RU2643922C1 - Hydrogel to substitute defects of biological tissue - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely, tissue engineering, and can be used for replacement, reconstructive and regenerative surgery. A hydrogel containing by mass %: sodium alginate – 5–10; D-asparagine – 0.01–1.0; sodium silicate or potassium silicate – 0.05–1.0; the rest is highly purified water are described.

EFFECT: injection makes it possible to obtain on the 40th day of intramuscular injection, a fine-celled construct with single cells located in the walls of its cells, cells with elongated nuclei that are similar in morphology to those of myocytes are seen on the border of the implant.

1 cl, 1 ex, 2 dwg

Description

The present invention relates to medicine, namely to tissue engineering and regenerative medicine, and can be used for replacement, reconstructive and regenerative surgery.

Extensive damage to internal organs is restored to a limited extent, in their place, as a rule, a connective tissue scar is formed, which is due to the low proliferative activity of mature cells, features of the architectonics of damaged biological tissue and features of the intercellular matrix in the postnatal period of ontogenesis. In particular, the limited ability of the striated skeletal muscles to self-regenerate is noteworthy, which justifies the need to develop methods for its artifact restoration.

A promising direction for the restoration of extensive defects in biological tissues, including muscle, is their filling with a hydrogel, which, on the one hand, can serve as a scaffold for proliferating cells, and on the other, be a carrier for various drugs and even stem cells (Sevastyanov V.I. Technologies of tissue engineering and regenerative medicine // Bulletin of transplantology and artificial organs. 2014. No. 3. P. 93-108).

Known injection heterogeneous biopolymer hydrogel based on the hydrolyzate of embryonic or postnatal collagen-containing tissues of animal origin (Sevastyanov V.I., Perova N.V. Injection heterogeneous biopolymer hydrogel for replacement and regenerative surgery and method for its preparation // Russian Patent No. 2433828, 11/20/2011 . Bull. No. 32). The most important disadvantage of this hydrogel, as well as similar products that include collagen, is their unpredictable biodegradation in terms of time, depending on the individual characteristics of the body, often the lifetime of the hydrogel matrix is insufficient for the formation of a full-fledged regenerant.

Known bioactive resorbable porous 3d matrix for regenerative medicine obtained on the basis of polylactide (Sevastyanov V.I., Popov V.K. Bioactive resorbable porous 3d matrix for regenerative medicine and method for its production // Russian Patent No. 2533457, 11/20/2014. Bull. No. 32).

The most important disadvantage of this matrix is the need for its surgical implantation into the affected tissue or organ, while the injection of hydrogels is low invasive. The advantage of injectable hydrogels, in contrast to the proposed matrix, is their uniform filling of the entire volume of a biological tissue defect, as well as ease of use.

Biodegradable polymer hydrogel based on taxanes is also known (Vlasov G.P., Paltuev PM, Semiglazov V.F. . The proposed taxane-based hydrogel acts as an effective system for delivering drugs to the affected organ.

The main disadvantage of a taxane-based hydrogel is the impossibility of using it as a framework for cell proliferation and organ regeneration.

The hydrogel of chitosan carboxyalkylamide is also known (E. Lodge, Goucher F., Perrot J.P. Chitosan carboxylalkylamide hydrogel, its preparation and use in cosmetology and dermatology // Russian Patent No. 2476201, 02.27.2013. Bull. No. 6).

The most important disadvantage of this hydrogel is its failure as a construct for the proliferation of parenchyma cells of internal organs, it is used as a topical preparation for the treatment of skin burns.

The closest analogue of the proposed hydrogel is sodium alginate biogel with the addition of chitosan (Yusova A.A., Gusev I.V., Lipatova I.M. Properties of hydrogels based on mixtures of sodium alginate with other polysaccharides of natural origin // Chemistry of plant raw materials, 2014, No. 4, pp. 59-66). The composition of this biogel includes sodium alginate and succinylated chitosan.

Sodium alginate is a white powder, according to the chemical formula, it is a family of unbranched double copolymers: residues of β-D-mannuronic acid and α-L-guluronic acid, connected by 1 → 4 glycosidic bonds. When water is added, it turns into a gel-like mass.

