RU2678966C1 - Method for improving biocompatability of pericardial biomaterials for reconstructive surgery - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to a method for improving the biocompatibility of materials made from pericardium for reconstructive cardiovascular surgery, consisting in the fact that implantable tissues are pre-incubated in saline, containing sodium chloride 0.9 %, EDTA (0.5–2 mM), organic buffer HEPES (5–20 mM) at pH 7.0 for 4–6 hours, then the incubation in saline is maintained containing sodium chloride 0.9 %, HEPES 5–20 mM (pH 7.8), sodium deoxycholate 0.5 % for 30–48 h, then the tissues are washed from sodium deoxycholate in a solution containing 0.9 % sodium chloride, HEPES 5–20 mM (pH 7.8) and 20 % ethyl alcohol for 8 days with a 2-fold change of medium every day for fresh, followed by washing with ethyl alcohol during the day, and after such treatment, mechanical detachment of the fibrous layer of the pericardium from the serous layer is performed by its mechanical tension in opposite directions perpendicular to the plane of the pericardium.EFFECT: prevention of calcification of materials, reduction of their toxicity to cells and the body’s immune response, suppression of fibrous hyperplasia on both sides of the pericardial material, acceleration of its repopulation by the cells of the recipient.1 cl, 2 dwg, 2 ex

Description

The invention relates to medicine, in particular to methods for increasing biocompatibility and suppressing calcification of materials made from layered biomaterials, in particular from pericardium, for reconstructive surgery, primarily cardiovascular surgery.

For targeted tissue regeneration in various fields of regenerative medicine, biomaterials are used that are processed before implantation in the recipient's body in a special way. Different types of preimplantation preparation of materials for reconstructive and cardiovascular surgery are aimed at suppressing the immune response to materials, for example, by their decellularization, (Ann. Thorac. Surg. 2001, v. 71 (5 Suppl) p. S428-432; Ann Thorac Cardiovasc Surg 2009; 15: 362-367; J. Heart Valve Disease 2011; v. 20, p. 341-347; Ann Thorac Surg, 2011; 92: 131-137), to suppress their ability to pathological calcification. It is known that the biocompatibility of materials is also determined by the properties of their matrix, the composition and spatial arrangement of the components of the matrix (architectonics). In particular, this refers to fragments of allo- and xenogenic pericardium that are widely used for reconstructive surgery. The serous layer of the pericardium (p. Serosum) has low cell adhesion, and the outer fibrous layer of the pericardium (p. Fibrosum) has increased cell adhesion, which is important to prevent adhesions when using these materials in cardiovascular surgery. Given the polar properties of layered biomaterials, in particular the pericardium, methods for their processing are being developed to increase the biocompatibility of materials for reconstructive surgery created on their basis.

A known method of preparing bioprosthetic materials based on the pericardium, in which to increase their biocompatibility, the fibrous layer of the pericardium is removed using a laser or mechanical devices, for example, a dermatome, to reduce the adhesiveness of this part of the material and, accordingly, to suppress unfavorable fibrous hyperplasia (U.S. N, 8.846.390 B2, 09/30/2014).

The disadvantage of this method is that neither mechanical nor laser treatment can accurately remove only the fibrous layer and maintain the serous, and reliable removal of the fibrous layer together with part of the medial layer is associated with an excessive decrease in the thickness of the biomembrane and its strength properties. Another disadvantage is that the removal of the fibrous layer by a laser is associated with damage to the structure of the tissue matrix in the removal zone, and a violation of the structure will cause an immune response to the implant, excessive fibrogenesis, aseptic calcification and destruction of the material. The same drawback will also occur with the machining of a dermatome, milling cutter and other mechanical means claimed in this patent.

A known method of preparing collagen-coated secondarily serous membranes made of pericardium, in which the pericardium is treated with ethanol, then the pericardial fibrous layer is removed with a scraper, after which the material is processed to remove cells, lipids, non-collagen proteins, then coated with various combinations of pure collagen to suppress cell adhesion and increasing the biocompatibility of the material (US patent 2013/0226314 A1, 29-08-2013).

The disadvantage of this method is that in this method it is also unclear how to remove the fibrous layer and preserve the medial and serous layer, since there are no criteria for such removal, and collagen coating is not critical to suppress the adhesion of the material to cells. Another disadvantage is the mechanical damage to the matrix when removing the fibrous layer with a scraper, as in the first analogue (US patent N 8,846,390 B2, 09/30/2014). In addition, coating with unstructured native collagen is also capable of causing a tissue reaction to the implant, resorption and the construction of new fibrous tissue, and cannot suppress the reaction to damaged collagen with such a hard removal of the fibrous layer of the pericardium.

