RU2431504C2 - Method for preparing composite chitosan and collagen resorbable matrixes for human skin cell culture - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemistry of high-molecular compounds, namely to preparing film and spongy chitosan and collagen materials effective in human skin cell culture and wound grafting. A method for preparing the composite chitosan and collagen resorbable matrixes for human skin cell culture involves preparation of solutions polysaccharide chitosan and protein collagen, mixing them in preset proportions and formation of film and spongy matrixes of mixed polymer solutions. For this purpose, chitosan and collagen solutions in the concentration 1.0-4.0 wt % in a general solvent (aqueous 2 % acetic acid) are prepared, mixed in preset proportions, and the film and spongy matrix materials are formed from the prepared solutions. An amount of collagen in polymer mixtures makes 2.5-10 wt % (of chitosan). Further, films and sponges are heated to temperatures within 50-100°C for 1.0-5.0 h in an atmospheric environment.

EFFECT: use of the declared method allows preparing the film and spongy resorbable composite materials of natural polymers effective in human skin cell culture.

2 cl, 6 ex, 2 tbl

Description

The method relates to the chemistry of natural macromolecular compounds, namely the production of composite materials based on chitosan and collagen in the form of films and sponges that are capable of absorbing in the human body and suitable for growing human skin cells on them. The invention allows to obtain composite matrix materials suitable for biomedical purposes, when using as a common solvent two natural polymers (chitosan and collagen) environmentally friendly 2% aqueous solution of acetic acid. In order to increase the stability of matrix materials in the culture fluid, the time of their resorption on the surface of the wound and increase the adhesion of cells to the surface of the material, they are heated, resulting in chemical crosslinking of polymer macromolecules.

Natural polymers, such as chemically modified chitin, chitosan, gelatin, silk fibroin and synthetic polymers, such as polylactone (polylactic acid) [D. Ozdemir, G. Schoukens, O. Goktepe, F. Goktepe, are used as polymer matrices for growing human cells. . Preparation of Di-Butyryl-Chitin Scaffolds by Using Salt Leaching Method for Tissue Engineering and Their Characteristics. // Journal of Applied Polymer Science. 2008. Vol. 109. P. 2882-2887; M.N.V. Ravi Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A.J. Domb. Chitosan Chemistry and Pharmaceutical Perspectives. // Chem. Rev. 2004. Vol. 104. P.6017-6084; H. Yamada, Y. Igarashi, Y. Takasu, H. Saito, K. Tsubouchi. Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts. // Biomaterials. 2004. Vol.25. P. 467-472; A.Prokop, A.Jubel, H.J. Helling, T. Eibach, C. Peters, S.E. Baldusc, K.E. Rehm. Soft tissue reactions of different biodegradable polylactide implants. // Biomaterials. 2004. Vol.25. P. 259-267].

The closest in essence is a method of producing matrix materials based on chitosan and collagen intended for growing epidermoid cancer cells [N. Shanhugasundaram, P. Ravichandran, P.N. Reddy, N. Ramamurty, S. Pal, K. Panduranga Rao. Collagen-chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells. // Biomaterials. 2001. Vol.22. P. 1943-1951]. Pre-prepared solutions of chitosan and collagen with a concentration of 0.6-1.4% (mass) in glacial acetic acid. The resulting polymer solutions are mixed in predetermined proportions, mixed and cast onto glass plates. In order to reduce the swelling of the resulting composite films in the culture medium, a solution of glutaraldehyde in toluene is introduced into the solutions of polymer mixtures, with which chemical crosslinking of chitosan and collagen macromolecules is carried out. Composite materials are obtained with a collagen content of 30.0-70.0% (mass).

Significant and obvious disadvantages of the method are the use of glacial acetic acid polymers as a solvent and an additional step in the process of chemical crosslinking of polymer macromolecules with glutaraldehyde dissolved in toluene. Glacial acetic acid in contact with human skin causes severe chemical burns, and its vapors irritate the mucous surfaces and eyes of a person. Toluene is a toxic product, according to the degree of impact on the human body, it belongs to the 3rd hazard class and at high concentrations its pairs act narcotically. Therefore, control over the absence of toluene in the resulting matrix materials is required. The high collagen content in matrix materials leads to a deterioration in the strength characteristics of the resulting matrices.

The technical task and the positive result of the proposed method is to obtain film and sponge composite absorbable matrices based on chitosan and collagen from polymer solutions in a solvent safe for human health, without the use of a chemical cross-linking reagent in a toluene solution and with a small content of collagen. The developed method is simple to implement. An aqueous 2.0% solution of acetic acid is used as a general polymer solvent, and to increase the stability of the matrices, instead of introducing a chemical cross-linking reagent (glutaraldehyde) into the polymer mixtures, heating the obtained materials at elevated temperature in the atmosphere is used.

