RU2330675C2 - Transplant for correction of connective tissue defects and method of production thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to production of genetically unmodified fibroblast cell cultures, multipotent mesenchymal stromal cells, marrow mononuclear leukocytes, and to production of combined transplant with matrix-carrier. Invention represents combined transplant for correction of connective tissue defects, characterised by that it contains cell cultures of marrow mononuclear leukocytes or multipatent mesenchymal stromal cells or human fibroblasts and matrix-carrier, produced by mixing of specified components, thus substratum is laminated with cell suspension of allogenic or autogenic cell culture at rate 5-10 million cells per 1 cm³, substrata with attached cells are wash out in Hanks' medium.

EFFECT: invention provides evident stimulating action on connective tissue growth within engraftment area.

6 cl, 1 ex

Description

The invention relates to the field of preparation of genetically unmodified cell cultures of fibroblasts, multipotent mesenchymal stromal cells, bone marrow mononuclear cells and obtaining a combined graft with a carrier matrix.

The study of the mechanisms of tissue and organ regeneration, the search for new technologies that could restore the lost function of an organ or system, led to the emergence of new directions that arose at the intersection of biotechnology and medicine - tissue engineering, regenerative medicine and organogenesis. These sciences study the creation of organs and tissues de novo. They are based on the principle of cell transplantation on carrier matrices.

The matrix carrier or matrix is a synthetic or biological complex to ensure mechanical strength of the structure with the desired properties, three-dimensional orientation of the cell culture applied to it. The main criteria for a biocompatible matrix to create a tissue engineering construct should be: the absence of cytotoxicity, the maintenance of adhesion, fixation, proliferation and differentiation of cells placed on its surface, the absence of an inflammatory reaction to the material and an immune response, sufficient mechanical strength in accordance with the purpose, bioresorbability by the usual metabolic pathways [1.4-10, 15-18, 20, 22, 25-27].

One of the first materials for tissue engineering was biodegradable synthetic biomaterials based on polymers of organic acids. Biodegradable polyesters are a family of biodegradable materials consisting of a chain of repeating residues of short-chain organic acids, such as lactic and glycolic. The composition of the polymer can include both one type of acid residue - PGA (Poly Glycolic Acid), PLA (Poly Lactic Acid), and their combination in various proportions PGLA (Poly Glycolic Lactic Acid) 30/70. In medical practice, these polymers are widely used in the form of suture material, reinforcing structures, nets for germinoplasty. The first commercial product was Vicryl suture from Ethicon Inc.

PGA is a solid thermoplastic material with a melting point of 225 degrees. Due to its high crystallinity (40-50%), it is insoluble in most organic solvents. Sufficient strength and ductility allow you to create various designs with a given mechanical strength - from non-woven, spongy materials to plates and screws for fixing bone fragments. Biodegradation of the polymer occurs by hydrolysis to carbon dioxide and water with a slight increase in the pH of the surrounding tissues [11], the residues are excreted as urine monomer. Mechanical strength lasts up to a maximum of 21-29 days with a weight loss of up to 40% by this period. The material does not possess cytotoxic properties, provides adhesion and supports cell proliferation, and also has osteoinductive properties to some extent.

PLA is a polymer of lactic acid residues. By its chemical, physical and biological properties it is close to PGA, however, it is closer to the body, as it is in it in the form of a monomer and participates in physiological processes. However, the best performance has a copolymer of PGA and PLA - PGLA. Depending on the percentage of lactic and glycolic acids, you can change the properties of the product, for example, ductility, strength, biodegradation period, etc.

For a long time, these polymers with various types of cells were tested. It was found that these materials can serve as a matrix for the creation of tissue-engineering structures of muscle, cartilage, bone and epithelial tissue. Polymers based on lactic and glycolic acids took a special place in reconstructive surgery and regenerative medicine. They are currently used to repair bone and cartilage defects in clinical practice [1.6–10, 15, 19, 25–27], and have been approved by the FDA (US Food and Drug Administration) as “first” safe biomaterials for tissue engineering [28].

Polylactone is another member of the polyester family. Translucent polymer with a melting point less than 60 degrees. The biodegradation period is about 3 years. These properties make it possible to obtain sufficiently ductile structures that provide long-term mechanical strength and support. A commercial medicine used in healthcare, Monocryl (Ethicon Inc.).

Polypropylene fumarate is a fairly strong plastic polymer, also intended for long-term support of the mechanical strength of the structure for at least 200 days. Biodegradation also occurs by enzymatic hydrolysis, but unlike previous materials does not affect the pH of surrounding tissues. Significant disadvantages include the formation of a fibrous capsule as a response to a foreign body, which is unfavorable in a number of situations [12, 14].