The closest adopted for the Prototype is a tissue treatment method for reconstructive cardiovascular surgery, including preliminary incubation of tissues prior to implantation in physiological saline with EDTA, followed by treatment with sodium deoxycholate monohydrate with non-ionic lipophilic detergent, which causes the death of donor cells, and followed by washing non-ionic detergent, in which a preliminary incubation of biomaterials is performed for 4-6 hours in physiological saline containing 0.5-2 mm EDTA and an organ HEPES buffer at pH 7.0, followed by incubation with sodium deoxycholate monohydrate for 30-48 hours at 37 ° C using physiological saline also containing HEPES organic buffer at pH 7.8, and tissue washes from deoxycholate in physiological saline containing 0 , 9% sodium chloride, HEPES (pH 7.8) and 15-25% ethyl alcohol, for 8 days at 37 ° C with a 2-fold change of medium every day for fresh, followed by washing from ethyl alcohol during the day (Patent for the invention of the Russian Federation No. 2499611 of 11/27/2013).

The disadvantage of this method, adopted as a prototype, is that after processing of the pericardial tissue in this way, not only the serous but also the fibrous layers remain and, therefore, the possibility of fibrous hyperplasia from one side of the biomaterial remains, which can subsequently contribute to the resorption of the material and thereby reduce its biocompatibility and durability.

An object of the present invention is to provide a method for preparing pericardium-based materials for reconstructive and cardiovascular surgery using sodium deoxycholate monohydrate, which, causing the death of donor cells and freeing tissue from immunogenic and calcification-inducing lipid components, not only prevents their calcification in the recipient's body , but also prevents fibrous hyperplasia on one side of the material and increases the biocompatibility of layered biomaterials.

The problem is achieved in that in the known method, including pre-incubation of biomembranes, in particular, the pericardium, before implantation in physiological saline with EDTA, followed by treatment with non-ionic detergent sodium deoxycholate, which causes the death of donor cells, and subsequent washing of the non-ionic detergent, in wherein the preliminary incubation of implants is performed for 4-6 hours in physiological saline containing 0.5-2 mM EDTA and HEPES organic buffer at pH 7.0, followed by incubation with 0.5% mono sodium deoxycholate idrate is performed for 30-48 hours at 37 ° C, also using physiological saline containing HEPES organic buffer at pH 7.8, and tissue washes from deoxycholate are performed in physiological saline containing sodium chloride (0.9%), HEPES ( pH 7.8) and 20% ethyl alcohol, for 8 days at 37 ° C with a 2-fold change of medium every day to fresh, according to the invention, after this treatment, mechanical separation of the pericardial fibrous layer from the serous and medial layers is performed its mechanical tension in opposite directions perpendicular to the plane of the pericardium.

The use of the indicated concentration of EDTA, sodium deoxycholate monohydrate, the selected treatment regimen for pericardial biomembranes, including their washing from the agents used, provides not only suppression of calcification ability, tissue decellularization, removal of lipid components, but also weakening of the medial layer to fibrous, which looks rather unexpected and this could not be predicted. Moreover, after processing the pericardial tissue in the manner adopted for the prototype, we found an increase in the tensile strength of the membrane in the tangential direction compared to the native pericardium by 2-4 times, despite the weakening of the connection of the serous layer with the fibrous one. Before processing the pericardium in this way, we were not able to carry out the mechanical separation of the fibrous and serous parts by pulling them in opposite directions. This explains why in the inventions taken as analogues, a laser, a dermatome and other mechanical devices are used to remove the fibrous layer. A simple alcohol treatment, as in analogue 2, is not enough to separate the fibrous layer without using a scraper. It remains unclear why the processing method adopted for the prototype, weakened the connection of the serous-medial and fibrous layers. Perhaps this is due to the chelation of calcium by EDTA, which, as we see, affects the strength properties of the membranes, but why this effect is so multidirectional remains unclear.

Below are examples of increasing the biocompatibility of membranes made from cattle pericardium, which was evaluated in a model of heterotopic implantation in experimental animals.

