CS269196B1 - Biocompatible collagenous material and method of its preparation - Google Patents

Description

EN 269 196 B1 1

The present invention relates to biocompatible collagen material and to epoeobic preparation thereof. Collagen, a relevant protein of connective tissue, bone, cartilage, skin and strength, has been used for a long time to serve the purpose of providing aids in medical applications. Several useful properties are used, such as low immunogenicity, gradual self-destructive degradation, wearability and appropriate physico-non-chan- nical properties, or even direct therapeutic supportive action in some pathological situations in fibrous structures. A collagenous, vigorous molded material with a varying degree of porosity, moisture content, or thirty-eight thsrapsutically effective drugs is used to create drainages, bullets, pads, or fillings in surgery, or even as a replacement for xenotransplates and reconstructive material in injuries and deep pathological changes. (Chvapil, M., Kronenthai BL, Van Winkles Medical and Medical Applications of Collagen. Jnt. Eev. Connective Tiseue Beecarch, 1973, 6, 1-61; Tanas IV, Burke JF, Deaigp et al. Bee, 1980,14, 65-81, Oliver RF, Barker H, Cooke A., Grante HA, Dermal Collagen Implants, Biomaterials, 1982, 3, 38-40, Doillpn CJ, Whyne CF, Berg RA, Olson BM, SilverF.H., Fibroblaet-collagen aponge interaotions in vivo, Scanning El.Micrography, 1984, III, 1313-1320, Chvapil M., Colagen Sponge: Theory and Practice of Medical Medicine, J. Biomed. Material Bas., 1977, 11, 721-741, Begalakis N., Flink, J., Sta-aikalis, JF, Burke, JF, Lannas, IV, Dissign of an Skin, Part II: J.Bio-Med. , 1980, 14, 511-528.

Despite the fact that for many years differently purified collagen has been regarded as a non-immunogenic material and is still not considered to be a potent immunogen, the above-mentioned literature shows that the choice of collagen source and its purification method is to be paid attention to. . Further questions related to the lack of problems associated with controlling the rate of collagen impalateate degradation by more precise control of the degree of sowing in the production of collagenous substance, and the issue of collagen fiber distribution; Especially applications requiring a new solution.

The above-mentioned disadvantages are solved by the collagen material of the present invention, which consists of one collagen type (I) comprising two distinct polypeptide chains </ 1 / I / 2 and </ 2 transversely sieved 1.5 up to 8 bonds per mole of collagen, wherein the non-collagenous protein content is at most 0.5% by weight. based on collagen, the calcium content does not exceed 1 mg / g of collagen and the ash content is not more than 0.5% by weight, the metal ions being in the form of a chelate complex. The collagen thus prepared, in contrast to known materials, is characterized by its ability to withstand the growth of microorganisms even under moist and non-sterile conditions, without the use of SalI bacteriostetics. The question of controlling the residence time of the implant in the tissue of the recipients can be, to some extent, controlled by reducing the lateral cross-linking of the collagen by the alkaline environment over a period of time, the longer the alkaline setting time, the shorter the residence time of the implant, respectively. It is its distribution by phagocytosis and enzymatic processes.

The essence of the preparation of the collagen material is that the collagenous silage is made up of 50% saturated to saturated calcium hydroxide solution at least three times in succession, then the binder is washed with water, neutralizing the aqueous solution containing 1 to 12% by weight. ammonium chloride or sulphate or sodium borohydride based on the weight of the ligament, then the binder is subjected to a non-collagenolytic effect of a protease such as pronase, trypsin, ohymotripsin, pepsin, panreatin or alkaline added in an amount corresponding to 10 to 10,000 casein units per 1 gcolagene at 20 to 37 ° C for at least 20 minutes, rewashed with water and subjected to the complexing agent selected from the group CS 269 196 B1 no nitrilotriacetic acid, sodium salt of this acid, ethylenediamino acid. tetraoot or other sodium carbonate for about 10 minutes and does not overcoat with water.

Grind the collagen mass and after acidifying to a pH of 2 to 4.5 and filter the fabric filter, eventually the acidified collagen mass after filtration to precipitate the fibrous form and rinse again with water. The fibrous or gel-like collagen mass can be frozen at -90 to -60 ° C and then freeze-dried and sterilized by temperature> 80 ° C or gamma irradiation, before exposure to freezing or after freezing. gaseous or liquid carbon dioxide feed below 0.02 to 0.6 MPa.

