by us is made of non-resorbable oxide ceramic foam system zirconium oxide - alumina. Composite matrices based on these compounds, as well as with the addition of hydroxyapatite and other components, are characterized by high biocompatibility [9], the absence of antigenic activity, significant mechanical strength, and also osteoconductive properties [10]. Osteoinductance is provided by the introduction of multipotent stromal cells of the human bone marrow into the graft.

Ceramic foam carriers obtained by duplicating a polyurethane foam base are characterized by an extremely high (85-95%) porosity value developed by a labyrinth-arched structure consisting of interconnected open pores of 50-200 microns in size. Such a carrier structure makes it possible to create optimal conditions for both cell cultivation, providing sufficient gas exchange and supply of nutrients, and for bone formation in vivo. The microporous surface of the matrix due to the adsorption of biologically active macromolecules provides effective attachment and spreading of cells. Significant proliferative activity of MSCs cultivated on the surface of ceramic foam carriers allows for a short period (7 days) to obtain a 5 × 5 × 5 mm matrix densely populated by viable, functionally active cells.

The resulting tissue engineering design (TEC) is not toxic to surrounding tissues, because the material does not have a cytotoxic effect. The osteogenic potential of the MSC adhered to the surface of the carrier determines the osteoinductive properties of the structure. The forming function of the TEC is ensured by the mechanical strength of the matrix.

Preparation of a ceramic foam carrier

A highly porous ceramic material with a cellular structure (open-cell foam ceramics) was obtained by duplicating a polyurethane foam base. This method allows to obtain material with an extremely high (85-95%) value of porosity, which is characterized by a developed labyrinth-arched structure, consisting of interconnected open pores of 50-5000 microns in size. Manufacturing technology includes the following operations.

1. Preparation of a ceramic suspension with a given content of α-Al ₂ O ₃ and ZrO ₂ powders and the required complex of rheological characteristics; suspension moisture content in the range of 27-29%.

2. Cutting the workpiece from polyurethane foam grade PPU-30-100 TU 6-05-5127-82 with a given cell size (the number of holes per linear inch is 60 ppi).

3. Etching (activation) of PPU-billets.

4. Impregnation of PUF-blanks with a ceramic suspension.

5. Removing excess ceramic slurry by compressing the workpiece with a degree of deformation of 85%.

6. Drying the workpiece in air at 110 ° C for 4 hours.

7. Burnout of foam at 400 ° C for 1 hour.

8. Sintering in the temperature range (1600-1700) ° C for 1 hour.

9. Finishing.

Heat treatment of alumina-zirconia system powders in the temperature range (100-1000) ° C leads to a decrease in the specific surface of the powders from (180-230) to (32-50) m ² / g and the formation of the crystalline structure of the powders with the appearance of alpha and traces gamma phase of alumina and tetragonal and monoclinic phases of zirconium oxide.

The porous skeleton formed from powders according to the proposed technology has a uniform pore size distribution, rounded shape of jumpers, which are good prerequisites for the formation of composite material by the method of subsequent impregnation. The resulting ceramic foam frame with a porosity of 82-84% has a compressive strength of about 50 MPa, which is comparable with the strength of lamellar bone tissue.

Samples of bioceramics in the form of three-dimensional blocks 0.5 × 0.5 × 0.2 cm in size with pore diameters from 50 to 100 nm and PPI = 60 (number of holes per linear inch) are used to create a tissue-engineering design.

Obtaining a culture of multipotent stromal cells (MSCs)

The source of MSCs is a suspension of bone marrow obtained by exfusion from the posterior ileal crest of the donors.

