RU2737358C1 - Super-thin ultrathin collagen membrane and method of production thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to tissue engineering, in particular to collagen membranes used as xenografts and discloses a collagen membrane from calamary biotissue and a method for production thereof. Collagen membrane of calamary biological tissue of Dosidicus Gigas family Ommastrephinae possesses tensile strength of 150-250 MPa, relative elongation at break of not less than 20 %, with thickness from 20 to 100 mcm, with thickness deviation from average value of not more than 5 %, with collagen content of not less than 98 % in terms of absolutely dry substance. Method of producing includes calamary carcass cutting into separate anatomical parts by mechanical method, with separation of covering mantle connective tissue, which is successively held in solution of neutral salt, in a slightly alkaline solution containing lipolytic and proteolytic enzymes, then washed with water, acid solution with high ionic strength, then an alkaline solution, distilled water and dried.

EFFECT: collagen membrane has biocompatibility, resorbability under physiological conditions, can be used as a xenograft, in tissue engineering, cell culture and cosmetic manipulations, virtually without restrictions.

1 cl, 1 ex

Description

The present invention relates to collagen membranes used as xeno grafts, matrix in tissue engineering, in cell culture growth, as well as mask bases for cosmetic manipulations.

Collagen membranes are traditionally obtained from the skin and other connective tissue structures (for example, the pericardium) of terrestrial animals, most often cattle and pigs.

Despite the fairly widespread use of these membranes, it turned out that they may contain pathogenic viruses, including the spongiform encephalopathy virus, which is fatal to humans. In addition, for large groups of the population, the use of xeno grafts from a particular animal species is not possible due to religious and ethical considerations.

In this regard, collagen membranes, obtained on the basis of connective tissue structures of marine hydrobionts, are acquiring more and more interest and significance. The environmentally cleaner environment for marine organisms to a large extent guarantees the absence of pathological factors that pose a danger to humans.

Fish skin is usually used as a source for obtaining collagen membranes.

To obtain a collagen membrane from fish skin, the original object undergoes special processing to destroy and remove extraneous cellular structures, as well as lipids, globular proteins, nucleic acids and other non-collagen components.

The resulting membrane is usually a collagen film with a thickness of about 0.2-0.4 millimeters, which is biocompatible and resorbable under physiological conditions - factors that determine the success of their use.

However, no less important characteristics of the collagen membrane, in addition to biocompatibility and resorbability, are its mechanical strength and plasticity.

The mechanical strength, characterized as the value of the ultimate fracture stress under linear deformation, for most of the known collagen membranes is in the range from 5 to 15 MPa, and the plasticity, estimated as the elongation at break, is at the level of 7-9%.

Despite the fact that collagen membranes with such characteristics can be used in practice, a rather urgent task is to obtain more durable and at the same time plastic collagen membranes, since high values of these indicators make it possible to expand the scope of application and reduce the risk of mechanical destruction of membranes, especially during surgical procedures and cosmetic manipulations.

Unfortunately, until recently it was not possible to solve this problem when using fish skin.

In this regard, it was proposed to use fish scales rather than skin as a raw material for obtaining a collagen membrane.

The known method of obtaining a high-strength collagen membrane, proposed by Tanaka, et. al. "High-strength collagen fiber membrane and manufacturing method thereof" (US Patent No. 9,315,562, Apr. 19, 2016.).

The method involves the use of fish scales as a collagen-containing raw material, mainly of the Tilapia species of the Cichlidae family.

The method is based on the extraction of collagen, followed by its release in the form of a hydrated fibrillar gel, dried between two layers of a moisture-proof material, in which the evaporation of the solvent occurs only from the open side surfaces.

Despite the fact that the resulting collagen membrane has a tensile strength of about 30 MPa, the membrane itself, as well as the method of its production, have a number of significant disadvantages.

First of all, the strength obtained according to the described method of the membrane does not fundamentally exceed the strength of the known analogs, and its plasticity, defined as the value of the maximum elongation at break, is no more than 8%.

In addition, the membrane is not uniform in thickness. Moreover, the difference in the thickness of the same sample can reach 30%, counting from the average value of this indicator. This leads to the unpredictability of the topology of its rupture, especially when the membrane is fixed with surgical sutures. In addition, the difference in thickness causes significant differences in the rate of resorption of individual sections of the membrane, which presents a certain problem in the case of its use as a resorbable xeno graft.