Ensuring the homogeneity of the collagen type is ensured by the choice of 31’ach collagen, preferably by achilles, instead of the more commonly used skin materials. It is known that co-lagen is composed of rod-shaped polymer molecules composed of three polypeptide chains, the so-called alpha chains, which are wound into one another in the form of a characteristic triple helix. The existence and stability of such a structure is due to the presence of glysine at each third position and the relative large amounts of amino acid residues, proline and hydroxyproline, also contribute to stability. Different, genetically determined types of collagens are found in various tissue structures. The number of all known types is still in the process, now we know 8 type groups from which the second part is divided into individual members of the type group. If the ligament is in normal physiological J situation, there is only one of the two known members of the collagen family of I, collagen, which contains two distinct polypeptide chains of < RTI ID = 0.0 &gt; Z1 / I / 2 &lt; / RTI &gt; and the organism (I / I / I) is found in pathological conditions only, or in vitro in cell cultures, tumors or tissue growth. However, the choice of collagen source alone does not ensure the problem of isologic activity of collagen preparations, but it simply simplifies it. In many cases, collagen-stimulated antigen formation is not significant in some aituaries, and may be less severe in the overall immunological resistance of collagen implant recipients. immunogenic capable of an unfavorable factor. In these contexts, two immunological properties of protein antigens are usually distinguished: antigenic speoifilae and immunogenicity. Antigenicity is the term used to describe the ability of a biscuit to react with secreted antibodies, while immunogenicity refers to the ability of a protein to stimulate the production of an antibody. Immunochemical assays in rhodium lead collagens revealed three distinct regions of collagen molecules characterized by antigenicity. The most important of these sanaohadxa regions in the terminal, C-cono telopeptide. the farther antigenic region! it is in the mid-ηβ j, the helical region of the molecule, but this is particularly active in the structural transformation resulting in a reduction in the alignment, that is, in the conversion of collagen to gelatin modification. Antigenic regions also occur in the N-cono telopeptide, and in particular, for and for collagen peptides cleaved in biosynthesis. The most important anti-nickel regions are those that are loaded in terminal peptidoohs. Since these regions are at the same time most sensitive, they are easily cleaved by non-collagenolytic xylase proteins, there is therefore a possibility of reducing the antigenicity of collagen preparations by enzymatic pathway. This possibility is used in the process of preparing the collagen material of the invention.

Unlike other methods of purifying collagen feedstock in the manufacture of a health-compliant material, the purification phase uses a step based on pronase, trypsin, chymotrypsin, or pathway, and a robust extraction of cleaved peptides of the remaining enzymes from the purified material. In terms of immunogenicity, the ability to induce antibody production, then this property is still unclear. Some workers believe that the cause may be in the heterogeneity of collagen types, since immunogenic reactivity is enhanced with the increasing number of collagen types found in the tested lolagen. Enzymatic cleavage of the terminal regions of immunogen CB 269 196 B1 3 does not diminish, as does the decrease in alignment in helical regions, e.g. It is important to observe and to modify the collagen often alongside significant increases in immunogenicity. For example, aethyldioyl, hydroxylamine, succinylaium, methylaium or other modifications on the acid and basic amino acid side chains of collagen have turned this material into a relatively strong immunogen. This suggests that chemical modification of the collagen side chains, often used in the preparation of collagen materials listed in the literature (Oliver BP, Barker H., Cooke A., Grande BA, Derme1 Collagen Implants, Biomateriala1982, 3, 38- 40). According to the invention, the requirement for the lowest molarity of native collagen molecule is respected, while respecting the requirement for as few collagen types as possible. On the other hand, such an approach precludes chemical treatments that ensure! otherwise inevitable microbial resistance to collagen derivatives by wet environments and staining of secondary contaminants by microorganisms. To solve the problem, the inventors have found that the substantial depletion of mineral collagen collagen, especially calcium and other salts of polyvalent cations, induces significant microbial resistance even in the absence of bacteriostatic agents, or aldehyde modification of collagen groups as is known from the art. preparation. Another advantage of the present invention is the ability to reproducibly regulate the degree of natural cross-linking of the produced collagen derivatives, by means of slow slow crosslinking. Thus, it is possible to define precisely the most important property of collagen substrates, which determines the rate of metabolic exchange or self-destruction of implants in vivo.