Monoanhydrides (poly- [trimellitylimidoglycine-co-bis (carbox-yphenoxy) -hexane], poly- [pyromellitylimidoalanine-co-1,6-bis (carboph-enoxy) -hexane]) are one of the most studied polymers at present. They are biologically resorbable by surface erosion for 28 weeks. A distinctive feature of these polymers is their high mechanical strength (up to 60 MPa), which allows them to be used for reconstructive surgery of bone and tendons. This allows you to create durable structures for a relatively long period [4, 13].

Polyorthoesters - polymers related to bioresorbable materials with a sufficiently high strength. Resorption is carried out by means of surface erosion for 150 days. A distinctive feature is the more pronounced osteoinductive properties, which allows them to be used in reconstructive orthopedics.

To create three-dimensional three-dimensional tissue equivalents, tissue-engineering constructs created in vitro are used. Combined cell transplants used in regenerative medicine are three-dimensional structures in the form of solid structures or gel-like forms, depending on the purpose and purpose of their application. For local stimulation of regenerative processes in damaged tissues, the creation of local three-dimensional volume formations (artifical tissues) using minimally invasive techniques, it is most preferable to use three-dimensional gel tissue-engineering structures [7], the injection of which into a given anatomical localization avoids additional undesirable trauma to the surrounding tissues that occurs during transplantation of solid structures, especially in places with a complex anatomical structure disorder of organs or tissues whose damage is undesirable.

Of particular importance is the structure, physicochemical, mechanical and biological properties of the carriers, such as biological inertness, mechanical strength, elasticity, lack of cytotoxicity and good adhesive properties of the carrier matrix. The carrier, which is included in the injection form of the combined cell transplant, must have both the properties of a liquid and a solid body, at the same time carry cells on its surface and pass freely through a sufficiently narrow lumen of the needle of the syringe or catheter to ensure the principle of minimally invasiveness. The elasticity of the carrier would allow, by changing its shape, not to damage, but to protect cells adhering to the walls of the needle from damage to the needle. Once in the tissue, the carrier must fix the cells in a principal place for some time, have good adhesive properties, stimulate the synthesis of extracellular matrix or the synthesis of biologically active substances.

An object of the present invention is to provide a combined graft for the most effective treatment of connective tissue defects.

To solve this problem, a group of inventions is proposed, united by a common inventive concept.

A tissue-engineering design for repairing defects in connective tissue, containing bone marrow mononuclear cells, is proposed. The source of human bone marrow mononuclear cells is a suspension of bone marrow (CMS) obtained by puncture of the posterior or anterior crest of the ilium or sternum, including during surgical interventions.

A tissue-engineering design for repairing defects of connective tissue is proposed, containing multipotent mesenchymal stromal cells of bone marrow or adipose tissue obtained from autologous or donor material, while the tissue is disaggregated; they are then cultured in the form of attached colonies in a growth medium containing fetal calf serum and glutamine, then they are repeatedly passaged in low density with a change in the composition of the medium, and cultivation is carried out avoiding the accumulation of mature stromal cells in the culture.

A tissue-engineering design for repairing connective tissue defects is proposed, which contains fibroblasts, which are obtained by lipoaspiration or biopsy of the skin and mucous membrane.

A tissue-engineering design is proposed that contains gelatin, various types of collagen and a demineralized bone matrix as a carrier matrix. The carrier matrix is used in the form of a gel, film and three-dimensional structure corresponding to a defect in connective tissue.

A method for producing a combined transplant is proposed, characterized by the fact that CMS processing in the laboratory is carried out in accordance with the recommendations of the “Instructions for the control of sterility of canned blood, its components, canned bone marrow preparations, blood substitutes and preservative solutions” - Ministry of Health No. I-42-4-85 01/01/1986

To reduce the total volume of the sample for further freezing of cells and removal of red blood cells, a fraction of mononuclear cells (nucleated bone marrow cells) is isolated. In order to increase the efficiency of isolation of nucleated elements, CD34 + / CD45 + cells, CFU-GM, CFU-HEMM, 10% hydroxyethyl starch is used as a sedimentation agent (Infucol HES 10%).

For this, a 10% HES solution is mixed with the sample in a ratio of 1: 1. The erythrocyte sedimentation occurs within 25 minutes, after which the supernatant is collected with a pipette, and the remaining cell suspension is subjected to repeated sedimentation. The supernatant recovered after erythrocyte sedimentation is washed twice from the sedimentation substance (HES) in RPMI 1640 medium by centrifugation at a speed of 1500 / min for 10 minutes. The cell pellet is collected with a pipette, an admixture of red blood cells is estimated.

