RU2530622C2 - Biotransplant for recovery of bone tissue volume in case of degenerative diseases and traumatic injuries of bones and method of obtaining thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, namely to cell transplantology for the regeneration of a bone tissue in surgical treatment of destructive, degenerative-dystrophic, traumatic or congenital affections of the bone tissue. A biotransplant contains a mixture of autologous multipotent mesenchymal stromal cells, cultivated in standard conditions, and multipotent mesenchymal stromal cells, differentiated in the osteogenic direction, taken in a weight ratio of 1:1 and in quantity of 10⁷ cells per 1 g of the weight of a matrix-carrier, which has in the basis of its structure a collagen-mineral complex, identical in the composition to natural bone material. A method of the biotransplant obtaining is based on the application of the culture medium AdvanceSTEM^TM/Antibiotic/Antimycotic Solution 100x with an addition of 10% of autologous serum of the patient's blood.

EFFECT: technical result of the invention consists in the provision of active angiogenesis with the formation of small capillaries, medium and large mature vessels in a young bone tissue together with the preservation of its osteoinductive properties; elimination of a risk of the immune response and an infection transmission caused by the application of embryonic calf serum; reduction of terms of the bone tissue recovery and its vascularisation.

2 cl, 2 dwg, 2 tbl

Description

The invention relates to medicine, namely to cell transplants for bone tissue regeneration in the surgical treatment of destructive, degenerative-dystrophic, traumatic or congenital bone tissue lesions.

In some cases, diseases or malformations lead to permanent loss of bone tissue in connection with the development of various pathological processes [Grigoryan A.S., Toporkova A.V. Problems of integration of implants into bone tissue (theoretical aspects). - M .: Technosphere, 2007, - 130 p.].

Surgical treatment of bone deficiency and defects is aimed at restoring lost tissue in its original histoarchitectonics and function. Currently, implant materials for targeted bone regeneration are widespread in widespread clinical practice. The effectiveness of bone restoration in this case during surgical treatment mainly depends on the properties of implant materials, which replace the defect.

Known materials for implantation in tissues based on resorbable and non-resorbable osteoinductive substances, which may be of mineral or biological origin. Thus, implantation in the subperiosteal region of collagen-based materials containing bone powder of hydroxyapatites, tricalcium phosphates using drugs - glycosaminoglycans [RU 2159101 C1, 25.03.99] is known.

Known biocompatible material for dentistry, including inorganic salts, collagen, sulfated glycosaminoglycans and water [RU 2155024 C1, 11.25.2000].

Various types of bone auto, allo, and xenografts are also widely used. It is known to use various types of cell cultures, often fibroblasts, as optimizers for wound healing in surgical periodontics [Fang B, Song Y, Lin Q, Zhang Y, Cao Y, Zhao RC, Ma Y. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft-vs.-host disease in two children. Pediatr Transplant. 2007 Nov; 11 (7): 814-7]. It is known to implant a culture of postnatal fibroblasts delivered to the area of a bone defect on a carrier - synthetic hydroxyapatite or on the dura mater of a human embryo [RU 2210352 C1, 08.20.2003].

It is known to use a collagen-based biological prosthesis to repair a bone defect, moreover, a hemostatic collagen sponge is used as a base on which autologous bone marrow is applied and impregnated with estrogen in a dose of 0.5 mg / kg [RU 2055535 C1, 03/10/1996].

The disadvantages of the above implants are due to low efficiency (limited indications for use), possible side effects. There is a direct correlation between the effectiveness of such implants and the type of defect, for example, cranial bones or long tubular bones, since the former are not capable of independent regeneration at all, and stimulation of regeneration in the latter is possible only with small defects with at least 3 supporting walls [Stosich MS, Mao J J. Adipose tissue engineering from human adult stem cells: clinical implications in plastic and reconstructive surgery. Plast Reconstr Surg. 2007 Jan; 119 (1): 71-83].

The use of autografts is always accompanied by an additional surgical field for its collection and does not guarantee survival in the transplant bed.