The advantages of the preparation of collagen materials according to the invention are related to the production of collagen foams intended for hemostatic and other purposes. Previously prepared materials of this type are based on collagen mass soaked in acid or base and freeze-dried under reduced pressure. These masses have a foam-closed structure, i.e. the individual sponge-like cores are separated from each other by a thin wall. Such a structure is suitable in all cases where the material serves for the purpose of clogging, but in some applications, the ability of the scalp or physiological scoop is required. liquid or controllable liquid permeability. For this purpose, the use of the methods of the invention may be based on the use of a water-soluble gas, preferably carbon dioxide gas, which is dissolved under pressure in a wet co-lager prior to freezing. 7 during the freeze-drying under reduced pressure, the walls of the voids in the foam are perforated by the expanding gas, so that the product has an open tuft capable of sucking in and leaking liquid. EXAMPLE 1 1 kg of purified bovine aoholic salt, stripped of end pieces and cut into pieces of about 1 cnP, are washed with water and extracted three times with 5 l of a solution containing 100 g / l of sodium chloride with occasional stirring, each for 24 hours at heat. Low: 10 ° C. The extracts are washed with distilled water twice each with 5 l of water for 4 hours. They are then extracted three times with 50% saturated calcium hydroxide solution for 24 hours at 10 ° C with occasional stirring. If the degree of cross-linking is determined from the denaturation temperature of the collagen in 6 M urea solution to be greater than 4 / mole, the alkali treatment is continued in the exchanged calcium hydroxide solution until the covalent sieving degree is reduced. After alkaline modification, neutralization is carried out by feeding 2 l of 25 g / l ammonium sulfate solution for 12 hours. After decanting, the pellets are washed with distilled water, washed twice with 5 l for 4 hours and then kept at 5 DEG C. for 24 hours in 10 l of redistilled water. The 51’achs are then blended in water using a stainless steel knife mixer at a temperature not exceeding 15 ° C. The fiber mixture is washed three times with distilled water and dewatered by decantation. The pulp is blended in 2 liters of water 4 EN 269 196 B1 with a pre-heatable 200 mg of Pronene (used Pronann P roinat product, pre-product of Streptomyces grisouo, supplied by Color, Heidelberg, NBR) and mild samie for 12 hours. Then, the fiber is decanted and washed five times with redistilled water for 5 hours. After the morning washing, the water left for 24 hours with the fiber does not reduce its conductivity by more than 36% and does not contain soluble ninhydrin detectable proteins. The collagen fiber is then acidified with acetic acid to pH 3.6 and blended in a swollen state at 5 ° C for 5 minutes, then 20 mg of ohjiatone III per g of collagen are added and neutralized with dilute sodium hydroxide at pH 7.0. The separated fibers are washed with redistilled water, 5 l each for 5 hours three times and 5 1 for 24 hours three times. The pulp is then partially dewatered by centrifugation and used to make collagen parts that are sterilized by gamma irradiation. EXAMPLE 2 1 kg of calico aohill sausage, cleaned and stripped from the ends of the wax shells, is washed with water, blended in an aqueous medium with a stainless steel knife mixer at a temperature below 20 DEG C. and washed several times with distilled water. The fiber is extracted with an aqueous solution of sodium chloride at a concentration of 10% by weight. at room temperature, three times 7 liters of extraction bath per 24 hours. Then the fiber is decanted in 10 l of water, drained by centrifugation and dispersed in 7 l of saturated calcium hydroxide solution. This solution is left for 48 hours, then centrifuged and the hydroxide solution replaced with a new one. Under the same conditions, the alkaline action is repeated twice to produce a cross-linking value of about 5 bonds per mole of collagen. This value depends on the type of application for which the collagen mass is determined. The pulp is then repeated by beads with redistilled water until the wash water is only slightly alkaline (pH Is less than 9.0) and neutralized with a solution of ammonium chloride and sodium bisulfate 1 : 1 at 25 g / L for 24 hours. The neutralized collagen is then dewatered by centrifugation, washed three times with redistilled water and then placed in a 3 l solution containing 5 g of pancreatin. The mixture was stirred at 35 ° C for 10 hours, then the pulp was drained, washed three times with redistilled water and extracted with 7 1 of redistilled water for 10 hours at 5 ° C. The extraction is repeated five times, the last extract no longer giving a positive reaction to nitrogenous substances by the ninhydrin colorimetric method. The pulp is then dispersed in a 3 L solution containing 1.5 g / L nitrilotriacetic acid and 5.5 g / L ethylenediamine tetraacetic acid disodium salt. After 24 hours, the solution is decanted, washed 5 times with 7 l of redistilled water for 10 hours. The last wash water after 24 hours of equilibration with collagen fiber does not increase the flow value of its conductivity by about 35%. substances. For this purpose, the pulp is acidified to pH 3.8 with acetic acid, cooled to 5 ° and mixed with stainless steel knives at this temperature. The resulting gel-like mass is filtered through a 3 kDa / cm 2 polyamide mesh filter, neutralized with dilute sodium carbonate solution, and the treated fiber is centrifuged. The fiber is then washed with redistilled water, stirred in 7 liters of redistilled water and left at 10 ° C. hours. After decantation, wash in 7 liters of water is repeated twice until conductivity? redistilled water no longer changes viao than 3% of the natural value. Tactile fiber can be used to form fibrous shapes, capillary tubes, spreading parts and flat shapes for medical purposes. Prior to drying or lyophilization into the material, the necessary amount of hyaluronate, antibiotics, immunodepressant agents and other pharmaceutical preparations may be added, the requirements of the particular medical situation. It is also possible to add these active substances to the collagen material in the form of a foam when the dry material is moistened or if a foam structure is desired. In this case, the fiber is again acidified with formic or acetic acid to pS 3.2 and a hyaluronic acid solution is added to the resulting gel.