An admixture of red blood cells in a concentrate of nucleated bone marrow cells (BSC) is not more than 5%. The concentrate of human bone marrow JSC is tested by the number of nucleated cells, their viability, sterility.

At the time of testing, but no more than 36 hours, the NSC samples are quarantined at temperatures up to 22 ° C, excluding contact with other samples.

The results of all studies characterizing a sample of the prepared bone marrow JSC preparation are entered into the Organization's database under a single identification number of the sample.

Highly purified dimethyl sulfoxide (dimexide) is used as a cryoprotectant, including in combination with hydroxyethyl starch (infucol HES, 10% solution for infusion, Serumwerk Bernburg).

Preparation of enclosing solutions is carried out in a box in an ice bath (to maintain the temperature + 4 ° C) in compliance with sterility.

For freezing and storage of cell suspension, 5 ml polypropylene cryovials manufactured by Corning (USA) are used.

Cryoprotective solution consists of 10% DMSO, 40% autologous serum and 2% HES (in final concentration). To prepare such a solution, 2.0 ml of autologous serum cooled to + 4 ° C is poured dropwise with constant mechanical stirring of 0.5 ml of dimethyl sulfoxide (DMSO) and 1 ml of 10% HES (calculated per 1 cryo-ampoule with a volume of 5 ml).

A freshly prepared enclosing solution, cooled to + 4 ° C, is slowly poured into 1.5 ml of cell suspension with constant mechanical stirring. After this, the cell suspension with the enclosing solution, cooled to + 4 ° C, is tightly closed with a lid, marked and placed in a cryobox.

From one bone marrow sample, at least 3 JSC cryo-ampoules and a satellite tube are frozen. The cryobox containing samples of the NSC with a satellite tube is transferred to the storage and subjected to smooth cooling to -80 ° C at a speed of 1 ° / min, after which the cryo-ampoules are immersed in liquid nitrogen for long-term storage.

Bone marrow concentrate is stored in a Dewar vessel in the liquid nitrogen phase until use.

Storage of samples and satellite tubes with a single identification number is carried out in a separate cryogenic storage case. Each SSC sample intended for cryo-freezing and cryopreservation is marked with an identification number indicating the concentration and composition of the cryoprotectant, the date of cryo-freezing, and the name of the laboratory.

A method of producing a combined cell transplant is characterized in that multipotent mesenchymal stromal cells are isolated from fetal or donor material, or from the recipient’s own tissues, the tissue is disaggregated, the resulting cell suspension is resuspended and cultured on growth medium containing transferrin, insulin, fibroblast growth factor and heparin, before the accumulation of mature stroma cells in the culture.

The following tissues are used as fetal material: bone marrow, thymus, liver, adipose tissue of human embryos of the 1st trimester of pregnancy.

The following tissues are used as donor material: bone marrow, thymus, liver, adipose tissue.

To obtain autologous MMSCs, tissues are used: bone marrow, adipose tissue.

Multipotent mesenchymal stromal cells are mainly obtained from donor bone marrow, but there are other sources - skeletal muscle, subcutaneous adipose tissue, fetal organs of hematopoiesis (liver, thymus, spleen). MMSCs from fetal organs of hematopoiesis and the methods for their preparation are practically not studied. The described methods for the growth of MMSCs from bone marrow and lipoaspirates make it possible to obtain heterogeneous populations of stromal cells, only a small percentage of which are stem cells. The cells of the mature stroma do not possess the property of differentiating into different cell lines and, thus, are therapeutically ineffective. After transplantation, stromal cells migrate mainly to the connective tissue and accumulate there. With standard passage at high inoculum doses, the proportion of mature stromal cells increases rapidly.

The use of a homogeneous (at least 80%) population of pluripotent MMSCs as a transplant can increase the effectiveness of treatment, increase the reproducibility of the results and avoid undesirable effects.

Multipotent mesenchymal stromal cells are isolated from fetal (bone marrow, thymus, liver, adipose tissue) or donor (bone marrow, adipose tissue, thymus) tissues. To isolate fetal cells using abortive material obtained by artificial termination of pregnancy in healthy women, previously examined for the presence of viral and bacterial infections.