Known material based on a composition comprising fibroblasts in the composition of hydroxyapatite [RU 2210352 C1, 08/20/2003]. The use of fibroblasts improves the therapeutic effect due to the stimulating activity of the cellular component, which helps to reduce the healing time of the wound surface, reduce the severity of postoperative gum recession and increase the duration of remission of a chronic disease. However, the use of hydroxyapatite as a carrier has the disadvantages indicated above, which causes a relapse of the disease in a significant percentage of cases, and is also accompanied by a decrease in the strength of the newly formed bone to mechanical stress. In addition, the cellular component of the material construction is used with insufficient efficiency due to the short shelf life of this composition (3-4 days) [Lendeckel S, Jödicke A, Christophis P, Heidinger K, Wolff J, Fraser JK, Hedrick MH, Berthold L, Howaldt H P. Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: case report. J Craniomaxillofac Surg. 2004 Dec; 32 (6): 370-3]. Thus, this material design is not optimal for maintaining cells in a viable state for a long time, which limits its widespread use in clinical practice.

Known bioengineered design for closing bone defects with restoration of bone tissue [RU 2416434 C1, 04/20/2011], which is a hybrid implant in the form of a porous polytetrafluoroethylene membrane with a multifunctional biocompatible non-resorbable coating (MBNP) doped with M-Ca-PCON or M-Ca-CON, where M is a metal selected from the series Ti, Zr, Hf, Nb, Ta, on the surface of which autologous or allogeneic stromal cells isolated from adipose tissue or bone marrow are passaged. The disadvantage is the use of animal blood serum for cell cultivation, which can lead to an immune response and the risk of infection transmission.

Known biotransplant for the treatment of degenerative and traumatic diseases of the bone tissue of the maxillofacial region [RU 2380105 C1, 01/27/2010], characterized in that it contains autologous or donor multipotent mesenchymal stromal cells (MMSCs) from bone marrow or adipose tissue of adult donors that are distributed in a fibrin clot at a concentration of 5-7 million cells per 1 ml, which is a polymerized platelet-rich plasma of the patient’s blood, and a carrier matrix, which has collagen a mineral complex identical in composition to natural bone material, or a biocompatible polymer. This biograft is selected for the prototype in the part of the “substance” object. The disadvantage is the insufficient efficiency of bone tissue restoration, and there is a risk of infection with prions and the occurrence of an immune response in a patient due to the use of fetal calf serum in the culture medium.

There is a method of obtaining a bioengineered design, which consists in the fact that stromal cells are isolated from the adipose tissue or bone marrow of the recipient, including progenitor cells capable of differentiating in the osteogenic direction, followed by cultivation and obtaining a cell population, which includes: washing the tissue in saline solution with antibiotics, tissue grinding, incubation in a solution of 0.1% collagenase at 37 ° C and constant stirring for 90 min, inhibition of isolated farms enta by adding 10% fetal calf serum (FCS), separation of mature adipocytes by centrifugation at 300 g for 10 min, washing the obtained cell pellet from the enzyme in DMEM (Sigma) containing 10% FCS, filtering the resulting cell suspension through a nylon filter and its separation by centrifugation at 400 g for 30 min at room temperature, triple washing of the obtained fraction of mononuclear cells in the form of a suspension in DMEM medium, while cell cultures were cultured until the first passaging in DMEM medium containing 20% serum mouths, then in DMEM medium containing 10% FCS, after which SCLC cultures are passaged onto the surface of MBNP of a hybrid implant, which is a porous polytetrafluoroethylene membrane with pore sizes of 200-500 μm, while MBNP is doped with M-Ca-PCON or M- Ca-CON, where M is a metal selected from the series Ti, Zr, Hf, Nb, Ta, 24 hours after passaging the surface of MBNP, the culture medium is changed to osteogenic and osteogenic stimulation is carried out using the following composition: DMEM medium, 10% FCS, 0.01 μM 1,25-dihydroxyvitamin D3 (Sigma), 50 μM ascorbate-2-phosph at (Sigma), 10 mM b-glycerophosphate (Sigma), when changing the medium every 3 days, then the cells were cultured in the induction medium for 14 days, after which the obtained bioengineered construct was used as intended [RU 2416434 C1, 04/20/2011].

