RU2499611C1 - Method for improving biocompatibility of valve and vascular grafts - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to methods for improving the biocompatibility of valve and vascular grafts ensured by reducing the immunogenicity and preventing the calcification. The method for improving the biocompatibility of valve and vascular grafts consists in pre-incubation of the tissue grafts in normal saline containing 0.9% sodium chloride, EDTA (0.5-2 mM), organic buffer HEPES (5-20 mM) at pH 7.0 for 4-6 hours, incubation in normal saline containing 0.9% sodium chloride, HEPES 5-20 mM (pH 7.8), 1% sodium deoxycholate for 30-48 h; the tissue grafts are washed from sodium deoxycholate in a solution containing 0.9% sodium chloride, HEPES 5-20 mM (pH 7.8) and 20% ethanol for 8 days with the medium replaced for the fresh one every day and washed from ethanol for one day.

EFFECT: method for improving the biocompatibility enables preventing the graft calcification, reducing their cell toxicity and the immune response to the tissue grafts.

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Description

The invention relates to medicine, in particular to methods for increasing biocompatibility and suppressing calcification of transplants of heart valves and blood vessels by their special treatment before implantation.

It is known that after transplantation of the valves of the heart and blood vessels, their calcification, degeneration and rejection take place, which significantly reduces the duration of their functioning. These limitations are one of the main causes of reoperations in adult patients and high mortality in patients in early childhood. Donor cells are considered in the scientific literature as one of the most significant causes of the immune response to transplants and their calcifications. In this regard, to reduce the immune response to the transplant and to prevent its calcification, methods are proposed for the treatment of transplants of heart and vascular valves, which ensure the destruction of donor cells (decellularization) in them before implantation into the recipient's body. Various approaches are used for the decellularization of heart and vascular valve transplants, including treatment with hypotonic shock, proteolytic enzymes, nucleases, detergents (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).

A known method of processing collagen-containing tissues, in which to increase the efficiency of tissue regeneration and reduce the immune response to transplanted tissue, they are processed prior to implantation in such a way that all non-collagen components, including cells and tissue matrix components such as elastin, proteoglycans, glycosaminoglycans, are eliminated using for this alkali, chelating agents, acids and salts (see: US Patent N 5993844, 30-11-1999). According to this invention, after the removal of non-collagen components, the structural organization of the collagen matrix, its ability to remodeling, which implies the migration of recipient cells responsible for maintaining tissue nativeness, are preserved. The proposed method does not use detergents and enzymes that can adversely affect the recovery of the collagen matrix after implantation.

The disadvantage of this method is that the proposed treatment causes the destruction / removal from the tissue matrix of not only cells, but also a number of proteoglycans, which play an important role in protecting tissue from calcification. It should also be noted that collagen is an essential component of the surface of myofibroblasts and smooth muscle cells, and therefore it is impossible to destroy cells without damaging the collagen of the tissue matrix. In addition, according to the specified method, all non-collagen proteins, and therefore elastin, are removed, which also indicates damage to the tissue matrix. As you know, damage to the tissue matrix induces an immune response, inflammation, fibrous degeneration. Thus, according to known biological concepts, the proposed treatment will damage the structure of the tissue matrix, and this, as is known, enhances the transplant rejection reaction and, ultimately, shortens its functioning.

A known method of decellularization of grafts of heart and vascular valves, based on the processing of biomaterials with a solution of trypsin (0.25%) and ethylenediaminetetraacetic acid (EDTA) (0.02%) at pH 7.4 and a temperature of 37 ° C for 1 day, followed by washing from these components and processing with a solution deoxyribonucleases (0.2 mg / ml) and riboxynucleases (0.02 mg / ml) (Eur J Vase Endovasc Surg, 2000, v.19, p. 381-386). Such treatment allows the destruction of donor cells and is considered as one of the main methods of decellularization, which suppresses the body's immune response to implanted tissues.

The disadvantage of this method is the fact that prolonged trypsin treatment causes not only the destruction of donor cells, but also damage to collagen fibrils, elastin and other tissue matrix proteins, which causes an increase in the immune response to the transplant. Moreover, there is experimental evidence of an increase in the calcification of grafts after treatment in such a way that casts doubt on the feasibility of its use to increase the biocompatibility of transplanted materials for cardiovascular surgery.