The disadvantages of this method include:

- the need for demineralization to remove the minerals contained in the scales

- the use of an extraction scheme for the isolation of collagen from demineralized raw materials, which significantly increases the duration of the process, requires large amounts of highly purified reagents and water, as well as the use of multiple centrifugation or filtration;

- formation of a membrane by drying a collagen gel between two layers of glass or polymer material, which is a long and practically not scalable procedure, and makes it extremely difficult to use the method.

The object of the present invention is a collagen membrane from marine aquatic organisms, which is a film of almost the same thickness, greater strength and plasticity, as well as a method for its production, devoid of the above disadvantages.

This goal is achieved by the fact that the covering biological tissue of the mantle of cephalopods, more precisely squids, specifically, the squid of the species Dosidicus Gigas of the family Ommastrephinae, is used as a raw material.

The Dosidicus Gigas squid is a large species of ten-armed squid, with an estimated biomass of about 10 million tonnes, while the catch in the Pacific Ocean alone ranges from 300,000 to 700,000 tonnes per year.

The bulk of dozidicus is processed to separate the mantle, the most nutritionally valuable component of squid. At the same time, already at the initial stages, the biological tissues covering the mantle are separated and practically not used.

The latter includes a layer of connective tissue that is directly adjacent to the mantle and is a special biological tissue, consisting of collagen fibers. This collagen biological tissue (to a certain extent functionally similar to fascia) is mechanically detached quite easily, and can be obtained in the form of a hydrated strong film of millimeter thickness, and an area of the order of several thousand square centimeters, depending on the size of the squid individual.

The main component of biological tissue is collagen, with an insignificant content of lipids, carbohydrates and minerals.

After cleaning biological tissue from non-collagen components, a thin strong membrane can be obtained, the geometric dimensions of which are close to the original fragment.

For this, the mechanically separated biological tissue is processed for 3-10 hours with an aqueous solution of neutral salt with an ionic strength of 0.2-0.5 M, for example, sodium chloride.

With this treatment, the biological tissue practically does not increase in volume, there is no significant change in its structure, at the same time, part of the globular proteins, peptides, nucleic acids and nucleotides, as well as low- and high-molecular carbohydrates pass into the saline solution.

When using saline solutions with a concentration below 0.2 M, a noticeable swelling of biological tissue is observed with a certain change in its native structure, and at concentrations above 0.5 M, the aggregation of collagen fibrils is promoted, with a significant increase in the rigidity of biological tissue, which negatively affects the completeness of extraction of non-collagen components.

A decrease in the processing time below the specified limit reduces the degree of extraction of non-collagen components, and its increase is not accompanied by its significant increase.

After processing, the biological tissue is removed from the saline solution and placed in a 5-10 fold excess of a weakly alkaline aqueous solution with a pH of 8-10, containing lipase and protease, in which it is kept for 3-4 hours at a temperature of 10-25 degrees. Celsius, with constant monitoring and maintaining the pH at the initial level. In the course of processing, lipids and non-collagen proteins are hydrolyzed, and the hydrolysis products pass into solution.

As a lipase, it is preferable to use an alkaline lipase, for example LIPOPAN F, lipazine, fluozyme GZH, as well as a neutral or alkaline protease, for example ENZYM, PROK, Superlotan A.

The selected pH range is close to optimal in terms of enzyme activity. At the same time, a decrease in the processing temperature below 10 degrees C increases the hydrolysis time, and its increase is above 25 degrees. C can lead to a change in the native structure of collagen and partial inactivation of enzymes.

After the end of the enzymatic treatment, the biological tissue is washed with water and treated for 30 minutes with 1 M acetic acid solution containing 10% sodium chloride to inactivate enzymes, and then with 0.1 M sodium bicarbonate solution to neutralize the acid, and distilled water to remove salts and other low molecular weight compounds.

The resulting moist purified biological tissue is extracted from the solution, dried in air or by sublimation.

