RU2729365C1 - Tissue-engineering structure for filling bone tissue of maxillofacial area - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to a tissue engineering structure for filling the bone tissue of the maxillofacial region. Construct comprises donor multipotent mesenchymal stromal cells on a carrier, which is represented by 150–200 mcm octacalcium phosphate granules with pore size of 1–5 mcm, on the surface of which there are incubated multipotent mesenchymal stromal cells obtained from the patient's gingival biopsy material and cultivated on a medium of aqueous sol of nanocrystalline cerium dioxide stabilized with citrate ions in concentration of (10–4) - (10–9) M, wherein concentration of multipotent mesenchymal stromal cells is 2 × 10⁷ cells per 1 g of carrier.

EFFECT: invention widens the range of equipment.

1 cl

Description

The present invention relates to the field of biotechnology and dentistry, namely, to the composition of a tissue-engineered structure for replenishing the volume of bone tissue in the maxillofacial region.

Significant atrophy of bone tissue complicates treatment with removable prostheses, and the aesthetic effect of prosthetics with non-removable orthopedic structures worsens. Dental rehabilitation of patients with the use of dental implants often becomes impossible due to the insufficient volume of bone tissue. Ensuring a sufficient volume of the alveolar part of the jaws before implantation is the main task of reconstructive interventions in order to normalize the functional state of the masticatory apparatus.

In reconstructive surgery, the technique of bone autotransplantation is most often used. However, this method has a number of limitations, especially when replacing large-volume bone tissue defects. The use of autografts, artificial osteoplastic materials, growth factors, platelet concentrates does not always give good results in clinical practice.

Often, the volume of bone tissue obtained is insufficient, repeated surgical intervention is required, and the trauma of surgery is noted. Thus, the development of effective means and methods of bone grafting, and the creation of optimal conditions for reparative osteogenesis is an urgent problem in surgical dentistry.

Fibrin enriched with leukocytes and platelets, which is an autologous fibrin clot (hereinafter referred to as L-PRF), is capable of releasing growth factors and leukocytes in the bone wound area, providing proliferative, angiogenic and anti-inflammatory effects. A reduction in the time of epithelialization of the sockets of the extracted teeth was noted when they were filled with a fibrin clot enriched with leukocytes and platelets.

Multipotent mesenchymal stromal cells (hereinafter MMSC) have been actively studied over the past decades; they are the optimal cell population for the creation of tissue-engineered structures. Autogenous cells do not come into conflict with their own immune system, do not cause allergic reactions. Multipotent mesenchymal stromal cells isolated from gingival biopsy specimens can differentiate in osteogenic, chondrogenic and adipogenic directions under the influence of induction factors. The presence of progenitor cells in MMSC merge with resident cells in tissues and, due to the expression of marker antigens, differentiate in a certain direction.

As a result of the conducted patent information search, the following sources were selected for further analysis.

Known pasty composite osteoplastic material (Patent RF2653480). In particular, the composition for stimulating the regeneration of the jaw bone tissue contains calcium hydroxide, barium sulfate and nanocrystalline cerium dioxide in isotonic solution. The ratio of the components in the composition is 40-42 wt. % calcium hydroxide; not less than 8 wt. % barium sulfate; 0.3-2 wt. % nanocrystalline cerium dioxide; the rest is up to 100 wt. % isotonic solution. The disadvantage of the proposed composition is the absence of biologically active substances in its composition, which significantly accelerate the regeneration process.

Known material for the restoration of bone structures (RF patent No. 2399387), which contains platelet-rich autoplasm of the patient, titanium nickelide powder with particle sizes up to 100 nm, colloidal 2.5% nanostructured silver with particle sizes up to 20 nm. The method of obtaining a known material consists in the fact that the patient's blood is centrifuged, the plasma is separated from the blood clot, the isolated clot is homogenized, colloidal nanostructured silver, nickelide powder and the plasma obtained after centrifugation are added to it.

A known method of obtaining myoblasts by culturing fibroblast-like cells isolated from the mucous membrane of the human oral cavity, including: a) biopsy from the mucous membrane of the human oral cavity; b) processing of biopsy material, which includes washing, grinding, enzymatic cleavage of gum tissue with proteolytic enzymes; c) culturing fibroblast-like cells isolated from disaggregated tissue on a suitable medium to obtain a culture of myoblasts; d) immunophenotypic testing of myoblast culture for immunophenotype and exclusion of viral and bacterial contamination. (US Pat. No. 2576842). This patent describes a method for obtaining myogenic cells (myoblasts, myoblast precursors) from a new, previously unknown source, namely, by culturing fibroblast-like cells isolated from the mucous membrane of the oral cavity (gums) of a person.

The materials listed above have several disadvantages:

- composite materials are resistant to resorption, contain compounds of unclear composition and biological effects;

- the materials do not provide the stability of the physicochemical conditions in the wound area, which makes possible abrupt changes in the rate and direction of the resorption and regeneration processes;

- do not provide a pronounced osteogenetic effect in the early stages of bone tissue regeneration.