The closest adopted for the prototype is a method of processing tissues of allograft heart valves, according to which, to reduce immunogenicity, suppress calcification, tissues are treated for 8-20 hours in a mildly alkaline buffer, preferably pH 8.0, then with proteolytic inhibitors, for which use ethylenediaminetetraacetic acid or aprotinin, after which they are incubated in a mildly alkaline buffer, preferably pH 8.0, in a solution of a nonionic detergent, preferably using a dodecyl solution sodium sulfate (from 0.03% to 0.1%) or sodium deoxycholate (from 0.5% to 2%) for 20-28 hours (mainly 24 hours), then repeatedly (mainly 3 times) wash fabrics from detergents with a moderately alkaline a solution (pH 8.0), Tris buffered and containing protease inhibitors, and then with the same solution, but without proteases, after which the tissues are incubated in a solution of deoxyribonuclease (0.005-0.1 mg / ml) and ribonuclease (0.0001-0.01 mg / ml) placed in a medium for cryopreservation and frozen for storage in liquid nitrogen (US Patent No. 7354749 B2, 04/08/2008).

The disadvantage of this method is that the sodium dodecyl sulfate solution, causing cell lysis, acts on the tissue matrix proteins and, according to known experimental data, increases the calcification of heart and vascular valve transplant tissues. Another disadvantage of this method is the use of Tris-buffered solution, since Tris promotes strong binding of deoxycholate to the tissue matrix, as a result of which deoxycholate cannot be washed even for 2-4 weeks with a daily change of medium, and this matrix remains toxic to cells. Another disadvantage of this method is the unreasonably wide range of EDTA (1-100 mm), because during processing at a concentration of EDTA of 5 mm or higher, damage to the tissue matrix occurs, which enhances the immune response to the tissue thus treated. The reason for this is the chelation of calcium ions, which play a significant role as ion bridges between different protein molecules in the native matrix structure. Therefore, incubation of tissues with EDTA at a concentration of 10 mM, declared as preferred, lasting for 14 hours will cause a violation of the structure of the matrix. Such disadvantages of the known method of treating transplanted tissues with nonionic detergents can cause complications in their surgical use, as indicated by some literature data (Europ. J Cardio-Thorac. Surg. 2012, v.41, p.1320-1325).

An object of the present invention is to provide a method for processing transplants for cardiovascular surgery using sodium deoxycholate, which, by causing the death of donor cells and freeing tissue from immunogenic and calcinogen-inducing lipid components, prevents calcification in the recipient's body and increases the biocompatibility of such transplants.

This object is achieved in that in the known method, including pre-incubation of tissues prior to 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, according to the invention, pre-incubation of the grafts 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 deoxychalate ohm sodium is carried out for 30-48 hours at 37 ° C, also using saline containing organic HEPES buffer at pH 7.8, and tissue washing from deoxycholate is performed in saline containing sodium chloride (0.9%), 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.

In a preferred embodiment of the method, 0.5 mM EDTA, 4 hours of treatment in an EDTA solution, 40 hours of treatment with deoxycholate and 20% ethyl alcohol in a washing solution are used.

The use of a low concentration of EDTA in the solution for pre-incubation, and a short pre-incubation time, protect the tissue matrix from damage associated with excessive calcium chelation. The use of HEPES organic buffer avoids the strong binding of deoxycholate in the tissue matrix that occurs when using phosphate buffer and especially Tris buffer. As a result of this binding, problems arise with washing the deoxycholate from the matrix, which causes the toxicity of such a matrix to cells. The increase in the time of treatment with deoxycholate allows for more efficient removal of lipid components from the matrix, which is necessary to suppress the calcification of the transplant and increases its biocompatibility. The use of ethanol in a washing solution and long-term washing are necessary for thorough removal of deoxycholate from the matrix, and accordingly, to prevent toxicity, which ultimately leads to increased biocompatibility.

Below are examples of the implementation of the proposed method for increasing the biocompatibility of transplants for cardiovascular surgery.