The possibility of using biomaterial is achieved at the following gestational periods: liver - 5-8 weeks, thymus - 18-20 weeks, bone marrow - 17-20 weeks, subcutaneous adipose tissue - 17-19 weeks. 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 1,500 × g mode 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 a 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 into new plates with a density of 10-30 cells per 1 cm ² in a growth medium additionally containing 10 μg / ml transferrin, 1 μg / ml insulin, 10ng / 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 ² and grown to a state of pre-confluency. The medium is replaced every 2 days. Subsequent passages are made in the same mode. An increase in the seed dose or a change in the composition of the growth medium leads to the rapid accumulation of mature stroma cells in the culture.

The method of obtaining tissue engineering design is characterized in that fibroblasts are obtained from fetus skin of the 1-2 trimester of pregnancy, the skin and mucous membranes of an adult, obtained as a result of plastic surgeries or biopsies, lipoaspirates. The skin is washed in Hanks' medium containing an antibiotic and antimycotic, crushed and enzymatically disaggregated in a 0.25% trypsin solution for 40 minutes under conditions of a CO ₂ incubator at a temperature of 37 ° C. Lipoaspirate is enzymatically disaggregated similarly. The cell suspension is diluted 1: 1 with Hanks saline, centrifuged at 1000 × rpm for 5 minutes and resuspended in DMEM / F12 growth medium containing 10% fetal calf serum, 2 mm glutamine. The suspension is plated on plastic Petri dishes with a diameter of 100 mm. The seeding density of the primary cell suspension is 500-1000 cells per 1 cm ² .

A method of obtaining a combined graft is characterized in that the carrier matrix is a substrate with a developed surface. Developed surface substrates (PSA) are prepared in advance (3-5 days) by soaking them in a DMEM culture medium.

As substrates, biomaterials based on collagen or gelatin or demineralized bone matrix are used.

PSAs are placed in 15-ml or 50-ml tubes and a cell suspension is layered on them at the rate of 5-10 million cells per 1 cubic meter. see PSA. In this case, the substrates should be completely covered with the culture medium. Autologous blood serum of the patient is added on Wednesday to 15-20% of the total volume.

The caps of the tubes are ajar. The tubes are placed in a CO ₂ thermostat and incubated at 37 ° C in an atmosphere of 5% carbon dioxide for 12-24 hours. Then the PSA is transferred to culture dishes and the cell attachment is analyzed by phase contrast microscopy.

Substrates with attached cells are carefully washed in Hanks' medium (collagen and demineralized bone matrix directly in the dishes; gelatin is transferred to test tubes, diluted and settled for 5-10 minutes, removing the supernatant). The procedure is repeated 2-3 times.

When using YSC bone marrow or adipose tissue, the concentration of cells should be increased to a maximum. The medium upon attachment of cells should not be strongly acidified. To do this, you can add a HEPES solution with a pH of 7.2-7.4 to a final concentration of 25 mM. After cell attachment, tissue engineering constructs can be incubated in culture dishes in DMEM culture medium with 10-20% autologous patient serum under standard conditions. At the same time, the medium should be regularly (at least 2 times a week) replaced.

Preparations based on gelatin, unlike other ITPs, can be used as an injection form and placed directly in sterile syringes.

Also, the fabric engineering structure is characterized in that the carrier is obtained by simple mixing of all its components at a temperature of 0 - + 4 ° C. If necessary, matrix proteins, growth factors, antibiotics and / or antiseptics are added to the main components of the carrier, and the amount of additives may vary depending on the clinical or experimental task and the volume of the output product, for example, fibronectin and / or laminin can be used as matrix proteins, and / or protein in a concentration of from 0.1 to 100 μg / ml, and preferably from 1 to 5 μg / ml, as biologically active substances - fibroblast growth factors and / or transforming growth factor α and β and / or f growth factor secreted by platelets and / or hepatocyte growth factor in concentrations from 1-300 ng / ml, and preferably from 1 to 100 ng / ml, or others, gentamicin in a concentration of from 0.08 to can be used as aseptic agents 0.4 mg / ml gel, and preferably from 0.1 to 0.2 mg / ml, and / or penicillin (or its derivatives), streptomycin (or its derivatives), any other broad-spectrum antibiotics. Upon completion of the carrier manufacturing process, a suspension of an allogeneic or autogenous cell culture of fibroblasts, bone marrow mononuclear cells, multipotent mesenchymal stromal bone marrow cells, adipose tissue or other origin is introduced into it.

Example 1

Cell culture. We used low-differentiated stromal adipose tissue from Wistar rats as a cell culture. Adipose tissue samples were taken from 11 animals. They were taken from the withers of an animal with a volume of 0.5 cm ³ and delivered to the laboratory. Of these, 10 samples were used for autotransplantation, and material from one animal was used for allotransplantation. The stromal fraction of poorly differentiated adipose tissue cells was isolated from the samples according to the standard protocol.