A known method of producing a biograft for the treatment of degenerative and traumatic diseases of the bone tissue of the maxillofacial region [RU 2380105 C1, 01/27/2010]. The platelet-rich blood plasma of the patient and the cell culture are obtained. The bone marrow obtained from an adult donor, including for autologous transplantation, can also serve as a source of MMSC; adipose tissue obtained from adult donors by lipoaspiration using a standard technique is also used to obtain MMSC. If the cells are not extracted from aspirates (bone marrow, lipoaspirate), but from dense tissues, such as the thymus, the organ is preliminarily crushed and enzymatically disaggregated. The suspension is filtered through a fine mesh stainless steel sieve. The cell suspension (aspirate) is diluted 1: 1 with Hanks saline, centrifuged in the mode of 1,500 × g for 10 minutes and resuspended in DMEM / F12 growth medium containing 15% fetal calf serum, selected for cell growth in low density, 2 mm glutamine. A cell suspension is plated on plastic Petri dishes with a diameter of 100 mm. The seeding density of the primary cell suspension is 1-20 million mononuclear cells per 1 cm ² depending on the source of excretion. After 1 day, non-adherent cells are removed, the growth medium is replaced. The cultures are incubated at 37 ° C in an atmosphere of 5% CO ₂ . After 6 days, growth of heterogeneous cell populations is observed. Once the culture reaches 50% confluency, the monolayer is trypsinized and replanted onto new plates with a density of 10-30 cells per 1 cm in a growth medium additionally containing 10 μg / ml transferrin, 1 μg / ml insulin, 10 ng / ml fibroblast growth factor - 2 and 8 U / ml heparin. After 7-10 days, dense colonies of small cells (7-10 microns in diameter) with a large number of mitoses are selected. The selected colonies are seeded in new plates with a density of 20 cells / cm and grown to a state of pre-confluency. The medium is replaced every 2 days. Subsequent passages are made in the same mode.

The method is as follows.

1. The creation of a suspension of cell culture and autoplasma. MMSCs intended for transplant preparation are pre-enzymatically disaggregated with trypsin and 5-7 million cells per 1 ml of platelet-rich plasma are added. A suspension of cells in an autogenous platelet-rich plasma is obtained by simply mixing the cell suspension and the plasma itself, and if necessary with the addition of all additional components with thorough mixing.

2. Preparation of the carrier matrix. The matrix in the form of blocks, chips or chips of material based on a collagen-mineral complex or biocompatible polymers (for example, polylactides, polyglycolides, polyhydroxyalkanates and the like, and combinations thereof) is washed with Hanks solution with cefazolin (1 g / l). In the container intended for the manufacture of the transplant, place a block, chips or chips of a given size and volume and not yet frozen plasma containing MMSC. A suspension of cells in the autoplasma is mixed with the material. Then, a thrombin solution of 50 U / ml in a 10% calcium chloride solution is added dropwise until it thickens. The polymerization process occurs at a temperature of 37 ° C for 30-40 minutes. Subsequently, the obtained tissue-engineering construction in the form of a biograft with a frozen fibrin clot is cultivated at 37 ° C for 3 to 30 days in an osteogenic culture medium, which may contain prednisone, vitamin C, dexamethasone, vitamin D (in the form of various isomers) depending on clinical tasks. Thus, the duration of the entire technological process of manufacturing the complex is from 3 to 30 days. The transplant obtained at this stage may be subject to storage at a temperature of 26-37 ° C in an atmosphere of air containing 2-7% CO ₂ and saturating humidity for 3 weeks.

The objective of the present invention is to increase the efficiency of recovery of bone deficiency.

The technical result of the invention is to ensure active angiogenesis with the formation of small capillaries, medium and large mature vessels in young bone tissue along with the preservation of its osteoinductive properties; eliminating the danger of an immune response and infection due to the use of fetal calf serum; reduction of bone restoration time.

The technical result regarding the “substance” object is achieved through the use of a mixture of MMSCs cultured under standard conditions and MMSCs differentiated in the osteogenic direction, taken in the ratio 1: 1 and in the amount of 10 ⁷ cells per 1 g of the weight of the carrier matrix.