Example 1

Fibrous hyperplasia of bovine pericardial fragments treated by the method adopted for the prototype was compared with the inventive method, in which the fibrous layer was removed after pericardial treatment. Pericardial fragments, according to fluorescence microscopic analysis, contained live cells before treatment. Before treatment, the fragments were incubated at + 4 ° C for 2 days in DME growth medium at pH 7.4, with antibiotics gentamicin (400 mg / l) and flucanazole (20 mg / l). After sterilization, the fragments were divided into three groups. The first group of fragments was treated using the prototype method, including preliminary incubation for 4 h in a solution containing 0.9% sodium chloride, 0.5 mM EDTA, HEPES organic buffer pH 7.0, then incubated for 40 h in a solution containing 0.9% sodium chloride and 0.5% sodium deoxycholate monohydrate, HEPES organic buffer pH 7.8, after which the fragments were washed in a solution containing 0.9% sodium chloride, HEPES organic buffer pH 7.8, 20% ethyl alcohol within 8 days with a 2-fold change of medium to fresh every day, and twice during the next day, washed from ethyl alcohol with a solution of 0.9% sodium chloride, pH 7.2. After processing, the fragments were placed in Dalbekko physiological saline, to which antibiotics gentamicin (50 mg / L) and flucanazole (5 mg / L) were added. The second group of bull pericardial fragments was initially treated as the first, and after the treatment was completed, the fibrous layer was removed by stratification of the fragments and placed in Dalbekko physiological saline, to which gentamicin (50 mg / l) and flucanazole (5 mg / l) were added. The third group consisted of untreated bull pericardial fragments. Biocompatibility and fibrous hyperplasia on the surface of the materials were studied in a model of subcutaneous implantation in Wistar rats. For this, fragments were implanted using the model of complete interstitial contact, while the samples were implanted without external chambers into three subcutaneous pockets formed along the rat spine (1 sample in one pocket bilaterally) under zolet / xylazine anesthesia. 13 weeks after implantation, the materials were explanted, the content of mineralized calcium was measured in them by absorption spectroscopy, and their histological analysis was performed. Fragments processed by the proposed method and the method adopted as a prototype were not calcified (0.5 ± 0.5 mg of calcium per 1 g of dry weight of the fragment. Untreated pericardial fragments contained a significant amount of mineralized calcium (25.7 ± 6.13 mg calcium per 1 g dry weight of the fragment.) A histological examination showed fibrous hyperplasia on the fibrous layer of the pericardium and the absence of such growth on the serous layer of the pericardium in the control fragments, which were processed using the method adopted for the prototype before implantation (Fig. 1a, b). In fragments processed by the claimed method, we did not find fibrotic hyperplasia on either side of the material (Fig. 1c, d). Thus, the claimed method completely suppresses not only calcification, but also fibrotic hyperplasia on both sides of the sheet material made of pericardium by the claimed method, thereby increasing its biocompatibility and prospects of use in reconstructive cardiovascular surgery.

Example 2

Compared the repopulation of recipient cells of pericardial fragments processed by the method adopted for the prototype, and the claimed method. Fragments were processed as in Example 1, and repopulation was also evaluated in the subcutaneous implantation model of Wistar rats. Histological examination showed that repopulation in the material obtained from pericardial fragments in the proposed manner is carried out more actively (Fig. 2b) than in the material obtained using the method adopted for the prototype (Fig. 2a).

Thus, the above examples show that the inventive method provides increased biocompatibility of materials made from pericardium for reconstructive and cardiovascular surgery in comparison with the method adopted for the prototype.

Claims (1)

A method of increasing the biocompatibility of pericardial biomaterials for reconstructive surgery, including preliminary incubation of tissues prior to implantation in physiological saline with EDTA, followed by treatment with sodium deoxycholate monohydrate with non-ionic lipophilic detergent, which causes the death of donor cells, and with subsequent washing of the non-ionic detergent in which pre-incubation is carried out, in which for 4-6 hours in physiological saline containing 0.5-2 mm EDTA and HEPES organic buffer at pH 7.0, the next incubation with sodium deoxycholate monohydrate is performed for 30-48 hours at 37 ° C using physiological saline also containing HEPES organic buffer at pH 7.8, and tissue washing from deoxycholate is performed in physiological saline containing 0.9% sodium chloride, HEPES (pH 7.8) and 20% ethyl alcohol, for 8 days at 37 ° C with a 2-fold change of medium every day to fresh, followed by washing from ethyl alcohol during the day, characterized in that after such processing they perform mechanical exfoliation of the fibrous layer erikarda (p.fibrosum) by its mechanical tension in opposite directions perpendicular to the plane of the pericardium.

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