Mononuclear cells were obtained from bone marrow. Tissue samples were taken from 5 animals and used for allotransplantation. Fibroblasts were isolated from rat skin. Of these, five samples were used for allotransplantation and five for autotransplantation.

In order to detect transplantable cells in the surrounding tissues, the cells were labeled with BrdU.

Carrier matrix. As a matrix, we used the Spongostan gelatin sponge (Johnson & Johnson, Inc.), crushed on a Silent Crasher M homogenizer at 15,000 rpm for 5 minutes. The particle size ranged from 70 to 150 microns in the form of thin, up to 3 microns in thickness, wavy structures.

Combined cell transplant. A suspension of particles obtained after homogenization of the gelatin sponge was combined with cell cultures at a rate of 5 × 106 per ml and incubated for one hour in a CO ₂ incubator, after which they were washed from the culture medium and packed under sterile conditions in 300 μl insulin syringes. Thus, three types of transplant were obtained, containing mesenchymal stromal cells, bone marrow mononuclear cells and fibroblasts.

Survival test. To determine the cell survival test as part of a tissue-engineering construct when passing through standard needles from Luer syringes of 0.6 mm in diameter and an insulin syringe needle (0.3 mm), we used a technique for counting living cells before passing through and after the needle.

Experimental study.

The experiment was performed on 43 female Wistar rats with an average body weight of 250 grams. Animals were divided into 3 groups (control, comparisons and main). The conclusion from the experiment of animals was carried out in accordance with the International Convention for the Protection of Animals by overdosing an anesthetic drug (Zoetil-50) on days 5-6, 12-21, and 27-35 after paraurethral administration of the transplant or only its matrix (without cellular component) into the urogenital and pelvic diaphragms between the vagina and urethra.

In the control group (10 animals), the rats were only given the transplant matrix without its cellular component, of which the 3rd was administered by paraurethral injection with an insulin syringe (the result was studied morphologically after slaughtering the animals on the 5th day) and 7th - paraurethrally through a small incision skin on the left between the vagina and urethra. Morphological research was carried out on the 14th (3 animals) and 35th day (4 animals).

In the comparison group (20 animals), rats received a paraurethral injection of a cell allograft. Morphological research was carried out after slaughter of animals on the 6th, 21st and 30th days.

In the main group (13 animals), a cell autograft was paraurethrally injected, and a morphological study was performed after the animals were slaughtered on the 5th, 12th and 27th days.

Methods of morphological research.

Excised sections of paraurethral tissue, including the urethra and the vaginal wall, 0.5-1 × 0.3-0.5 × 0.2-0.5 cm in size, were fixed in conventional formalin in 10% neutral formalin, dehydrated in alcohols and poured into paraffin. Test histological sections 4-5 μm thick were made and stained with hematoxylin and eosin. Based on the results of the study of these sections, the paraffin blocks were oriented so that the lumen of the urethra turned out to be perpendicular to the section. Next, step sections stained with hematoxylin and eosin were studied sequentially until paraurethral tissues were reached in histological preparations in the area of matrix or transplant administration. At the same level of oriented blocks, at least 10 sections were made for histological (staining with hematoxylin and eosin, van Gieson picrofuxin), immunomorphological (detecting Ki-67 antigen, type I and III collagen) and morphometric studies.

For immunomorphological (immunoperoxidase) studies, histological sections were placed on adhesive-coated (APES-acetone) slides. Endogenous peroxidase in dewaxed sections was blocked with 3% hydrogen peroxide. The antigens were unmasked according to the standard scheme, for 10-30 min in a microwave oven in a 0.1 M solution of nitrate buffer (pH 6.0). Used mouse monoclonal antibodies to type I collagen (Abcam, UK), type III collagen (clone HWD 1.1, Bio Genex, USA) and Ki-67 protein (Novocastra, UK). The nuclear non-histone protein Ki-67 is expressed only in the phases G1, S, G2 and M of the cell cycle and is a marker of proliferating cells. After incubation of sections with primary antibodies (working dilution of antisera - 1: 100-300, incubation time - 45-60 minutes at 37 ° C) they were treated with universal secondary biotilinized horse antibodies (Novocastra, UK). For subsequent visualization of the result of the antigen-antibody binding reaction, a streptavidin-peroxidase complex and a DAB solution (Novocastra, UK, BD Pharmingen, USA) were used. The final reaction product consists of small brownish granules staining cell nuclei (for Ki-67 antigen) and type I or III collagen fibers. After the immunoperoxidase reaction, the histological preparations were stained with hematoxylin, studied and photographed using a DM-LB light microscope (Leica, Germany) with a JVC video camera and a Pentium 4 computer.