Known biograft, selected as a prototype for the treatment of degenerative and traumatic diseases of the bone tissue of the maxillofacial region (RF patent No. 2380105), characterized in that it contains autologous or donor multipotent mesenchymal stromal cells (MMSC) from bone marrow or adipose tissue of adult donors, which are distributed in a fibrin clot at a concentration of 5-7 million cells in 1 ml, which is a polymerized platelet-rich blood plasma of a patient, and a carrier matrix (in the form of crumbs, shavings), based on its structure, a collagen-mineral complex, identical in composition natural bone material. The disadvantage is the insufficient efficiency of bone tissue restoration, and there is also a risk of prion infection and the emergence of an immune response in a patient due to the use of fetal calf serum in the culture medium. The same patent describes a method for producing a biograft for the treatment of degenerative and traumatic diseases of bone tissue according to claim 1, characterized in that MMSC isolated from bone marrow or adipose tissue are enzymatically disaggregated with trypsin and introduced at the rate of 5-7 million cells per 1 ml of autologous platelet-rich plasma, mixed, if necessary with the addition of additional components, the resulting suspension is mixed with the material of the carrier matrix, which is a collagen-mineral complex in the form of blocks, shavings or crumbs, with washed Hanks solution with cefazolin (1 g / L), then a solution of thrombin 50 U / ml in a 10% solution of calcium chloride is added dropwise until thickening and polymerized at 37 ° C for 30-40 minutes, the resulting graft is cultivated at 37 ° C for 3 to 30 days in an osteogenic culture medium.

The objective of the proposed technical solution is to increase the efficiency of surgical treatment of patients with a surgical profile by improving osteoplastic operations through the use of new tissue-engineered structures.

The technical result is the possibility of using fibrin enriched with leukocytes and platelets, obtained from the patient's blood by a known method, as part of a tissue engineering structure, the use of MMSC isolated from a biopsy of the patient's gum, the use of octacalcium phosphate (OCP) as a carrier, which has good resorption, biocompatibility, and has a developed porous surface, with cultivation on a medium containing citrated nanodispersed CeO ₂ , which has high antioxidant activity. The effectiveness of the application of the proposed tissue-engineering structure has been proved. The use of the structure in such a composition will significantly reduce the risk of complications and shorten the rehabilitation period for patients after surgical removal of jaw bone defects.

The technical essence of the proposed solution lies in the use of a tissue-engineered structure for replenishing the volume of bone tissue in the maxillofacial region, including donor multipotenous mesenchymal stromal cells, platelet-rich plasma of the patient's blood and a carrier. As a carrier, octacalcium phosphate granules with a size of 150-250 μm are used, on the surface of which multipotent mesenchymal stromal cells are incubated, obtained from a biopsy of the patient's gums, at the rate of 2 × 10 ⁷ cells per 1 g of carrier.

And also in using a method for obtaining a tissue-engineering structure, characterized in that the cultivation of multipotent mesenchymal stromal cells is carried out using an aqueous sol of nanocrystalline cerium dioxide stabilized by citrate ions at a concentration of (10-4) - (10-9) M, then obtained multipotent mesenchymal stromal cells are mixed with octacalcium phosphate granules at the rate of 2 × 10 ⁷ cells per 1 g of carrier and incubated at 37 ° C and 5% CO ₂ for 24 hours, after which the tissue-engineered construct is used as intended.

The use of the tissue-engineered construction under study accelerates the process of osteoreparation and promotes postoperative rehabilitation of patients with the following interventions: increasing the volume of bone tissue in width and height; sinus lift; wells conservation after removal; filling of bone defects during cystectomy.

The tissue-engineered construction is recommended for clinical use in reconstructive surgery.

The use of multipotent mesenchymal stromal cells isolated from the oral mucosa seems to be more convenient due to the availability of a tissue source and the minimal invasiveness of biopsy sampling.

The use of (L-PRF) tissue-engineered construction can reduce postoperative complications, shorten the patient's rehabilitation period in the postoperative period.

The proposed technical solution is illustrated by the following technique for obtaining a tissue-engineered structure.

1. Preparation of autologous fibrin clot (L-PRF) according to the standard method.

In patients from the cubital vein, venous blood was taken into sterile disposable glass vacuum tubes with a volume of 9 ml. Immediately after blood sampling, the tubes were placed in a centrifuge. The centrifugation process took place in a given mode: 1300 revolutions for 8 minutes. After that, three layers were formed in the test tubes - blood plasma, platelets and leukocytes in the structure of a fibrin clot, and erythrocytes deposited at the bottom of the test tube. Then, a fibrin clot rich in leukocytes and platelets was taken from the tubes using straight forceps, placed in a sterile dish and used immediately or within several hours. The fibrin clot (L-PRF) was finely dispersed and mixed with the tissue engineered construct.