A bone marrow aspirate is layered on a ficol solution separating gradient (density 1.077 g / ml) and centrifuged at 1300 rpm for 20 min at + 4 ° C. The interphase ring with nucleated cells is selected and centrifuged at 1100 rpm for 10 min at + 4 ° C. The supernatant is removed, and the cell pellet is suspended in culture medium (DMEM / F12 1: 1 (PanEco, RF) with the addition of 10% fetal calf serum (HyClone-Perbio, USA), 2 mM L-glutamine (PanEco) , RF) and 0.5 mg / ml amikacin (Synthesis of AKO, RF). A cell suspension is plated on plastic Petri dishes (Nunclon) with a diameter of 90 mm at a concentration of 10 ⁴ cells per 1 ml and incubated under standard conditions (temperature 37 ° C, under saturating with moisture in an atmosphere of 5% CO ₂₎ After 24 hours, nonadherent cells were removed and adherent cells were washed with culture cFe they are incubated until 75-85% confluency is reached. Subsequently, the medium is changed every 2-3 days depending on the growth characteristics of the culture. The first colonies appear after 7-10 days. Their growth dynamics and cell morphology are analyzed, and small-cell ones are selected according to the results fast-growing colonies for further propagation Selected cells are incubated until an incomplete monolayer is reached (no more than 50% confluency) and again scattered in the same density. Cultivation is carried out up to 3-4 passages until the required amount of cell mass is obtained (within 4-5 weeks).

At all stages of isolation, immunophenotypic characterization of MSCs is performed by specific, satellite, and negative markers (vimentin, CD 45, HLA-ABC, CD 29, CD 44, CD 49d, CD 71, CD 73, CD 90, CD 105, CD 106, CD 54) using a FACS Caliber flow cytometer (BD Biosciences, USA).

Preparing the carrier matrix

Ceramic foam carriers are subjected to sterilization by γ-irradiation at a dose of 2.5 Mrad. This sterilization method does not cause changes in physico-mechanical and biomedical properties. Then the media is washed with saline and dried in a stream of air.

Methods of populating MSC samples

Prepared samples are placed in 12 or 24-well plates (Nunk, Denmark). To prepare the cell suspension, MSCs at passage 2–3 were removed from Petri dishes with trypsin-Versen solution, centrifuged at 1100 rpm for 10 min at + 24 ° С, and the precipitate was resuspended in the culture medium. The resulting suspension with a cell concentration of 1 to 2 million in 1 ml of medium is applied to the samples at the rate of 100 μl of suspension per 100 mg of sample. Due to the pronounced capillary properties of the carrier, the suspension quickly penetrates inside. Incubated for 60 min under standard conditions to initiate the attachment of cells to the walls of the pore channels and culture medium is added. After a day, the samples are transferred to new plates. The change of culture medium is carried out every two days.

Assessment of cytocompatibility of carrier matrices

The cytotoxicity of the samples was assessed using an MTT test based on the ability of living cell mitochondria succinate dehydrogenase to restore light yellow MTT tetrazolium (3- (4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide) to insoluble, dark-colored formazan extractable using organic solvents (DMSO). The test results showed that ceramic foam samples of the zirconium oxide - alumina system did not have a toxic effect on MSCs in the in vitro system. The absence of carrier degradation eliminates the likelihood of a toxic effect in longer in vivo experiments.

Samples having an open-cellular structure with a porosity of 82-84% and a rough relief are characterized by a large specific surface area. Due to this, the matrices have a pronounced capillary effect - a cell suspension applied to previously prepared samples immediately penetrates inside. The microporous matrix structure formed by microparticles of oxide compounds (sizes from 200 to 900 nm) promotes the adsorption of biologically active macromolecules from the culture medium, which stimulate cell attachment and spreading. According to electron microscopy, cells on ceramic foam carriers look well spread over the surface of the matrix, tightly adjacent to it. Thus, the structured surface of the test samples, as expected, is a favorable basis for cell adhesion.

Quantitative and visual assessment of MSC growth revealed a significant proliferative activity of cells upon cultivation on the surface of ceramic foam carriers of size 5 × 5 × 5 mm. The average time for doubling MSCs was about 72 hours. This allows for a short period (7 days) to obtain a matrix densely populated by cells. The survival rate of cells cultured in the samples is at least 95%. Moreover, the distribution of cells in the volume of the carrier is quite uniform. Morphologically, the cells adhered in the back of the carrier do not differ from those located on the periphery - they have a similar elongated or polygonal shape and are very flat. These results indicate the effective colonization of MSC carriers throughout the volume and good cell survival in the central regions, which suggests the possibility of relatively simultaneous and efficient bone formation in the depth and along the periphery of the carrier.