Conducted generally accepted negative and positive control procedures for the reagents and tissues used in the processing of parallel sections. A negative control consisted in carrying out a reaction with the exception of primary (specific) antibodies by replacing them with an non-immune reagent (bovine serum), and a positive one in the use of histological preparations of breast cancer.

The ratio of the number of cells with the expression of the Ki-67 antigen to the number of cells with unpainted nuclei in their nuclei was calculated morphometrically in paraurethral tissue in the area of matrix or transplant administration (the result was expressed as a percentage). Cell counting was performed in each histological section, in at least 20 fields of view, with a magnification of the microscope × 400 (lens × 40, eyepiece × 10). Statistical processing of the material was carried out, calculating the average value of the indicator, the standard deviation, the error of the mean. The significance of differences in the indicators between the groups of observations was compared using Student's criterion. The differences were considered significant at a 5% significance level (p <0.05) according to student table.

Results and discussion.

Microscopy of a combined cell transplant. During phase-contrast microscopy of the tissue engineering construct in a liquid medium, it was noted that most of the cells were concentrated on particles of a gelatinous carrier from 5 to 50 units per particle in the form of single cells or their clusters.

Survival test. As a result of the test, up to 97% of the living cells were preserved when passing through a needle 0.6 mm in diameter and 93% through a needle 0.3 mm in diameter (insulin syringe).

The results of the experiment.

1.5-6th day.

In the observations of the control group (on the 5th day), small foci of hemorrhage with moderate lymph and macrophage infiltration mixed with neutrophilic leukocytes remained in the area of injection of the acellular matrix. In such areas fragments of the injected matrix were detected in the form of complexes of eosinophilic structures, among which there were single fibroblast-like cells, lymphocytes and macrophages. Separate vessels were dilated and full-blooded. The proliferative activity of connective tissue cells and inflammatory infiltrate at the lesion sites was minimal; nuclei of 6.7 ± 1.3% of the cells expressed Ki-67 antigen.

In contrast to the control, in the observations of the comparison group (the 6th day after the injection of an allogeneic cell transplant), microcirculatory disturbances in the form of dilated full-blood vessels and swelling of the tissue around the allograft were pronounced. The latter was detected in the form of compactly located eosinophilic structures with massive infiltration by groups of cells with moderately polymorphic rounded nuclei, as well as lymphocytes, macrophages and fibroblast-like cells. Perifocal cell infiltration was expressed. In the composition of the infiltrate, along with macrophages, lymphocytes and neutrophilic leukocytes, isolated labrocytes and groups of eosinophilic leukocytes were distinguished. Around the allograft, fibroblast activation and an increased number of dilated full-blooded capillaries were observed. Ki-67 antigen was expressed by nuclei of 11.4 ± 2.2% of connective tissue cells, vascular walls and infiltrate in areas perifocal to the allograft, as well as cells localized among particles of its matrix.

In the observations of the main group (5th day after the injection of the autologous cell transplant), microcirculatory disorders and the degree of cell infiltration were similar to those found in the comparison group, but less pronounced. The morphology of the cellular and matrix components of the autograft did not differ from the allogeneic. Around the autograft a granulation tissue was formed with many newly formed capillaries. The proliferative activity of cells in the autograft and surrounding connective tissue, including infiltrate cells, was significantly higher than in the observations of the comparison group. Expressed the Ki-67 antigen of the nucleus of 33.5 ± 6.9% of such cells.

Thus, in contrast to the impact on the tissue of an isolated acellular matrix transplant, which was traumatic in nature, allo and autografts modulated the local activity of various cell populations, and, apparently, the graft cells remained viable. Already on the 5-6th day, the introduction of an allograft led to a 1.7-fold increase (compared with the control) in the proliferative activity of connective tissue cells perifocal to the graft, along with a pronounced, possibly, immune response to its allogeneic cellular component. The morphology of the cellular and matrix components of the autograft did not differ from the allogeneic, but the proliferative activity of the cells of the autograft and the surrounding connective tissue was 3.4 times higher than with allograft. At the same time, microcirculatory and immune responses to the autograft were less pronounced. There were no specific features in the tissue distribution of type I and III collagen, but they were revealed among the matrix fragments of allo and autografts, which indirectly indicates the synthetic activity of the graft cells.

2. 12-21th day of the experiment.