Claims (6)

5 a note of 1.5% per weight of dry collagen. 5% by weight of pharmacy-like preparations can also be added to the gel. After concentrating to gel, freeze-dry and lyophilize under reduced pressure. The resulting product is in the form of a closed foam, whereby pharmacologically active substances are excreted slowly throughout the healing process. The hyaluronate addition gel formed can also be used to prepare the open foam so that it is saturated with gaseous carbon dioxide in a sealed pressure vessel after cooling to 5 ° C and then suddenly frozen to below -40 ° C. The frozen material, optionally formed into the desired final state, is subjected to freeze-sublimation under reduced pressure. It is sterilized after drying in a hot air sterilizer for 30 minutes at 115 ° C. The tactose is obtained by collagen formations formed by open foam and optionally supporting pharmaceutical preparations delivered either prior to freezing or together with the moistening saline prior to application. The collagen material of the present invention can be found widely used mainly in medicine. Acid and water-impregnated, it forms a high viscosity gel-like mass. It can be used as a carrier and sorbent for pharmacologically active formulations and supportive substances in the healing process and for the preparation of artificial skin, xerotransplants, and replacements in ocular, vascular, and internal surgery. PEEDMET VTNÁLEZU A biocompatible collagen material comprising a single-collagen type (I) comprising two "different polypeptide chains l1 / L / g 2, cross-linked with 1.5 to 8 bonds per mole of collagen, wherein the non-collagenous protein content is at most 0.5% by weight based on collagen, the calcium content does not exceed 1 mg / g of co-gene, the ash content is 0.5% by weight and the metal ions are in the form of a chelate complex. 2. A process for preparing collagen material according to claim 1, wherein said collagen material is treated with 50% saturated to saturated calcium hydroxide solution at least three times in succession to collagenous connective tissue of Slia, then washed with water, neutralized with a 1% aqueous solution. % to 12 wt. ammonium chloride or sodium sulfate or sodium bisulfate based on the weight of the binder, then the binder is subjected to a non-collagenolytic effect of a protease such as propionase, trypsin, chymotripsin, pepsin, pancreatidine or alkalysis added in an amount corresponding to 10 to 10,000 casein units per g of collagen at at a temperature of 20 to 37 ° C for at least 20 minutes, again washed with water, exposed to the complexing agent selected from the group consisting of nitriolotriacetic acid, sodium salt of this acid, ethylindiaaminotetraacetic acid or its sodium salt after at least 30 minutes. minutes and finally the collagen mass is washed with water. 3. The method of preparing collagen material according to claim 1 or 2, wherein the collagen material is ground after acidification to a pH of 2 to 4.5 and filtered through a fabric filter. 4. A process for preparing collagen material according to any one of claims 1 to 3, wherein the acidified collagen mass is filtered into the fibrous form after filtration by neutralization or displacement of sodium chloride and rinsed again with water. 5. The method of preparing collagen material according to items 1 to 4, wherein the fibrous or gel-like collagen mass is frozen at -10 to -60 [deg.] C., dried by lyophilization and sterilized by providing a temperature higher than 80 [deg.] C. and / or gamma irradiation. . 6. A method for preparing collagen material according to any one of claims 1 to 5, wherein the collagen mass is subjected to gaseous or liquid carbon dioxide at a pressure of 0.02 to 0.6 MPa prior to freezing or freezing.