The specified task and the result are achieved in that the method involves the preparation of solutions of a polysaccharide - chitosan and protein - collagen concentration of 1.0-4.0%) (wt.) In a common solvent (aqueous 2% solution of acetic acid), mixing them in given ratios and molding from solutions of polymer blends of film and sponge matrices. The films are formed by casting the solution through a die with an adjustable gap on a glass substrate and subsequent evaporation of the solvent. The production of spongy materials is carried out by casting a solution of polymer mixtures onto a polymer substrate, followed by freezing at T = -45 ° C and freeze drying until the solvent is completely removed. In order to increase the stability of matrix materials in the culture fluid, the time of their resorption on the surface of the wound and increase the adhesion of cells to the surface of the material, they are heated in the temperature range of 50-100 ° C for 1.0-5.0 hours in atmospheric atmosphere. The amount of collagen in polymer mixtures is 2.5-10% (by weight of chitosan).

Example 1. Obtaining a composite film containing 1.0% collagen (by weight of chitosan).

3 g of chitosan is dissolved in 97 g of an aqueous 2% solution of acetic acid and a 3% solution of chitosan is obtained. 0.5 g of collagen is dissolved in 99.5 g of an aqueous 2% solution of acetic acid and a 0.5% solution of collagen is obtained. To 100 g of a 3% solution of chitosan, 6 g of a 0.5% solution of collagen are added with stirring over 1 hour. The resulting polymer mixture solution is dialyzed against water in order to reduce acetic acid in the polymer solution. The solution of the polymer mixture with a die is applied to a glass substrate (or poured into Petri dishes), and then dried at room temperature until the solvent is completely removed. The resulting composite film contains 1.0% (wt.) Collagen (table 1, example 1).

Example 4. Obtaining a composite film containing 10.0% collagen (by weight of chitosan).

3 g of chitosan is dissolved in 97 g of an aqueous 2% solution of acetic acid and a 3% solution of chitosan is obtained. 0.5 g of collagen is dissolved in 99.5 g of an aqueous 2% solution of acetic acid and a 0.5% solution of collagen is obtained. To 100 g of a 3% solution of chitosan, 60 g of a 0.5% solution of collagen are added with stirring over 1 hour. The resulting polymer mixture solution is dialyzed against water in order to reduce acetic acid in the polymer solution. The solution of the polymer mixture with a die is applied to a glass substrate (or poured into Petri dishes), and then dried at room temperature until the solvent is completely removed. The resulting composite film contains 10.0% (wt.) Collagen (table 1, example 4).

Table 1 The deformation and strength characteristics of composite films based on chitosan and collagen Examples Collagen Content (wt.%) Film properties Young's modulus, E, GPa Tensile strength, σ _p , MPa Elongation at break, ε,% Specific surface, m ² / g one 0 5.2 96.0 13.3 1,261 2 2.5 6.8 131.2 11.1 - 3 5,0 7.0 140,4 14.1 - four 10.0 7.2 148.0 14.2 1,412

Examples 2-3. Obtaining composite films is carried out analogously to example 1 and 4.

Example 5. Obtaining a spongy matrix material containing 10.0% collagen (by weight of chitosan).

The polymer mixture solution is prepared analogously to examples 1 and 4. The prepared solution is poured into glass or plastic vessels (or Petri dishes), frozen at -45 ° C and subjected to freeze drying. The resulting sponge material has the characteristics of:

- porosity - 34.5%;

- apparent density - 27.2 kg / m ³ ;

- specific surface area - 7.22 m ² / g;

- moisture absorption - 50.0%.

Example 6. Improving the stability of matrices in a culture medium by heat treatment.

In order to give the matrices stability in the culture medium, increase the adhesion of the cells to their surface and the time of the resorption of the matrices on the wound surface, the films and sponges are heated in the temperature range of 50-100 ° C for 1.0-5.0 hours in atmospheric environment.

A change in the deformation-strength characteristics of matrices and their solubility in water is shown by the example of chitosan films (Table 2).

table 2 Change in the deformation-strength characteristics and water solubility of chitosan films depending on the time of their heating. Additional film processing Film properties Young's modulus, E, GPa Tensile strength, σ _p , MPa Elongation at break, ε,% Solubility in water,% Freshly formed film 4.7 122.0 38,0 one hundred Warm up 3 hours at 100 ° C 6.4 158.1 22.3 0

After additional heating of the films, their strength and Young's modulus (elasticity) increase, indicating an increase in the stiffness of the films, as well as a loss of solubility in water.

Claims (2)

1. A method of producing composite absorbable matrices based on chitosan and collagen for growing human skin cells, characterized in that the film matrices are prepared from a mixture of solutions of chitosan and collagen with a total concentration of 1.0-4.0 wt.% In a 2% aqueous solution acetic acid and formed on a glass substrate; to obtain sponge matrices, solutions of mixtures of chitosan with collagen are frozen at -45 ° C and subjected to freeze drying, while the amount of collagen in polymer mixtures is 2.5-10% by weight of chitosan. 2. The method according to claim 1, characterized in that in order to give the matrices stability in the culture medium, to increase the adhesion of the cells to their surface and the time of the resorption of the matrices on the wound surface of the film and the sponge, they are heated in the temperature range of 50-100 ° C for 1, 0-5.0 h in atmospheric conditions.