Example 1

Compared the biocompatibility of the walls of the aortic pig treated with the claimed method and the method adopted for the prototype. For this purpose, NIH 3T3 cells (fibroblasts obtained from mouse embryos) were seeded on treated materials and their viability after 1 day and 3 days of cultivation was assessed. Cells were obtained from the All-Russian collection of cell cultures of the Institute of Cytology RAS. According to fluorescence microscopic analysis, pig fragments of the aortic wall contained live smooth muscle and endothelial cells before treatment. Before treatment, the fragments were incubated at 4 ° C for 2 days in DME culture medium at pH 7.4, with antibiotics gentamicin (400 mg / l) and flucanazole (20 mg / l) to ensure sterility. Then the fragments were divided into two groups. The first group was treated by the method adopted for the prototype, including pre-incubation for 14 hours in physiological saline containing 0.9% sodium chloride, 10 mm EDTA, Tris-HCl buffer (50 mm, pH 8.0), followed by incubation in physiological saline, containing 0.9% sodium chloride, 1% sodium deoxycholate, Tris-HCl buffer (20 mM, pH 8.0), for 24 hours, washing the sodium deoxycholate with the above EDTA solution with a 3-fold change of medium every 6 hours and subsequent washing the same solution without EDTA. After that, the fragments were placed in Dalbecco's nutrient medium, to which 10% of cattle fetal serum was added, and the antibiotic gentamicin (50 mg / L) and antimycotic flucanazole (5 mg / L). Another batch of fragments was treated by the claimed 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 tissue fragments were incubated for 40 h in a solution containing 0 , 9% sodium chloride, 1% sodium deoxycholate, 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 for 8 days with a 2-fold change of medium to fresh every day, and twice during the next s ducks were washed from ethyl alcohol in a solution of 0.9% sodium chloride, pH 7.2. After such a multi-stage incubation, fragments of the second group were placed in a culture medium identical to that used for the first group. NIH 3T3 cells were grown in Dalbecco growth medium with 10% serum and with antibiotic gentamicin 50 μg / ml and fluconazole. After reaching the monolayer, the cells were detached from the culture vials with trypsin solution and plated on the surface of the fragments of the control (prototype) and experimental (inventive method) groups. A day after plating, a microscopic fluorescence analysis of cell viability was performed by staining with nuclear dyes, one of which penetrates into living cells and, binding to DNA, fluoresces in the blue-green region of the spectrum (Hoechst 33342), and the other binds to DNA only in dead cells and fluoresces in red. Fluorescence microscopic analysis showed that in the experimental group after 1 day and three days, 97 ± 2% of the cells were alive. In the control group, 45 ± 3% were alive after 1 day, and after 3 days, 11% ± 2% of the cells remained alive. This indicates that the claimed method increases the biocompatibility of the walls of the aorta with living cells.

Example 2

Compared the biocompatibility of the walls of the pulmonary artery of a pig, processed by the claimed method, or by the method adopted as a prototype. As in example 1, cells of the NIH 3T3 line were sown on the processed materials and their viability was assessed after 1 day and 3 days of cultivation. Fragments of the walls of the pulmonary artery, like pig aorta, according to fluorescence microscopic analysis, contained live smooth muscle and endothelial cells before treatment. Before treatment, the fragments were incubated at 4 ° C for 2 days in DME culture medium at pH 7.4, with antibiotics gentamicin (400 mg / l) and flucanazole (20 mg / l) to ensure sterility. Then the fragments, as in example 1, were divided into two groups. The first group was treated by the method adopted for the prototype, including pre-incubation for 14 hours in physiological saline containing 0.9% sodium chloride, 10 mm EDTA, Tris-HCl buffer (50 mm, pH 8.0), followed by incubation in physiological saline, containing 0.9% sodium chloride, 1% sodium deoxycholate, Tris-HCl buffer (20 mM, pH 8.0), for 24 hours, washing the sodium deoxycholate with the above EDTA solution with a 3-fold change of medium every 6 hours and subsequent washing the same solution without EDTA. After that, the fragments were placed in Dalbecco's nutrient medium, to which 10% fetal cattle serum was added, and antibiotics gentamicin (50 mg / L) and flucanazole (5 mg / L). Another batch of fragments was treated by the claimed method, including pre-incubation for 4 hours in a solution containing 0.9% sodium chloride, 0.5 mm EDTA, HEPES organic buffer pH 7.0, then incubated for 40 hours in a solution containing 0.9 % sodium chloride, 1% sodium deoxycholate, 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 for 8 days from 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 incubation, fragments of the second group were placed in a culture medium identical to that used for the first group. NIH 3T3 cells grown in Dalbecco growth medium with 10% serum and with antibiotic gentamicin 50 μg / ml and fluconazole were detached from the culture vials with trypsin solution and plated on the surface of fragments of the control (prototype) and experimental (inventive method) groups. One day and 3 days after seeding, according to microscopic fluorescence analysis, in the experimental group, 97 ± 2% of the cells seeded on the surface of the fragments were alive. In the control group, 54 ± 4% were alive after 1 day, and after 3 days 14% ± 2% of the cells remained alive. Thus, this example also shows that the claimed method increases the biocompatibility of the walls of the pulmonary artery for living cells.