The use of the developed TECs to make up for long bone defects involves the manufacture of carriers of various sizes and shapes. The selected structure of ceramic foam carriers and cultivation conditions allow us to effectively populate and grow MSCs on carriers with a diameter of 18 mm. The cells inside such carriers do not morphologically differ from those cultured on smaller samples, are also fairly evenly distributed over the volume of the sample, and their survival rate is about 90%. Thus, the possibility of efficient cultivation of cells at a distance of up to 900 μm from the edge of the carrier is shown, and therefore, the manufacture of grafts to replace defects up to 18 mm thick is justified. It should be noted that TEC transplantation of such large sizes requires the use of additional methods for the induction of angiogenesis in order to increase cell survival in vivo.

The cultivation of MSCs on carriers with sizes from 5 mm to 18 mm in cross section is carried out under standard conditions and does not require the creation of any additional conditions, for example, perfusion of the medium (as recommended when creating other tissue-engineering structures). Experimental data indicate that the selected carrier structure with macropores ranging in size from 50 to 200 microns provides sufficient gas exchange and supply of cells with nutrients, without requiring the creation of special cultivation conditions.

The total production time of the graft from the moment of bone marrow collection to the receipt of a fully populated MSC of the ceramic foam matrix is on average about 6-7 weeks and is determined by the concentration and proliferative activity of MSC in the bone marrow suspension and the size of the carrier, setting the required number of cells for population.

The graft is a carrier densely populated by MSCs, cultured from 1 to 3 passages, in depth and along the periphery. From one carrier, 200 to 500 thousand cells are immobilized. Vitality, determined by the exclusion of staining with 0.4% trypan blue, is at least 90%. The functional activity of MSCs is confirmed by the ability to directed differentiation into mesodermal lines in inductive media. Immunophenotypic analysis demonstrates the expression of stromal markers CD90 and CD 105 in 60-90% of cells and the lack of expression of the marker CD34.

The effectiveness of the proposed fabric engineering design

Experimental study

Animals

We used sexually mature male outbred rats weighing 180-200 g. The animals were kept on a standard laboratory diet. 24 hours before surgery, the animals did not receive food. Conducted 3 series of experiments on 72 animals.

Experimental models

The developed methodology of operations in all series of experiments was the same with the observance of asepsis rules.

Work with laboratory animals was carried out in accordance with the order of the USSR Ministry of Health No. 755 of 08/12/1977. To conduct an experimental study, permission was obtained from the Bioethics Commission of Remetex CJSC (Minutes No. 3 dated 02.21.2008). Surgical interventions were performed under ether anesthesia, before and after the operation, animals were administered non-narcotic analgesics.

To study the reparative processes of bone tissue during transplantation of a tissue-engineering construct, a defect was applied in the area of the cranial vault of animals. Before surgery, the hair from the brain of the rat skull was sheared. In the area of the parietal and inter-dark parietal bones of the skull, a 3-cm-long skin incision was made. Fascia and muscles were dissected in layers, removed to the sides and exposed the surface of the bones, the periosteum was bluntly peeled. A bone defect was applied in the middle of the cranial vault, using a circular dental cutter, cutting a rounded hole with a diameter of 7 mm without damaging the dura mater, which was indicated by the absence of heavy bleeding from the cerebral venous sinuses. To stop bleeding from the endosteum and cancellous bone, gauze swabs were used. In the bottom of the defect, a tissue-engineering structure (TEC) or a carrier matrix without cells was placed on the surface of the dura mater, so that its edges fit snugly against the edges of the defect, after which the defect was closed with the periosteum and the wound was sutured in layers. Anesthesia and surgery lasting 15-20 minutes, all animals tolerated satisfactorily without early and late complications.

To study the repair of soft tissues, neoangiogenesis, germination of TECs by the tissues of the recipient, TECs were injected subcutaneously in the back of the rat. To do this, the hair on the back of the animal was sheared and made two cuts, one in the withers area and the other 4 cm more caudal than the first cut. The fascia was diluted in a blunt manner and the TEC was immersed in both formed beds, after which the wound was sutured.

Series of experiments

During the study, 3 series of experiments were conducted.

1. Group 1a — TEC (a carrier matrix based on foam ceramics and MSCs pre-cultured on it) were introduced into the bone defect of the skull, n = 12.

