RU2504406C1 - Method for making bioresorbed small-diameter hybrid vascular graft - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and tissue engineering, and may be used in cardiovascular surgery for small-vessel bypasses. A vascular graft is made by two-phase electrospinning with the staged introduction of the ingredients into the polymer composition.

EFFECT: making the bioresorbed small-diameter vascular graft possessing the improved biocompatibility ensured by using the polymer composition of polyhydroxybutyrate (PHBV) with oxyvalerate, and epsilon-polycaprolactone with type IV collagen, human fibronectin and human fibroblast growth factor (hFGF) additionally introduced into the composition.

2 cl, 1 ex

Description

The invention relates to the field of medicine and tissue engineering, and can be used in cardiovascular surgery when performing shunt operations on vessels of small diameter.

Cardiovascular diseases are the main cause of mortality and disability of the population, while ischemic lesions of the vascular bed are leading in this area (coronary heart disease, Lerish’s syndrome, etc.). One of the effective methods of treating this pathology is bypass surgery using the vessels of the patient himself (autografts) or using grafts based on synthetic polymers. At the same time, the use of autografts is limited due to their fragility and the difficulty of selecting intact vessels of the desired diameter, suitable for shunting. And the use of grafts based on synthetic polymers leads to induced immunological reactions that contribute to the development of chronic inflammation and thrombosis. These shortcomings led to the need to search for new materials to create vessels based on tissue engineering. At the same time, there are currently no functionally reliable vascular prostheses of small diameter (no more than 5 mm) that could be used for coronary artery bypass grafting and reconstruction of small diameter arteries of various vascular pools.

A known polymer composition for the manufacture of biodegradable medical devices and coatings for medical purposes, in particular vascular stents, where a biodegradable polymer obtained from N-substituted-4-aza-caprolactone (4-aza-caprolactone-based polymeric compositions useful for the manufacture of biodegradable medical devices and as medical device coatings: pat. 8,137,687 USA. No. 12 / 064,108; Fil. 08/09/06; Pub. Date 01/03/07.). Moreover, the functional suspension group applied to the ring nitrogen ensures its amphiphilic properties, and any drug or biologically active substance can be included in the polymer.

A disadvantage of the known polymer is the use of a hydrophobic polymer, which significantly reduces its biological compatibility. Cell adhesion on the surface of the material is significant

A known method of creating a biodegradable vascular implant with buffering a bioresorbable polymer (Biodegradable vascular device with buffering agent: pat. 7,803,182 USA. No. 10 / 856,459; Fil. 05/28/2004; Pub. Dat 09/28/2010), which includes the inclusion of one or more structural components made from degradable biopolymers. At the same time, the buffering agent is released during the polymer resorption and allows maintaining the pH of the tissues surrounding the implant, which in turn can ensure the absence of a non-specific inflammation reaction that can affect the patency of the structure.

The disadvantage of this method is the release of acidic resorption products of polymers (lactic and glycolic acids) into the surrounding tissues, which causes acidification of the surrounding tissues, causing the development of chronic inflammation and thrombosis of the vascular implant. This circumstance requires an additional introduction of a buffering agent into the structure, which complicates the production technology and increases its cost. An important drawback of the method is the rejection of the use of autologous cells to form the inner surface of the vascular implant, which would avoid the reaction of non-specific inflammation and thrombosis.

Closest to the claimed is a method of forming three-dimensional tissue-engineering structures based on biocompatible and biodegradable polymers with the introduction of biologically active substances (growth factors and chemoattractants) into the copolymer skeleton (Engineering of strong, pliable tissues: pat. RE 42.575 USA. No. 11 / 529.691; Fil 08.28.2006; Pub. Date 07.26.11.). The fabricated polymer framework is pre-implanted into the subcutaneous tissue to form the cell layers of the future implant, after which the graft is implanted into the cardiovascular network. A synthetic porous matrix containing growth factors and chemoattractants, in the process of decay, contributes to the attraction and organization of autocytes of various origins. It is this method that we accept as a prototype.