Succinylated chitosan is a carboxyl-containing water-soluble derivative of chitosan obtained by the N-acylation reaction by treating chitosan with succinic anhydride in its acetic solution. The heteropolymer of D-glucoseamine and N-acetyl-D-glucoseamine, connected by a 1,4-β-glycosidic bond, chitosan has antibacterial, antifungal, antiviral and immunomodulating properties, can be used as a component of gel compositions, as a drug delivery agent.

The main disadvantage of the biogel based on sodium alginate with the addition of chitosan is: the absence of additives that affect cell proliferation, which contributes to the population of the gel implant with fibroblasts. So, intramuscular administration of a biogel based on sodium alginate and succinylated chitosan is accompanied by the formation of a connective tissue capsule around the implant, which makes it difficult to populate the parenchyma of the involved organ with its cells.

Objectives: To improve the regeneration of biological tissues by introducing a hydrogel forming a framework for the regeneration of the affected organ in the area of the lesion site. To develop a biocompatible hydrogel resistant to spontaneous biodegradation, controlled by mechanical properties and architectonics formed in the tissues, after the introduction of the gel, tissue engineering construct.

The hydrogel for replacing defects in biological tissues, as the main component, includes sodium alginate with modifying additives (D-asparagine and sodium silicate (or potassium silicate), which, unlike biogel based on sodium alginate with the addition of chitosan, allows you to control the rate of biodegradation, mechanical properties and morphology formed in the tissues, after the introduction of the gel, tissue engineering construct.

The hydrogel proposed for filling defects in biological tissues, including large muscle defects, consists of (in wt.%):

- sodium alginate from 5% to 10% by weight

- D-asparagine from 0.01% to 1% by weight

- sodium silicate (or potassium silicate) from 0.05% to 1% by weight

- the rest is highly purified water.

The dispersion of the mass fractions of the components of the drug is due to the difference in the requirements for structural and mechanical characteristics of the hydrogel implant. The increase in the content of sodium alginate within the proposed wt.% Allows to increase the density, strength, and also to reduce the size of the cells formed in the tissues, after the introduction of the hydrogel, construct. By varying the percentage of sodium alginate, it is possible to fill in defects of different tissue properties and mechanical properties.

With an increase in the content of D-asparagine, which has antiproliferative properties, the time interval from the implantation of the hydrogel to its colonization with cells increases, which prevents its encapsulation by the connective tissue membrane and inhibits the massive population of the resulting construct with proliferating fibroblasts.

With an increase in the content of sodium silicate (or potassium silicate) in the construct formed after implantation of the hydrogel, the content of silicic acids increases, which increase the strength and significantly extend the lifetime of the polymer framework formed in the tissues, which is designed to populate the regenerating tissue cells.

The technical result of the invention is the intramuscular administration of a hydrogel based on sodium alginate, D-asparagine and sodium silicate (or potassium silicate), which manifests itself on the 40th day from the start of the study by the presence of a fine-meshed construct, with single cells located in the walls of its cells, at the border cells with elongated nuclei that are similar in morphology to myocytes are visible, there are no signs of the formation of a fibrous capsule around the implant.

Thus, the use of implants based on the proposed alginate gel modified with sodium silicate (or potassium silicate) with the addition of D-asparagine is promising for the development of a new method for the treatment of gross muscle tissue defects.

The main components of the hydrogel are sodium alginate, D-asparagine and sodium silicate (or potassium silicate):

Sodium Alginate - Chemical formula: (C6H7O6Na) n. It is a salt of alginic acid, a natural polysaccharide extracted from red and brown seaweed. When finished, it looks like a light beige powder that absorbs water perfectly. It is the hygroscopicity of sodium alginate that makes it possible to effectively use it as a moisture retaining agent, as well as a gelling agent, stabilizer, and substance for encapsulating drugs.

D-asparagine - The chemical formula of D-asparagine: C4H8N2O3. D-asparagine is an odorless white crystalline powder, soluble in water, the chemical name is the dextrorotatory isomer of aspartic acid monoamide.