2. Group 1b — a carrier matrix (based on ceramic foam) without cells was introduced into the bone defect of the skull, n = 12.

3. Group 2a - TIC (matrix-carrier based on foam ceramics and MSCs pre-cultured on it) were injected subcutaneously in the back region, n = 12.

4. Group 2b — a carrier matrix (based on ceramic foam) without cells was injected subcutaneously in the back region, n = 12.

5. Group 3a - TIC (a support matrix based on polylactic acid and MSCs pre-cultured on it) were injected subcutaneously in the back region, n = 12.

6. Group 3b - a matrix of the carrier (based on polylactic acid) without cells was injected subcutaneously in the back, n = 12.

The animals were removed from the experiment in all series on the 21st and 60th day of observation by cervical dislocation under deep ether anesthesia.

Material analysis

In the first series of the experiment, the isolated bone-marrow objects were fixed in 10% neutral formalin and decalcified in a 25% Trilon B. solution. The fragments of the obtained tissues were poured into paraffin and serial sections were prepared according to the generally accepted technique. Sections were stained according to Mallory. The preparations were examined using a Zeiss Axioplan-2 microscope; photography was performed with a Hitachi HV-C20A digital camera.

To prove the reproducibility of damage to the bone tissue of the skull for the experimental and control groups, the total regenerate area was determined using the Scion Image 3.0 morphometric program.

The use of one-way analysis of variance demonstrated the absence of statistically significant differences between groups (achieved confidence level p = 0.975). Thus, the reproducibility of damage was shown, which led to the correctness of the comparison of the shares of the structural components of the regenerate.

To conduct morphometric analysis on microphotograms in Scion Image 3.0, the entire regenerate and its components were isolated: TEC, connective tissue and bone part of the regenerate integrated with TEC, inside which mature bone regenerate was taken into account.

The area of the regenerate and each of the components of the regenerate was determined in pixels and converted into arbitrary units (1 conventional unit = 10 pixels).

The obtained values of the areas of the structural components of the regenerate were correlated with the value of its total area. These relative values were used in the statistical processing of the material, which was carried out using the program Statistica 6.0.

If the distribution in the subgroup was not normal, the data was transformed. For comparison of means used student's criterion.

Experimental Results

The twenty first day of observation

In the experimental and control groups, on the 21st day after the traumatic injury, no hematomas and signs of an inflammatory reaction were detected in the defect area. TEC and regenerate completely filled the space between the fragments. Edges of fragments devoid of osteocytes underwent active resorption.

Bone callus consisted of two parts: connective tissue and bone. Loose unformed connective tissue was located between the edges of the TEC and the maternal bone, while it grew into the region of the TEC. The connective tissue in the regenerate was characterized by a high density of proliferating cellular elements. The cells had a fusiform, polygonal or rounded shape and were surrounded by a fibrous intercellular matrix. Around the blood vessels were dense clusters of fibroblast-like cells, which acquired a rounded and flat shape and formed unevenly colored primary bone beams.

Bone regenerate structures located randomly between areas of connective tissue were represented by a network of bone trabeculae. Reticulofibrotic bone tissue occupied an insignificant part of the regenerate between the TEC and the maternal bone. The surface areas of the regenerate were represented by a network of immature bone trabeculae with an unevenly colored matrix and loose connective tissue and capillaries of blood vessels in the intertrabular spaces. In the experimental group (1a) at these periods of observation, the regenerate was mostly represented by loose fibrous connective tissue and, to a lesser extent, bone regenerate, as well as in the control group, but there were more sites of reticulofibrotic bone in the experimental group. In deeper regions of the regenerate, the newly formed bone tissue underwent compaction as the bone trabeculae thickened, the cell density in the intercellular substance decreased, and the bone matrix was more intensely stained.

Thus, on the 21st day of the experiment, active intramembranous reparative osteogenesis was observed in all groups. The bone defect was completely filled with TEC and regenerate tightly welded with it; the latter was represented by loose connective tissue and reticulofibrotic bone tissue. The differences between the experimental and control groups consisted in the rate of reparative processes, that is, in the dynamics of an increase in the proportion of the bony part of the regenerate and a decrease in the proportion of loose connective tissue.