In the observations of the control group (on the 14th day of the experiment), microcirculatory disturbances and inflammatory changes were not observed. Only in 2 cases out of 3 revealed small perivascular lymph and macrophage infiltrates. In all the observations, the formation of foci of sclerosis in the area of the injury with mild lymph and macrophage infiltration and single mast cells was characteristic. Some macrophages of the infiltrate contained hemosiderin. Small fragments of the introduced matrix, and without any cellular reaction, were found only in 1 of 3 cases. The proliferative activity of connective tissue cells and infiltrate in the foci of sclerosis was low. The core antigen Ki-67 was expressed 2.0 ± 1.7% of the cells. Noticeable changes in the distribution of collagen of types I and III were not detected, except for their accumulation in the foci of sclerosis.

In the observations of the comparison group (on the 21st day of the experiment), proliferative processes prevailed in the areas of the connective tissue perifocal to the allograft and productive cellular reactions. Microcirculatory disturbances were not expressed. In the preserved allograft fragments, along with the matrix, groups of polymorphic cells with basophilic nuclei, lymphocytes, macrophages and fibroblast-like cells were detected. Around the allograft, granulation tissue infiltrated by lymphocytes and macrophages with an admixture of labocytes grew. Macrophage groups contained hemosiderin. Similar infiltrates were observed around the vessels and at a distance from the allograft. The proliferative activity of allograft cells was as high as the cells of the surrounding granulation tissue. Expressed the Ki-67 antigen of the nucleus of 23.3 ± 5.8% of the cells. The accumulation of collagen of both types I and III was expressed on the periphery of the allograft and in the peripheral areas of the connective tissue. A positive reaction to these types of collagen of the cytoplasm and the cell membrane of a part of the cells localized between fragments of the allograft matrix indicated the possibility of their synthesis not only by local tissue cells, but also introduced by allogeneic cells.

In the observations of the main group (on the 12th day of the experiment) proliferative changes predominated both from the side of the autograft cells and from the surrounding connective tissue. The surviving fragments of the autograft were surrounded by many newly formed dilated vessels. Elements of the autograft matrix were separated by a dense cell infiltrate, represented by moderately polymorphic cells with round, less extended nuclei. These cells were morphologically similar either to lymphocytes and monocytes, or to fibroblasts, and their clusters extended far beyond the boundaries of the preserved fragments of the autograft matrix. Peripheral to the autograft there was a marked proliferation of granulation tissue. Lymphocytes, in contrast to the observations of the comparison group, were rarely detected. The core antigen Ki-67 was expressed in 38.7 ± 4.1% of the cells. As in the comparison group, the accumulation of type I and III collagen was expressed along the periphery of the autograft and in the regions of the connective tissue perifocal to it. A positive reaction to these types of collagen of the cytoplasm and cell membrane of a part of the cells localized between fragments of the autograft matrix indicated the possibility of their synthesis by introduced autologous cells.

Thus, on the 12-21st day of the experiment, an increased proliferative activity of allo- and especially (1.7 times higher) cells of autografts, as well as of the connective tissue surrounding them, was noted. Around the grafts, granulation tissue grew, and allografts were characterized by its lympho- and macrophage infiltration mixed with labrocytes and perivascular infiltrates similar in cellular composition. An accumulation of type I and type III collagen was noted, mainly perifocal to allo and autografts and between fragments of their matrix. A positive reaction to these types of collagen of the cytoplasm and the cell membrane of a part of cells localized between fragments of the autograft matrix indicated the possibility of their synthesis by introduced allo - and autologous cells.

3.27-35th day of the experiment.

In the observations of the control group (on the 35th day of the experiment), only single small foci of sclerosis were detected in the paraurethral tissue, sometimes with groups of siderophages. Elements of the transplanted matrix were not detected. The lumen of the urethra in its width and shape did not change. The proliferative activity of the cells of the paraurethral connective tissue did not significantly differ from the indicator observed for the 14th day of the experiment in the observations of the control group. The core antigen Ki-67 was expressed 1.8 ± 0.6% of the cells. The distribution of type I and type III collagen also corresponded to that described for observations of the same control group.

In the observations of the comparison group (on the 30th day of the experiment), focal excess growth of the paraurethral connective tissue was characteristic, which led to compression and narrowing of the urethral lumen. In place of allografts, the cellular and matrix components of which were no longer visualized, among foci of scar tissue with slight lymph and macrophage, sometimes with an admixture of neutrophilic leukocytes, infiltration revealed groups of closely spaced and dilated thin-walled vessels. In such areas, collagen of types I and III accumulated. Perivascular lymph and macrophage infiltrates were also noted. The proliferative activity of connective tissue cells in the foci of sclerosis was low. The core antigen Ki-67 was expressed 3.8 ± 1.5% of the cells.