As clinical dental practice has shown, the use of biografts (tissue engineering structures), which include MMSCs differentiated in the osteogenic direction, does not satisfy specialists in terms of qualitative indicators of the formed young bone, namely, angiogenesis indicators. Since it is known from the literature and from the results of our own research that MMSCs have high regenerative potential, primarily due to their ability to exert a paracrine effect on surrounding cells, that is, due to the production of growth factors, cytokines and trophic factors by these cells, it seemed necessary to use MMSCs cultivated under standard conditions and possessing the above properties as a component of a biograft. Also, to increase the efficiency of bone formation in its composition, MMSCs differentiated in the osteogenic direction were used. Using a combination of cells as part of a biograft allows you to simultaneously solve two main problems in the regeneration of bone tissue: trophism due to the formation of vessels and capillaries (MMSCs cultured under standard conditions) and the formation of mineralized bone tissue at the site of a bone defect (MMSCs differentiated in osteogenic direction) . In Fig.1 and Fig.2: 1 - newly formed bone trabeculae, 2 - vascular structures (brown staining), and 3 - granulation tissue, indicating inflammation. Significant formation of bone beams and mature, consisting of 3 membranes of blood vessels, is noted.

The data of our clinical studies, followed by histological analysis, indicate that the use of such a combination significantly increases the content of forming bone tissue and the formation of large blood vessels in comparison with tissue-engineering structures based on MMSCs cultured under standard conditions.

To evaluate the characteristics of newly formed bone tissue, a morphometric assessment of the bone area was performed on random histological sections stained with hematoxylin (n = 270). The preparations were photographed using an Axiovert 40 microscope (Zeiss, Germany), an AxioCam MRC color digital camera (Zeiss, Germany), and AxioVision 4.6 software. Morphometric analysis was performed using the MetaMorph 7.1.0.0 software (Universal Imaging Corporation, USA).

As a result of a morphometric analysis of bone tissue formation (table 1) using biotransplants on a KollapanG matrix carrier, it was shown that the use of a mixture of MMSC cells cultured under standard conditions (AdvanceSTEM ^TM (HyClone) / Antibiotic / Antimycotic Solution 100x (HyClone) with the addition of 10% autologous blood serum of the patient), with MMSC cells differentiated in the osteogenic direction in AdvanceSTEM ^™ (HyClone) / Antibiotic / Antimycotic Solution 100x (HyClone) medium and 10% autologous blood serum of the patient with the addition of ascorbic acid and dexamethasone , and taken at a ratio of 1: 1 in the amount of 10 ⁷ cells per 1 g of the weight of the carrier matrix, significantly increases the content of forming bone tissue compared with the use of tissue-engineering structures based on MMSCs cultured under standard conditions (AdvanceSTEM ^TM (HyClone) / Antibiotic / Antimycotic Solution 100x (HyClone) and 10% autologous patient serum).

Table 1

* p <0.05

Vascular counting and statistical analysis were performed (table 2). The number and size of vessels and capillaries was evaluated on random histological sections immunohistochemically stained with antibodies to the CD31 endothelial cell marker. Vascular counting was performed using MetaMorph 5.0 (Universal Imaging) and Adobe PhotoShop (Adobe Systems). Vascular density was calculated on sections in 4-5 fields of view (field of view 1.107 mm ² ) in 3 random sections for each biopsy sample at a magnification of 100x. Moreover, all the vessels were divided into 3 groups: capillaries (CD31-positive formations without clearance or less than 20 microns in length), medium-diameter vessels (CD31-positive formations from 20 to 40 microns in length), large vessels (with a diameter of more than 40 microns); the number of vessels in each group was counted separately. Statistical analysis of the data was carried out using the Statistica 6.0 program, the nonparametric Mann-Whitney test was used.

table 2

* p <0.01

The calculation of newly forming large vessels showed that as a result of the use of a mixture of MMSCs cultured under standard conditions and MMSCs differentiated in the osteogenic direction on the KollapAn-G carrier matrix, the number of large vessels significantly increased compared to the use of MMSC biotransplant cultivated under standard conditions, in combination with KollapAn-G. From the results obtained, it can be concluded that the use of tissue-engineering design based on a mixture of MMSCs taken in a ratio of 1: 1 in the amount of 10 ⁷ cells per 1 g of the weight of the carrier matrix — CollapAn-G is reliably optimal in terms of stimulating angiogenesis .

The experimental data obtained by the authors indicate that the ratio of multipotent mesenchymal stromal cells cultured under standard conditions and multipotent mesenchymal stromal cells differentiated in the osteogenic direction, 1: 1 and the number of cells 10 ⁷ per 1 g of carrier matrix weight are optimal from the point of view of stimulating the general processes of regeneration and formation of blood vessels and the formation of young mineralized bone 3 months after transplantation of tissue einzhenernoy design.