The results of the morphometric study also revealed statistically significant intergroup differences.

In the group with the introduction of TIC into the bone wound, an intensive increase in the share of the bone part of the regenerate was observed (38.4 ± 4.7% versus 20.1 ± 2.3%) with a corresponding decrease in the proportion of connective tissue (62.2 ± 9.7% against 81.3 ± 12.1%) compared with the control group (p <0.0001). The appearance of signs of bone compaction was also noted, in some sections mature compact bone tissue was revealed, the proportion of which was 1.78 ± 0.8%, while it was absent in the control group.

Thus, based on the data of morphometric studies, it can be argued that on the 21st day after traumatic bone damage under the influence of the introduction of a carrier matrix with pre-cultured MSCs in the bone wound, there is an increase in the share of the bone part of the regenerate and a decrease in the share of connective tissue. Acceleration of reparative processes is also expressed in the fact that in the experimental group at this time there were signs of bone marrow maturation.

60th day of observation

On day 60 of the experiment, as well as 21, there were no signs of transplant rejection in the area of traumatic injury. The space between the TEC or the carrier matrix was completely filled with regenerate. In this case, a tight fit of the bone tissue to the surface of the implant was observed with interposition of loose fibrous connective tissue and bone tissue. That allows us to argue about effective osseointegration between TECs and maternal bone. Part of the bone regenerate underwent active resorption, and the bone acquired the physiological shape and size of the original bone.

Bone callus at these times consisted mostly of bone tissue. Only insignificant areas of loose connective tissue remained in the intertrabular spaces of the reticulofibrotic bone. Reticulofibrotic bone tissue was represented by wide powerful bone trabeculae, intertrabular spaces in the form of narrow channels filled with bone marrow and blood vessels.

Around the blood vessels one could see the formation of concentrically located bone plates, which were more intensely stained. In animals that were injected with TECs, there were more such sites with a more intensively stained bone matrix, which indicates active maturation of bone regenerate. In terms of maturity, the regenerate was approaching the original bone, however, the edges of the fragments of the original bone were still resorbed and empty bone lacunae could still be seen.

Thus, on the 60th day of the experiment, restoration of the bone structure was noted, the regenerate was almost completely represented by bone tissue, which could provide support function. The rate of reparative processes in all groups decreased, the processes of mineralization and remodeling of bone tissue in accordance with the functional load of the bone organ came to the fore.

After 2 months from the start of the experiment in the experimental group, the rate of reparative processes remained higher than in the control group. In the control group, some animals still observed areas of loose fibrous connective tissue (4.6 ± 1.1%). The bone regenerate remodeling processes in the experimental group proceeded noticeably faster, the share of compact bone tissue in this group was 57.3 ± 7.9% versus 31.5 ± 4.6% in the control group (p <0.0001). In the control group, the intertrabular spaces were filled with bone marrow; in the experimental group, the intertrabular spaces were already significantly smaller, and they were presented in the form of narrow channels with blood vessels passing through them.

findings

1. Transplantation into the bone defect region of the matrix carrier based on the ceramic foam system of zirconium and aluminum oxides does not cause pathological changes in the surrounding tissues and does not cause changes in the general condition of rats.

2. The transplanted carrier matrix based on the ceramic foam system of zirconium and aluminum oxides effectively engraft in the defect area and integrates with the surrounding bone tissues. Between the transplant and the maternal bone tissue, full bone tissue is formed.

3. Transplantation of a carrier matrix based on ceramic foam system of zirconium and aluminum oxides with pre-cultured bone marrow MSCs accelerates reparative processes and more effective osseointegration.

4. The results obtained provide an experimental justification for the use of allogeneic transplantations of prenatal multipotent mesenchymal stromal cells and chondroblasts in the treatment of extensive bone defects in clinical practice.

Thus, the zirconium oxide – alumina systems proposed for the creation of a TEC PC matrix are not toxic to surrounding tissues, because do not have a cytotoxic effect. Due to their structure, carriers are characterized by high capillarity and adhesiveness, which ensures the efficiency of their colonization of MSCs. High proliferative activity allows for a short time to obtain a carrier densely populated by viable, functionally active cells. Moreover, all these characteristics are preserved in cells cultured at a depth of 900 μm from the surface of the carrier without creating additional diffusion of the medium. Sufficient mechanical strength of the carriers and the ability to simulate the necessary shape determines their application for the restoration of bone defects.

