RU2360690C1 - Method for making collagen implants - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: parent material represents tendons, ligaments, fascias, pachymeninx, pericardium, animal and human cartilages. Mechanically cleaned tissue is fragmented by required dimension and treated with mixed Triton x-100 1% and 3% sodium deoxycholate in an ultrasonic bath at temperature 30°C within 3-6 hours with the solution changed twice. Then the tissue is washed in running water, processed with sodium hydroxide 0.1M within 10 hours, processed with a solution containing hydrochloric acid 1M and sodium chloride 1M within 10 hours, processed with a solution containing sodium chloride 1M and phosphate buffer 0.1M within 10 hours, washed in phosphate buffer 0.1M, washed triply in distilled water, lyophilised, packed and sterilised by radiation technique.

EFFECT: high-biocompatible collagen implants to be used for surgical treatment of tendoligamentous apparatus and damages of other soft tissues, as barrier membranes, as a carrier of cells, medical products and biologically active substances.

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Description

The invention relates to medicine, namely to reconstructive surgery, and is intended to obtain biological plastic material used in the surgical treatment of injuries of ligaments, tendons and other soft tissues, for closing wound surfaces, as barrier membranes, and also as a carrier of biologically active substances , cells, medicines.

A known method of producing collagen from bone tissue. According to this method, bone tissue is mechanically cleaned, sawn into wafers with a thickness of 0.1 to 2.0 cm, washed with a 0.1 M solution of phosphate buffer with a pH of 5.8-6.0, and digested in a solution of active 0.125-0.325% papain at 60 ° C for 24 hours, washed with 5 volumes of water at 60 ° C, treated at room temperature for 10-14 hours with 0.4 N alkali solution, washed with running water, treated with 3% hydrogen peroxide for 4 hours , then degreased in a mixture of ethanol / chloroform in a ratio of 1: 2, decalcification is carried out in 0.5-1.0 N hydrochloric acid, they are washed with purified water, ethanol and dried at room temperature, packaged and sterilized by the radiation method (RF Patent No. 2273489 of May 2, 2006), which was chosen as the prototype, since it is closest in its technical solution to the proposed invention.

The disadvantages of this method is that it is applicable only to obtain collagen from bone tissue, and the use of papain during processing leads to a change in the native structure of collagen, which in turn leads to a decrease in the mechanical strength of collagen and makes it more accessible for cellular enzymes, which leads to to accelerate the resorption of the implant in the body of the recipient.

The objective of the invention is to obtain collagen implants suitable for operations on soft tissues.

The technical result of the proposed method is to obtain collagen implants with a high degree of biocompatibility, which can be used in surgical treatment to restore the integrity of ligaments, tendons, to close wound surfaces, as barrier membranes between tissues of various kinds, as a carrier of biologically active substances, cells, medicines.

The technical result is achieved by using soft tissue formations of connective tissue as a raw material, after mechanical cleaning, tissue fragments are sequentially treated with a mixture of Triton x-100 solution and sodium deoxycholate solution, treated with an alkali solution, treated with an acid and sodium chloride solution, lyophilized, packaged and sterilized by radiation method.

Soft tissue formations of connective tissue, such as tendons, ligaments, fascia, dura mater, pericardium, cartilage of humans and animals, such as pigs, cows, rams, etc., can serve as material for collagen implants.

Before starting chemical treatment, tissue fragments of the required size are mechanically cleaned from surrounding soft tissues and blood.

As is known, transplantation antigens of connective tissue are concentrated mainly on the surface of cell and basement membranes. By their chemical nature, antigens contained in connective tissue are mainly glycoproteins, proteoglycans, sialoproteins, lipoproteins. The intercellular substance, structured collagen, has a much lower species specificity compared to other body proteins. Thus, the entire processing process is aimed at removing highly antigenic components of the tissue and at the same time preserving the collagen structure, which determines the mechanical properties of the implants.

For this, after mechanical cleaning, the material is treated with a mixture of surface-active substances (surfactants) of a 1% solution of Triton x-100 and a 3% solution of sodium deoxycholate in an ultrasonic bath at a temperature of 30 ° C for 3-6 hours with a double change of solution. This surfactant composition most effectively removes cells, cell and basement membranes, lipoproteins and saponified fats from tissue, but other surfactants can be used. After surfactant treatment, the material is washed with running water.

After that, the material is placed in a 0.1 M solution of sodium hydroxide (NaOH). At this stage, proteins located on the surface of collagen, glycoproteins, sialoproteins and glycosaminoglycans are removed from the material. The treatment is carried out in an ultrasonic bath for 10 hours at room temperature.

Then the material is treated with a solution containing 1 M hydrochloric acid (HCl) and 1 M sodium chloride (NaCl) for 10 hours. An acid treatment allows the removal of residues of non-collagenic acid-soluble proteins, glycoproteins and glycosaminoglycans, nucleic acids from the material, and the presence of salt avoids collagen swelling and accelerates the washing out of non-collagenic proteins from the material. For greater efficiency, the process is carried out in an ultrasonic bath at room temperature.