Example 3

Compared the biocompatibility of the walls of the aorta of a pig, processed by the claimed method and the method adopted for the prototype, in a model of heterotopic implantation under the skin of rats. According to fluorescence microscopic analysis, pig fragments of the aortic wall contained live smooth muscle and endothelial 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 two groups. The first group was treated by the method adopted for the prototype, including pre-incubation for 14 hours in physiological saline containing 0.9% sodium chloride, 10 mm EDTA, Tris-HCl buffer (50 mm, pH 8.0), followed by incubation in physiological saline, containing 0.9% sodium chloride, 1% sodium deoxycholate, Tris-HCl buffer (20 mM, pH 8.0), for 24 hours, washing the sodium deoxycholate with the above EDTA solution with a 3-fold change of medium every 6 hours and subsequent washing the same solution without EDTA. After processing, the fragments were placed in Dalbecco's nutrient medium, to which 10% fetal cattle serum was added, and antibiotics gentamicin (50 mg / L) and flucanazole (5 mg / L). Another batch of fragments was treated by the claimed method, including pre-incubation for 4 hours in a solution containing 0.9% sodium chloride, 0.5 mm EDTA, HEPES organic buffer pH 7.0, then incubated for 40 hours in a solution containing 0.9 % sodium chloride, 1% sodium deoxycholate, 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 for 8 days from 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 such a multi-stage incubation, fragments of the second group were placed in a culture medium identical to that used for the first group. The third group consisted of untreated fragments of the walls of the aorta. Graft calcification was studied by subcutaneous implantation in rats. For this, the fragments were placed one by one in porous steel chambers (pore diameter 50 μM), and then chambers were implanted under the skin of Wistar rats under thiopental anesthesia. After 1.5 months, the fragments were explanted from rats and the content of mineralized calcium was measured in them by atomic absorption spectroscopy. Visual inspection showed that in the control group of fragments processed by the method used as a prototype, there was a pronounced proliferation of fibrous capsule around the samples, which indicates toxicity and an increased immune response to the material in comparison with experimental fragments processed with the claimed method. Fragments processed by the proposed method and the method adopted for the prototype were not calcified (0.5 ± 0.5 mg of calcium per 1 g of dry weight of the fragment). Untreated aortic walls were significantly mineralized and contained 85 ± 7 mg of calcium per 1 g of dry fragment weight. Thus, the claimed method completely suppresses calcification in a heterotopic model of subcutaneous implantation in rats, and does not cause a strong immune system response, which is observed for control fragments processed by the method, taken as a prototype.

Thus, the above examples show that the inventive method provides an increase in the biocompatibility of transplants for cardiovascular surgery in comparison with the method adopted for the prototype.

Claims (1)

A method for increasing the biocompatibility of heart and vascular valve transplants, including preliminary incubation of tissues before implantation in physiological saline with EDTA, followed by treatment with non-ionic lipophilic detergent sodium deoxycholate, which causes the death of donor cells, and subsequent washing of the non-ionic detergent, characterized in that the preliminary incubation 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 de sodium xicholate 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 15-25% 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.