From the description of the examples in the prototype method, it follows that the biograft is installed after a certain period of time (exact dates are not indicated, they are called only months, so it can be assumed that a month later) after CT, which is done 3 months after the biotransplant is introduced into the defect area . Histological examination of the newly formed bone tissue is carried out with the installation of an implant, i.e. after another month (thus, it takes about 4 months from the moment of transplantation of the tissue-engineering structure to the installation of the implant). At the same time, CT is not a reliable method of research, and histological examination is considered the "gold standard". The results of a histological examination of young bone tissue formed using the tissue-engineering construct proposed in this study, the transplantation of which is carried out 3 months after its introduction, shows its readiness for implant placement, which is recorded in the medical records. Thus, the recovery time of the defect in the present invention, compared with the prototype method, is reduced by a month, due to the presence in the proposed biotransplant MMSCs cultured under standard conditions, which due to paracrine activity stimulate the regeneration, formation of blood vessels and trophism.

The technical result in the part of the “method” object is achieved due to the fact that autologous blood serum of the patient is prepared, after centrifugation, the cell suspension is planted in an amount of 1 × 10 ⁵ cells per Petri dish in a standard AdvanceSTEM ^™ / Antibiotic / Antimycotic Solution 100x culture medium supplemented with 10 % autologous blood serum of the patient; after the first passage, the cells are removed from the substrate and divided into 2 equal parts: one part of MMSCs is continued to cultivate under standard conditions, and the other is differentiated in the osteogenic direction for 2 weeks, then the cells are removed from the substrates and mixed in a 1: 1 ratio; after which they are mixed with the matrix carrier in the amount of 10 ⁷ cells per 1 g of the weight of the matrix carrier and incubated for one day at 37 ° C in an atmosphere of 5% CO ₂ .

The combination of the main nutrient medium AdvanceSTEM ^™ (HyClone) / Antibiotic / Antimycotic Solution 100x (HyClone) and 10% autologous blood serum of the patient during MMSC cultivation provides: a) higher proliferative activity of cells in the culture without negative impact on the level of apoptosis; b) obtaining a culture with better, in comparison with using DMEM + AC medium, indicators of preservation of multipotent properties, in particular, with a significantly higher potential in terms of inducing osteogenic differentiation and the ability of cells to proliferate under these conditions (Pat. No. 2418855 of the Russian Federation, publ. May 20, 2011).

The use of AdvanceSTEM ^™ (HyClone) / Antibiotic / Antimycotic Solution 100x (HyClone) and 10% autologous blood serum of the patient during cultivation of MMSC eliminates the risk of infection, in contrast to the prototype, which uses DMEM / F12 growth medium containing 15% fetal calf serum .

The cell suspension is planted in an amount of 1 × 10 ⁵ cells, since this number is optimal for cell proliferation in culture.

MMSCs are divided into 2 equal parts in order to cultivate one part of the cells under standard conditions, and the other part to differentiate in the osteogenic direction.

MMSCs mixed with the carrier matrix are incubated overnight at 37 ^° C in an atmosphere of 5% CO ₂ , since under these conditions a high degree of cell adhesion to the carrier matrix is ensured.

Description of the drawings explaining the invention

Figure 1 - decalcified cryosections of biopsies obtained after the introduction of tissue engineering design based on MMSC and CollapAn-G. Immunohistochemical staining with vascular detecting antibodies CD31; preparations are stained with hematoxylin, eosin. The scale cut is 100µm.

FIG. 2 - decalcified cryosections of biopsies obtained after the introduction of tissue engineering constructs based on a combination of MMSCs cultured under standard conditions and MMSCs differentiated in the osteogenic direction (1: 1 ratio), on a CollapAn-G carrier Immunohistochemical staining with CD31 antibodies; preparations are stained with hematoxylin, eosin. The scale cut is 100µm.

The method is as follows.