References

Claims (5)

1. A biograft based on a highly porous ceramic material of the zirconium oxide-alumina system and multipotent stromal cells of the human bone marrow for the restoration of extended bone defects, characterized in that it contains autologous or donor multipotent mesenchymal stromal cells (MMSCs) from the bone marrow and a carrier created by the bone marrow based on the highly porous ceramic material of the zirconium oxide-alumina system using the technology of duplication of a polyurethane foam base, at m the carrier is densely seeded with MMSCs cultivated from 1 to 3 passages, while from one carrier from 200 to 500 thousand cells are immobilized, the vitality of which is at least 90%, and the functional orientation is confirmed by the ability to directed differentiation into mesoderm lines and expression of stromal markers is demonstrated CD90 and CD 105 in 60-90% of cells and lack of expression of the marker CD34. 2. A biograft based on foam ceramic carriers of the zirconium oxide-alumina system and multipotent mesenchymal stromal cells of the human bone marrow for the restoration of extended bone defects according to claim 1, characterized in that bioceramic samples in the form of three-dimensional blocks of size 0 are used as a carrier 5 × 0.5 × 0.2 cm with a pore diameter of 50 to 100 nm and PP1 = 60 (the number of holes per linear inch), and the powders of the alumina – zirconia system are heat treated in the temperature range le (100-1000) ° C, which leads to a decrease in the specific surface of the powders from (180-230) to (32-50) m ² / g and the formation of the crystalline structure of the powders with the appearance of alpha and traces of the gamma phase of aluminum oxide and tetragonal and monoclinic phases of zirconium oxide, and thus, the porous skeleton formed from powders according to the proposed technology has a uniform pore size distribution, rounded shape of jumpers. 3. A biograft based on ceramic foam carriers of the zirconium oxide-alumina system and multipotent stromal cells of the human bone marrow for the restoration of extended bone defects according to claim 1, characterized in that the source of MSCs is bone marrow suspension obtained by exfusion from the posterior iliac crest donors. 4. A biograft based on ceramic foam carriers of the zirconium oxide-alumina system and multipotent stromal cells of human bone marrow for the restoration of extended bone defects according to claim 1, characterized in that it is a carrier densely populated by MSCs cultured from 1 to 3 passages, in depth and on the periphery. 5. A method of producing a biograft based on foam ceramic carriers of the zirconium oxide-alumina system and multipotent stromal cells of the human bone marrow for the restoration of extended bone defects according to claim 1, characterized in that the obtained bone marrow aspirate is layered on a ficol solution separating gradient (density 1.077 g / ml) and centrifuged at 1300 rpm for 20 min at + 4 ° С, the thus obtained interphase ring with nucleated cells was selected and centrifuged at 1100 rpm for 10 min at + 4 ° С, while the supernatant is removed, and the cell pellet is suspended in the culture medium (DMEM / F12 1: 1, with the addition of 10% fetal calf serum or autologous serum, 2 mM L-glutamine and 0.5 mg / ml amikacin, the cell suspension is plated on plastic Petri dishes at a concentration of 10 ⁴ cells per 1 ml and incubated under standard conditions (temperature 37 ° C, in conditions of saturation humidity in the atmosphere with 5% CO ₂ ), then the loose cells are removed, and the attached cells cells are washed with culture medium and incubated until reaching 75-8 5% confluence, in the future, the medium is changed every 2-3 days depending on the growth characteristics of the culture, while cultivation is carried out up to 3-4 passages until the required amount of cell mass is obtained, while the ceramic foam carriers are sterilized by γ-radiation at a dose of 2 , 5 Mrad, then the carriers are washed with physiological saline and dried in a stream of air, prepared carrier samples together with the obtained cell suspension with a cell concentration of 1 to 2 million in 1 ml of medium are applied to samples from p counts of 100 μl of suspension per 100 mg of the sample, incubated for 60 min under standard conditions to initiate the attachment of cells to the walls of the pore channels and add culture medium, the samples are transferred to new plates during the day, and then the culture medium is changed every two days .