In essence, it is an air-dry collagen film (membrane) containing, in terms of absolutely dry matter, at least 98% collagen, with virtually no lipids, as well as low and high molecular weight carbohydrates.

The complex of physical and mechanical properties of the collagen membrane significantly exceeds the properties of known analogues. So the ultimate stress at break reaches 250 MPa, with a relative elongation of more than 20%.

Air-dry collagen membrane, has a thickness of 20 to 100 microns, with a deviation from the average value of no more than 5%.

The process of obtaining a collagen membrane, according to the proposed method, excludes the stages of extraction and deposition of collagen, and is characterized, in general, by simplicity, short duration, does not require the use of expensive reagents and complex equipment, and can be easily scaled up.

Below is a specific example of the application of the invention.

The squid Dosidicus Gigas is cut into anatomical parts, with the separation of the layer of connective biological tissue immediately adjacent to the mantle, which is washed with water.

100 g of biotissue, about 950 sq. cm, is placed in a fivefold excess of 0.5 M sodium chloride solution, in which it is kept for 8 hours, after which it is removed from the saline solution and washed with 2 liters of water.

The washed biological tissue is transferred into an aqueous solution of sodium bicarbonate with a pH of 8, containing Lipopan F lipase and Enzym protease, in amounts providing lipase and protease activity in the solution at a level of 50 and 10 IU, respectively, kept in it for 4 hours at a temperature of 20 ° C , in the mode of periodic stirring and pH-staging, and then it is removed from the solution and washed with 1 liter of water.

The washed biological tissue is placed in a 1 M solution of acetic acid containing 10% sodium chloride, in which it is kept for 10 minutes, and then in a 0.2 M sodium bicarbonate solution to neutralize the acid, and washed with distilled water until the medium pH is neutral.

The washed biological tissue is dried in air at a temperature of 40 ° C.

The result is a collagen membrane, which is a thin flat translucent sheet with an area of 820 sq. cm, 50 microns thick, with a thickness difference of 1.5 microns, with a tensile strength of 220 MPa, and a relative elongation at break of 23%, containing 98% collagen in terms of absolutely dry matter.

The collagen membrane is biocompatible, resorbable under physiological conditions, characterized by increased physical and mechanical characteristics, can be used as a xeno graft, in tissue engineering, cell culture cultivation and cosmetic manipulations, practically without restrictions.

Claims (9)

1. Collagen membrane made of squid biological tissue, with a tensile strength of 150-250 MPa, a relative elongation at break of at least 20%, a thickness of 20 to 100 microns, with a thickness deviation from the average value of no more than 5%, with a collagen content of at least 98% in terms of absolutely dry matter. 2. Collagen membrane according to claim 1, characterized in that the biological tissue is used as a biological tissue covering the mantle of the squid. 3. Collagen membrane according to claim 1, characterized in that a giant squid of the species Dosidicus Gigas of the family Ommastrephinae is used. 4. The method of obtaining a collagen membrane from squid according to claim 1, which consists in the fact that the squid is cut into separate anatomical parts mechanically, with the separation of the integumentary connective tissue of the mantle, which is sequentially kept in a solution of neutral salt, in a weakly alkaline solution containing lipolytic and proteolytic enzymes, then washed with water, a solution of acid with high ionic strength, then with an alkaline solution, distilled water and dried. 5. A method according to claim 4, characterized in that an aqueous solution of sodium chloride with an ionic strength of 0.2-0.5 M is used as a solution of a neutral salt, and the holding time in it is 3-10 hours. 6. The method according to claim 5, characterized in that an aqueous solution of sodium bicarbonate with a pH of 8-9 is used as a weakly alkaline solution, containing lipase and protease in amounts providing lipase and protease activity at the level of 50 and 10 IU, respectively. 7. The method according to claim 5, characterized in that the treatment with a weakly alkaline solution is carried out in the pH-staging mode for 3-4 hours at a temperature of 10-25 ° C. 8. The method according to claim 4, characterized in that a 1 M acetic acid solution containing 1-2 M sodium chloride is used as a solution of an acid with a high ionic strength, and a 0.1 M sodium bicarbonate solution is used as an alkaline solution. 9. A method according to claim 4, characterized in that drying is carried out in air at a temperature not exceeding 40 ° C.