To prepare an autologous blood serum of a patient, blood sampling in an amount of 80 ml is carried out in test tubes with a gel for obtaining blood serum (for example, Vacuette # 455071 BD, USA); when forming a blood clot, the blood tubes are centrifuged at 400 rcf for 10 minutes; serum (yellow supernatant) is collected in a test tube and filtered sequentially through 0.45 micron and 0.22 micron filters; at the same time, adipose tissue intended for isolation of MMSCs is crushed with vascular scissors until a suspension of small pieces of no more than a few cubic millimeters in size is obtained and enzymatically disaggregated by mixing with enzyme solutions, for example, type I collagenase (e.g., Worthington Biochemical, USA) and dispases (e.g. firms Invitrogen Corporation, Germany) with a ratio of tissue volume (in ml) to the volume of the enzymatic solution (in ml) 1: 2. After incubation in a red cell lysis buffer, filtration through nylon membranes with a pore size of 40 microns, and centrifugation, a cell suspension of 1 × 10 ⁵ cells per 100 mm diameter Petri dish is placed in a standard AdvanceSTEM ^™ / Antibiotic / Antimycotic Solution 100x culture medium (company manufacturer of HyClone) with the addition of 10% autologous patient blood serum and grown in a CO ₂ incubator (5% CO ₂ ) at 37 ° C until the first passage. After that, MMSCs are removed from the substrate and divided into 2 equal parts: one part of the cells continues to be cultured under standard conditions (AdvanceSTEM ^™ / Antibiotic / Antimycotic Solution 100x / 10% autologous patient blood serum), and the other part is differentiated in the osteogenic direction by adding standard cultivation medium allowed by the Ministry of Health and Social Development of the Russian Federation for retail sale in pharmacies of preparations: ascorbic acid at a concentration of 50 mg / l, regist. number: P N002218 / 01 of 03/18/2008 and Dexamethasone at a concentration of 0.1 μm / mol: P N012237 / 02 of 08/04/2006. Cultivation is carried out in a confluent monolayer for 2 weeks, changing the medium to fresh every 3 days. Then all MMSCs are removed from the substrates and mixed in a 1: 1 ratio. Before use, the carrier matrix (collagen-mineral carrier, for example, CollapAn-G granules) is wetted in a small volume of AdvanceSTEM ^TM medium. In a container intended for the manufacture of a biograft, cells are mixed with a carrier matrix in the amount of 10 ⁷ cells per 1 g of the weight of the carrier matrix and incubated for 24 hours at 37 ° C in an atmosphere of 5% CO ₂ . The duration of the entire biotransplant manufacturing process is from 20 to 30 days. The resulting transplant should be used within 4-8 hours.

Claims (2)

1. A biograft for restoring bone volume in degenerative diseases and traumatic injuries of bones, containing autologous multipotent mesenchymal stromal cells of the patient and a carrier matrix having, based on its structure, a collagen-mineral complex identical in composition to natural bone material, characterized in that it contains a mixture of multipotent mesenchymal stromal cells (MMSCs) cultured under standard conditions and multipotent mesenchymal stromal cells etok differentiated into osteogenic direction taken in the ratio 1: 1 and in an amount of 10 ⁷ cells per 1 g weight of the carrier matrix. 2. A method of obtaining a biograft for restoration of bone tissue volume during degenerative diseases and traumatic injuries of bones according to claim 1, which consists in preparing the adipose tissue obtained from a patient, crushing it, enzymatically disaggregating it; the resulting suspension is filtered, centrifuged and resuspended in culture medium; the MMSC suspension is plated on Petri dishes with a diameter of 100 mm, incubated at 37 ° C in an atmosphere of 5% CO ₂ until the first passage, differentiated in an osteogenic culture medium with the addition of ascorbic acid and Dexamethasone; cultivation is carried out in a confluent monolayer for 2 weeks, changing the medium to fresh every 3 days; the carrier matrix, which is a collagen-mineral complex, is softened and mixed with a cell suspension, characterized in that an autologous blood serum of the patient is prepared, after centrifugation, the cell suspension is planted in an amount of 1 × 10 ⁵ cells per Petri dish in a standard AdvanceSTEM ^TM / Antibiotic / Antimycotic Solution 100x with the addition of 10% autologous blood serum of the patient; after the first passage, the cells are removed from the substrate and divided into 2 equal parts: one part of MMSCs is continued to cultivate under standard conditions, and the other is differentiated in the osteogenic direction for 2 weeks, then the cells are removed from the substrates and mixed in a 1: 1 ratio; after which they are mixed with the matrix carrier in the amount of 10 ⁷ cells per 1 g of the weight of the matrix carrier and incubated for one day at 37 ° C in an atmosphere of 5% CO ₂ .

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