In observations of the main group (on the 27th day of the experiment) similar to the comparison group, but more pronounced changes were revealed. The lumen of the urethra was sharply narrowed due to compression by an increased volume of paraurethral connective tissue, rich in blood vessels. The severity of lymph and macrophage infiltration was less than in the comparison group. In addition, among foci of mature (scar) connective tissue, groups of lipocytes or striated myocytes were detected in combination with multiple newly formed small vessels. It cannot be ruled out that these areas of adipose and muscle tissue are the result of certain variants of differentiation of transplanted autologous cells, but perhaps such cells are of local origin. Along with mature connective tissue, foci of granulation tissue with active neoangiogenesis were encountered. The proliferative activity of cells was different in the foci of scar and granulation tissue. So, the Ki-67 antigen of the nucleus was expressed in 1.3 ± 0.2% of the cells of the scar tissue, but 15.5 ± 3.7% of the granulation tissue (including those with the inclusion of groups of lipocytes and myocytes). The accumulation of type I and type III collagen was especially pronounced in scar tissue and around conglomerates of dilated vessels at the site of autografts.

Thus, on the 27-35th day of the experiment, in contrast to the control observations, in the comparison groups and the main group, a pronounced focal excessive growth of connective tissue was revealed, leading to compression and narrowing of the urethral lumen. In place of allo and autografts, the components of which were no longer determined, foci of scar tissue rich in type I and III collagen were formed, with conglomerates of dilated thin-walled vessels. In observations with allografts, focal lympho- and macrophage, often perivascular infiltrates remained, and with autografts foci of granulation tissue with active neoangiogenesis, groups of lipocytes and striated myocytes were detected. The latter may indicate different differentiation of transplanted autologous cells.

Thus, the developed combined graft based on cells and a carrier matrix retains its biological and physical properties after administration without significant loss of functionally active cell units.

An experimental morphological study of the results of transplantation into paraurethral tissues of 43 female rats of an injection combined cell transplant containing allogeneic and autologous cells showed that transplant cells, especially autologous ones, have a pronounced activating effect on the proliferation of local cell populations of connective tissue and, apparently , for a long time, up to 20 days or more, retain their viability, proliferative and synthetic activity. Microcirculatory and immune responses to an autograft are much less pronounced than to an allograft. Autologous cell transplantation reveals foci of granulation tissue with active neoangiogenesis, groups of lipocytes and striated myocytes. The latter may indicate different differentiation of transplanted autologous cells or the effect of an autograft on the proliferation and differentiation of local progenitor cells of different lines. In the outcome of the experiment, on the 27-35th day, there is a pronounced focal growth of connective tissue in the area of administration of both allo and autografts.

Claims (6)

1. A combined graft for restoration of defects in connective tissue, characterized in that it contains cell cultures of bone marrow mononuclear cells or multipatent mesenchymal stromal cells or human fibroblasts and a carrier matrix obtained by mixing these components, while the cell suspension is layered on the substrate from an allogeneic or autogenic cell cultures at the rate of 5-10 million cells per 1 cm ³ , substrates with attached cells are washed in Hanks medium. 2. The combined graft according to claim 1, where the carrier matrix is made in the form of a gel, a crushed sponge, a film and a three-dimensional structure corresponding to a defect in connective tissue. 3. The combined graft according to claim 1, where the carrier matrix further includes antiseptics selected from the group: silver, copper, gold ions and broad-spectrum antibiotics. 4. The combined graft according to claim 1, where the source of mononuclear cells is a suspension of bone marrow obtained by puncture of the posterior or anterior crest of the ilium or sternum, including during surgical interventions. 5. The combined transplant according to claim 1, where the multipatent mesenchymal stromal cells of bone marrow or adipose tissue are obtained from autologous or donor material. 6. A method of producing a combined transplant for repairing defects of connective tissue according to claim 1, characterized in that the cell suspension consisting of bone marrow mononuclear cells or multipatent mesenchymal stromal cells or human fibroblasts at the rate of 5-10 million cells per 1 cm ^{3 of} substrate layered on a carrier matrix, which is a substrate with a developed surface based on collagen, gelatin or demineralized matrix, including matrix proteins selected from the collagen group, rin, fibronectin, laminin in a concentration of from 0.1 to 100 μg / ml and, if necessary, fibroblast growth factors and / or transforming growth factor α and β and / or growth factor secreted by platelets and / or hepatocyte growth factor, then substrates with attached cells are washed in Hanks medium and subjected to cryo-freezing in a solution consisting of 10% DMSO, 40% autologous serum and 2% HES.