2. Preparation of an aqueous sol of nanocrystalline cerium dioxide stabilized with citrate ions.

This sol was prepared by dissolving 0.24 g of citric acid in 25 ml of a 0.05 M aqueous solution of cerium (III) nitrate, which was then rapidly added with stirring to 100 ml of a 3 M ammonia solution and then held for 2 hours. Transmission electron microscopy has shown that the sol consists of weakly aggregated, almost isotropic CeO2 particles 2-3 nm in size. The pH of the sol was in the range of 7.2-7.4. The average hydrodynamic radius of CeO2 nanoparticles, determined by dynamic light scattering using a submicron particle size analyzer N5 "BeckmanCoulter" (USA), was 2-3 nm. (Shekunova T.O. Synthesis, biological and photocatalytic activity of cerium dioxide sols stabilized by citrate ion / T.O.Shekunova, D.O. Gil, O.S.Ivanova, V.K. Ivanov, Yu.D. Tretyakov // Nanosystems: physics, chemistry, mathematics. 2013. T. 4. No. 1. P. 83-89).

3. Cultivation of autologous MMSCs of the oral mucosa.

The gingival biopsy was placed in a standard growth medium for transportation, followed by culturing the cell population from it. Then incubation is carried out at + 4 ° C for at least 8 hours. An aqueous sol of nanocrystalline cerium dioxide stabilized by citrate ions in concentrations (10-4) - (10-9) M was used as a culture medium.

Expansion of cells was carried out according to the standard method of culturing MMSC. When the monolayer reached 70-80% confluency, the cells were washed twice with a sterile solution of phosphate-buffered saline, removed from the surface of the vials with 0.05% trypsin-EDTA solution (incubation for 2-3 minutes), trypsin activity was blocked by adding 3-5 ml of washing medium ... Detached MMSCs were collected in sterile tubes, centrifuged and subcultured at a density of about 10 ⁴ cells per cm ² . Then MMSCs were cultured until the required amount was reached, but not more than 4 passages.

4. Preparation of a tissue-engineered construction on an octacalcium phosphate carrier for transplantation.

Synthetic octacalcium phosphate granules with a size of 150-250 µm were placed in a Petri dish (d = 94) so that they formed a line. The cell suspension (1 ml) was transferred dropwise along the line onto OCP granules at the rate of 2 × 10 ⁷ cells per 1 g of carrier. Then, Petriautologous MMSCs were incubated in a dish at 37 ° C and 5% CO ₂ for 24 hours. (it is necessary to mix granules with MMSC at the rate of 2 × 10 ⁷ cells per 1 g of carrier)

After incubation, the composite tissue-engineered construct, consisting of a carrier (octacalcium phosphate) and autologous gingival MMSCs, was washed three times from the culture medium with a sterile solution of phosphate-buffered saline and resuspended in 0.5 ml of saline, placed in sterile tubes with a number corresponding to the donor code, and transported to the clinic in a medical thermal container at room temperature.

To ensure optimal cell viability, the tissue-engineered construct is recommended to be used within 12 hours from the moment the cells are applied to the carrier.

Based on immunophenotyping data, counting colony-forming units, culture data, characteristics of the composite tissue-engineered structure, a passport of the cell product and tissue-engineered structure is drawn up before each use.

conclusions

- multipotent mesenchymal stromal cells obtained from a biopsy of the patient's gums are characterized by high proliferative and secretory activity, as well as the ability to differentiate in the osteoblastic direction;

- improvement of the properties of materials based on octacalcium phosphates can be carried out using biotechnology, which makes it possible to form a layer of mesenchymal cells on the surface of granules, isolated and cultured from a biopsy of the oral mucosa;

- the volume of newly formed reticulofibrous and lamellar bone tissue in the peripheral and central zones of bone defects exceeds the indicators of the control groups due to the pronounced osteoinductive effect of the tissue-engineered structure, due to which they induce the formation of bone regenerate not only from the edges of the bone defect, but also in its center;

- optimized conditions for the formation of chemical bonds between the OCP carrier and MMSC of gingival origin make it possible to create a single complex - tissue-engineered osteoplastic material;

- the use of tissue-engineered construction helps to reduce the time of epithelialization of postoperative wounds, sockets of extracted teeth;

- octacalcium phosphate has no cytotoxic effect on MMSC of gingival origin.

Claims (1)

A tissue-engineered structure for replenishing the volume of bone tissue in the maxillofacial region, including donor multipotent mesenchymal stromal cells on a carrier, which is used as granules of octacalcium phosphate 150-200 μm in size with a pore size of 1-5 μm, on the surface of which multipotent mesenchymal stromal cells are incubated, obtained from a biopsy specimen of the patient's gums and cultured in an aqueous sol medium of nanocrystalline cerium dioxide stabilized by citrate ions at a concentration of (10-4) - (10-9) M, while the concentration of multipotent mesenchymal stromal cells is 2 × 10 ⁷ cells per 1 g of carrier ...