Sodium Silicate - Chemical formula: Na2SiO3. It is a white fine powder without a specific taste and smell. Dissolving in water, forms a viscous solution. In dilute solutions, sodium silicate decomposes into anions of silicic acid and sodium cations. Silicon acids are formed under the action of sodium silicate acids, the degree of polymerization of which depends on the conditions of the chemical reaction.

Potassium Silicate - Chemical formula: K2SiO3. It is a white finely divided hygroscopic powder without a specific taste and smell. Dissolving in water, forms a viscous solution. In dilute solutions, potassium silicate decomposes into silicic acid anions and potassium cations. Silicon acids are formed under the action of potassium silicate acids, the degree of polymerization of which depends on the conditions of the chemical reaction.

The drug is obtained by dissolving 0.5-1 g of D-asparagine powder in 50 ml of highly purified water in a thermostat at 40 ° C, then 5-10 g of sodium alginate powder is added to the resulting mixture with constant stirring, the resulting mixture is kept in a thermostat at 45 ° C during the day. Then, 0.05-1 g of sodium silicate (or potassium silicate) is dissolved in 30 ml of highly purified water. Then, the prepared solution is mixed, the resulting mixture is adjusted to 100 wt.% With highly purified water and processed in a homogenizer at 10 thousand rpm for 30 minutes, with obligatory cooling of a glass of a homogenizer in an ice bath. At the final stage, the preparation is kept in a thermostat at 45 ° C for 12 hours, then the hydrogel is ready for use.

The drug was tested on 24 white non-linear male rats with an average weight of 272 ± 15 grams, the obtained hydrogel was injected into the left femoral muscle of the rats by intramuscular injection, and then, under the influence of calcium cations contained in biological fluids, its polymerization occurred, with the formation of a cellular construct. Euthanasia was performed 40 days after the start of the study by decapitation of pre-anesthetized rats. Muscles were fixed in a 10% neutral solution of paraformaldehyde. Samples were passed through isopropanol, followed by pouring into paraffin. Paraffin blocks were cut into sections with a thickness of 10 μm on a microtome MPS-2 (USSR). Micropreparations were stained with hematoxylin and eosin.

On micropreparations of muscle tissue obtained from rats that received an intramuscular injection of the drug, single cells with elongated nuclei (presumably myocytes) appeared in the bridges of the resulting mesh structures of the frame at the interface with the gel with muscle; There were no signs of a connective tissue capsule around the implant. The experimental results are confirmed in Fig. 1 and 2, on which the cellular structure of the skeleton formed after the introduction of the hydrogel is traced, located in the cell wall region of the cell skeleton with elongated nuclei.

Example 1. A non-linear male rat, weighing 265 g, received an injection of 0.5 ml of the drug into the muscles of the right thigh. For surgery, zoletil-xylazine anesthesia was used. Euthanasia was performed on day 40, after which, with the help of exarticulation, the right hind limb was separated from the corpse, then the skin and lower leg were removed from it. The thigh muscles were fixed in a 10% neutral solution of paraformaldehyde. Samples were passed through isopropanol, followed by pouring into paraffin. Paraffin blocks were cut into sections with a thickness of 10 μm on a microtome MPS-2 (USSR). The obtained histological sections were stained with hematoxylin and eosin, then, after dehydration in a series of alcohols, the micropreparation was placed under a coverslip using polystyrene resin. The prepared micropreparation was photographed using a Levenhuk-230 camera (USA). The experimental results are confirmed by Fig. 1 and 2, where fig. 1 shows an area of a net skeleton formed from an injected preparation and adjacent fibers of striated skeletal muscle tissue. Single cells with elongated nuclei (presumably myocytes) are visible on the border with the muscle, in the ligaments of the reticular framework, the photo was taken under magnification at X40 (stained with hematoxylin eosin). In fig. Figure 2 shows a mesh framework with single cells located in the walls of its cells; individual sections are traced morphologically similar to emerging muscle fibers; photo was taken under X100 magnification (stained with hematoxylin eosin).

Claims (5)

Hydrogel for the replacement of defects in biological tissues, containing sodium alginate, characterized in that the hydrogel has, wt.%: - Sodium alginate - 5-10, - D-asparagine - 0.01-1.0, - Sodium silicate or potassium silicate - 0.05-1.0, - The rest is highly purified water.

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