After treatment with acid, it is necessary to bring the pH of the material to neutral and wash off the residual solutions. For this, the material is sequentially placed first in a solution containing 1 M NaCl and 0.1 M phosphate buffer for 10 hours, then washed in a 0.1 M phosphate buffer solution, and then washed three times with distilled water.

After chemical treatment is completed, the material is lyophilized, packaged and sterilized by the radiation method.

Our studies of the composition of the obtained implants show that the fat content in them is about 0.7%, the glycosaminoglycan content is less than 1%, and the collagen content is 93% of the dry weight of the sample. In the study of histological sections and according to electron microscopy, it was found that the samples were missing any cells and the collagen structure has a typical connective tissue appearance.

An example implementation of the method.

The human dura mater is mechanically cleaned of the accompanying soft tissues and blood and cut into 2 × 3 cm fragments. 100 grams of the material is placed in an ultrasonic bath, filled with 1 liter of a solution containing 1% Triton x-100 and 3% sodium deoxycholate, and treated for 3 hours at a temperature of 30 ° C with a double change of solution, the solution is drained, the workpieces are washed in running water, then 1 liter of 0.1 M sodium hydroxide (NaOH) solution is poured and treated for 10 hours at room temperature, the solution is drained and fragment you fill in with 1 liter of a solution containing 1 M hydrochloric acid (HCl) and 1 M sodium chloride (NaCl), and treat for 10 hours at room temperature, the solution is drained and pour 1 liter of 0.1 M phosphate buffer solution with 1 M NaCl, treated at room temperature for 10 hours, then washed with 1 liter of a 0.1 M solution of phosphate buffer, then washed three times with distilled water.

The material thus treated is lyophilized, packaged and sterilized by radiation.

An example of the practical use of the collagen implant from the dura mater (TMO) as a barrier membrane obtained by the described method.

Patient K., 46 years old, medical history No. 1365. The patient went to the MONIKI clinic for chronic periodontitis 37, the absence of 36 and 35. On the roentgenogram of the destructive changes in the bone tissue in the projection of the absent 35, 36 and roots 37 were not detected. A treatment plan is proposed: dental implantation according to a two-stage technique in segments 35 and 36, as well as in segment 37 simultaneously with removal of 37 and replacement of the bone cavity with demineralized bone chips with overlapping of the area with a 1.5 x 2 cm TMO barrier membrane. Before the operation, a clinic was performed X-ray and laboratory diagnostics, there are no contraindications to surgery.

Progress of the operation: under local anesthesia, an incision of the mucosa along the alveolar ridge in the region of absent 35 and 36, around the root 37 and additional vertical incisions, flaps were removed, 37 was removed, meticulous curettage of the socket, wound toilet with antiseptic solutions, dental implants were installed in segments 35 (4 13 mm), 36 (5 by 13 mm) and the place of the removed 37 (5 by 13 mm), where the bone cavities are made with demineralized bone chips mixed previously with a solution of 30% lincomycin hydrochloride, the implantation area is blocked by a barrier with a TMB membrane pre-soaked for 20 minutes in physiological saline and saturated with the patient’s blood, the flaps were mobilized and fixed with sutures. In the postoperative period, antibacterial, anti-inflammatory therapy was carried out, healing by primary intention, subjectively significant edema and pain were not observed. X-ray control was carried out after the operation immediately, after 1, 3 and 5 months. 5 months after the operation, the formation of full bone tissue in the plastic zone, complete resorption of the membrane from TMW was noted. The result of the use of a TMO membrane during simultaneous dental implantation should be considered satisfactory, namely, complete resorption after 5 months and the formation of full bone tissue under it.

Thus, the use of the proposed method allows to obtain collagen implants with a high degree of biocompatibility, which can be used for surgical treatment of the tendon-ligamentous apparatus and damage to other soft tissues, as barrier membranes for separating one type of tissue from another, as a carrier of cells, drugs and biologically active substances.

Claims (1)

A method of manufacturing collagen implants, including mechanical cleaning, treatment with an alkali solution, treatment with an acid solution, preservation and sterilization, characterized in that tendons, ligaments, fascia, dura mater, pericardium, cartilage of humans and animals are used as starting material, the workpieces are treated with a mixture 1% solution of Triton x-100 and 3% solution of sodium deoxycholate at a temperature of 30 ° C for 3-6 hours with a double change of solution, treated with 0.1 M sodium hydroxide solution for 10 hours, processed Pipeline solution containing 1M hydrochloric acid and 1M sodium chloride for 10 hours, treated with a solution containing 1 M sodium chloride and 0.1 M phosphate buffer for 10 h, washed with 0.1 M phosphate buffer solution, washed with distilled water three times.