The disadvantage of this method is the need for a two-stage implantation, which includes, at the first stage, subcutaneous implantation of a biodegradable structure, and at the second stage, the installation of an implant populated with autocells in the final location. As a result, the patient requires additional surgical interventions that increase the risks of complications and the development of adverse outcomes. The disadvantage of the proposed method is the need for a long (from 3 to 6 months) subcutaneous implantation for the full population of the polymer matrix with autocytes.

The technical result of the invention is the creation of a bioresorbable vascular implant of small diameter with enhanced biocompatible properties through the use of a polymer composition based on polyhydroxybutyrate with hydroxyvalerate (PHBV) and epsilon-polycaprolactone with the addition of type IV collagen, human fibronectin and human fibroblast growth factor (bFGF).

Epsilon-polycaprolactone (PCL), which is a product of the oil industry and is a cyclic monomer with high strength and elasticity, acts as a structure-forming polymer in the manufacture of the proposed implant. In addition, PCL is capable of bioresorbing in an animal or human body with an average biodegradation period of up to 3 years.

Polyhydroxybutyrate with hydroxyvalerate (PHBV), introduced into the composition of the polymer composition, belongs to the bacterial polymers of the polyhydroxyalkanoate (PHA) class and has high biocompatibility to body tissues, is capable of biodegradation with the formation of non-toxic decomposition products. It is known that the inclusion in the composition of the polymer composition of a copolymer of microbial origin of PHBV with an oxyvaleriate level from 8.5% to 37% allows solving the problem of cytotoxicity when creating a hybrid vascular implant based on pure polycaprolactone. The polymer composition based on PHBV and PCL is resorbed in the body with the formation of safe components - hydroxybutyric acid, water and carbon dioxide, which allows them to be used in various ratios when creating bioresorbable medical constructions.

In addition, the introduction of PHBV into the composition allows to improve the adhesive properties of the implant, which is a critical factor in the manufacture of vascular grafts using cell technology.

The main task when choosing the ratio of these polymers in the composition is to maintain the bioresorption rate in the body sufficient to complete the reconstruction of its own new vessel in place of the resorbable implant, and to take into account the features of the method for manufacturing the tubular structure of a future small diameter vascular implant.

The studies of physical and mechanical characteristics, in vitro cytotoxicity and biodegradation of bioresorbable polymer structures in vivo were carried out on the basis of the Department of Experimental and Clinical Cardiology of the Research Institute of Complex Problems of Cardiovascular Diseases SB RAMS.

It was found that the following ratios of polymers in a solvent are optimal: 3% -8% PHBV and 10% -14% PCL. In the proportion of PHBV: PCL in solution, the dry weight of the polymers relative to each other will look like this: from 23.1: 76.9 (this is if the final concentration in the solution is 3% PHBV and 10% PCL) to 36.4: 63, 6 (if the final concentration in the solution is 8% PHBV and 14% PCL) with intermediate variations. The obtained non-toxic biocompatible biodegradable copolymer compositions of various compositions have an adequate bioresorption rate and physicomechanical properties to create small diameter vascular prostheses that can be used both as a cell-free prosthesis and as a matrix for creating a hybrid vascular graft using cell technology and tissue engineering.

The main characteristics of polymer structures are illustrated by research data, where Table 1 shows the rates of biodegradation of polymer structures during subcutaneous implantation of Wistar rats over 8 months.

Table 1 Polymer constructions 1 month 2 months 3 months 4 months 5 months 6 months 7 months 8 months 5% PHBV + 10% PCL - - ± + + + + + 7.5% PHBV + 10% PCL ± + + + + + + ++

It should be noted that up to 8 months of observation, bioresorption of these polymer structures was determined only microscopically.

Analysis of the cytotoxicity of the resulting matrices based on different compositions of the polymer composition was carried out on the basis of the state of multipotent mesenchymal stromal cells (MMSCs) planted on the matrices, cultured on matrices for 7 days. In this case, the relative number of cells in the state of early apoptosis, late apoptosis and necrosis was evaluated.

table 2 Relative number of MMSCs viable in apoptosis and necrosis Test Series No. Matrices Live MMSK,% Early apoptosis MMSC,% Late apoptosis MMSC,% Necrosis of MMSC,% one 5% PHBV + 10% PCL 73.5 25.1 1.3 0.2 2 7.5% PHBV + 10% PCL 72.8 24.9 1.7 0.5 3 10% PCL (sample for comparison) 43.7 54,4 1.3 0.5

Thus, the studies demonstrate the suitability of bioresorbable tubular polymer structures based on PHBV and PCL for the creation of a vascular implant of small diameter and confirm the prospects of their use in cardiovascular surgery.

In addition, protein components are introduced into the composition of the polymer composition that optimize cell adhesion and promote the reconstruction of neointima of the vascular implant based on autologous cells, which will avoid immunological conflict, increase hemocompatibility and long-term patency of small diameter vascular implants. Type IV collagen and fibronectin, which are introduced into the composition, form an analogue of the basement membrane during the formation of the inner surface of the tubular structure, and the growth factor bFGF is designed to activate chemotaxis of the body’s own fibroblast-like cells in the wall of the implant, to form the walls of the vessel.

The manufacture of a bioresorbable tubular polymer implant of small diameter is carried out by the method of two-phase electrospinning, which allows you to create the most porous tubular structure due to the random distribution of polymer threads on a winding collector with a diameter of 2-6 mm. The most optimal for populating the implant with cells is a pore size of 30-150 microns both when using cell technologies in vitro and through natural blood flow in vivo.

The formation of filaments from a polymer composition with introduced protein substrates occurs in a strong electric field arising between two electrodes of opposite charge. When the polymer solution leaves the syringe through a needle, the polymer solidifies and forms a fiber, which is wound on a pin collector of a selected diameter, being randomly located and forming porosity.

In addition to the formation of porosity, the advantage of this method of performing a tubular framework is a low temperature regime, which allows combining polymers with heat-sensitive protein components while preserving the bioactivity of the latter. Sealing protein substrates into a polymer fiber ensures the preservation of their functions both during sterilization of a tubular polymer structure and during the long term bioresorption after implantation in the body.

The electrospinning process is divided into 2 phases, due to the need to introduce three biologically active substances of multidirectional action into the polymer solution, followed by their localization in the inner and outer layers of the tubular polymer structure.

The method is as follows. The polymer composition is made by mixing the dry substance epsilon-polycaprolactone (polycaprolactone, PCL) with a molecular weight of 80,000 Kda and polyhydroxybutyrate with hydroxyvalerate (PHBV) with a molecular weight of 2,307 Kda with an inclusion of hydroxyvalerate from 8.5% to 37%. in the proportion of PHBV: PCL in dry weight solution - 23.1-36.4: 76.9-63.6. Chloroform acts as a solvent of polymers, the amount of which will depend on the desired final volume of the solution (for example, from 10 ml to 100 ml). The mixing of polymers in a solvent is carried out at room temperature on a magnetic stirrer until the polymers are completely dissolved.

To create a bioresorbable tubular polymer structure, the following modes are used: voltage - 10-50 kV, polymer solution feed rate - 1-10 ml / hour, distance between the needle and the collector - 1-20 cm, collector rotation speed - 10-300 rpm .

The first stage of electrospinning is that type IV collagen at a concentration of 100 μg per 1 ml of polymer solution and human fibronectin at a concentration of 10 μg per 1 ml of the composition are added to the finished polymer solution. The threads of the polymer composition containing the feeder components are wound on a collector of the selected diameter for 10-15 minutes until the implant wall thickness is 75-125 μm, thereby forming its inner surface.

The outer wall of the bioresorbable implant is formed at the second stage of electrospinning, for which a human fibroblast growth factor (bFGF) is introduced into the polymer solution at a concentration of 0.01 μg per 1 ml of polymer solution and the threads are continued to be wound in the specified electrospinner mode until the tubular wall 300- 500 microns.

Below are the results of physical and mechanical tests of the obtained samples made from a polymer composition based on PHBV and PCL with protein components introduced into the composition that optimize cell adhesion.

Table 3. Test Series No. Matrix synthesis time (PHBV + PCL) Diameter of a matrix, mm σ, MPa □% Emod, N / mm ² one 5% PHBV + 10% PCL 30 min 2.0 2.0 ± 0.28 284.7 ± 11.53 4.62 ± 0.81 2 7.5% PHBV + 10% PCL 30 min 3.0 1.75 ± 0.09 369.1 ± 51.43 4.18 ± 0.42 3 5% PHBV + 10% PCL 60 min 4.0 1.4 ± 0.05 287.9 ± 21.45 2.31 ± 0.23 Note: σ is tensile stress; □ - elongation; Emod is the modulus of elasticity.

The following is an example implementation of the method.

Example 1. The manufacture of the polymer framework of the vascular implant with a diameter of 4 mm based on 3% PHBV and 10% PCL. Why do weighed dry polymers at the rate of 0.3 g of PHBV (with the inclusion of hydroxyvalerate 37%) and 1.0 g of PCL, 10 ml of chloroform is introduced as a solvent. The mixing of the ingredients is carried out on a magnetic stirrer until the polymers are completely dissolved. The electrospinning process is carried out with the following parameters: voltage - 25 kV, polymer solution feed rate - 1 ml / h, distance between the needle and the collector - 15 cm, collector rotation speed - 150 rpm.

To carry out the first phase of electrospinning, 2.5 ml of a polymer solution (¼ of the total volume of a polymer solution) is supplemented with a type IV collagen solution at a concentration of 100 μg / ml and a human fibronectin solution at a concentration of 10 μg / ml. We fill the first sterile syringe with the finished composition and start the process of manufacturing the polymer matrix by the method of two-phase electrospinning. Formed nanowires for 7.5 minutes are randomly wound on a pin with a diameter of 4 mm until the syringe is completely empty, after which the feeding device is turned off and the syringe is removed.

The second stage of electrospinning is carried out using a composition prepared from the remaining 7.5 ml of the polymer composition (¾ of the total volume of the polymer solution) and an additionally introduced solution of human growth factor bFGF at a concentration of 0.01 μg / ml. The resulting composition is filled into the second sterile syringe and, without changing the electrospinning parameters, they continue to form nanowires, which are randomly washed over the first (inner) layer of the implant wall until the second syringe is completely emptied.

Thus, the inner diameter of the resulting vascular graft is 4 mm, and its porous wall consists of two layers (inner and outer), each of which contains the corresponding components necessary for the formation of its own vessel at the implantation site.

Claims (2)

1. A method of manufacturing a bioresorbable hybrid vascular implant of small diameter, comprising the use of a biodegradable polymer composition obtained by mixing polyfibroxybutyrate with a molecular weight of 2307 Kda in chloroform with the inclusion of hydroxyvalerate from 8.5% to 37% and epsilon-polycaprolactone with a molecular weight of 80,000 Kda and performed electrospinning, where the pore size between randomly spaced threads is 30-150 μm, characterized in that the ratio of polymers in the dry mixture of the PHBV composition: PCL is 23.1-36.4: 76.9-63.6, while in the first phase of electrospinning, type IV collagen at a concentration of 100 μg per 1 ml of solution and human fibronectin at a concentration of 10 μg per 1 ml of the composition are added to the polymer solution and in the second phase of electrospinning is carried out using a polymer composition supplemented with a fibroblast growth factor at a concentration of 0.01 μg per 1 ml of solution. 2. The method according to claim 1, characterized in that the inner diameter of the manufactured vascular implant is from 2 to 6 mm, while the thickness of the inner wall is 75-125 μm, and the total wall thickness of the implant is 300-500 μm.