Method for making bioresorbed small-diameter hybrid vascular graft

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

 

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

Cardiovascular diseases are the leading cause of death and disablement, while ischemic lesion of the vascular bed are leading in this area (ischemic heart disease, syndrome Leriche and so on). One of the most effective ways of treatment of this pathology is carrying out shunting operations using vessels of the patient (autotransplants) or with the use of grafts based on synthetic polymers. At the same time, the use of autotransplants restricted due to their fragility and complexity of selection of intact vessels of the desired diameter, suitable for bypass surgery. And the use of grafts based on synthetic polymers leads to induced immunological reactions, contributing to the development of chronic inflammation and thrombosis. These drawbacks have led to the search for new materials to create vessels based tissue engineering. At present, there are no functionally reliable vascular prostheses of small diameter (5 mm), which could be used in coronary artery shunter is the training and the reconstruction of the arteries of small diameter different vascular regions.

Known polymer composition for the manufacture of biodegradable medical devices and coatings for medical purposes, in particular vascular stents, where the structure-forming substances adopted 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. 09.08.06; Pub. Date 01.03.07.). When this functional pendant group attached to the ring nitrogen, provides its amphiphilic properties, and the composition of the polymer may include any drug or biologically active substance.

The disadvantage of this polymer is the use of a hydrophobic polymer, which greatly reduces its biological compatibility. Adhesion of cells on the surface of the material is very difficult.

There is a method of creating a biodegradable vascular implant with buferizovannyiy bioresorbable polymer (Biodegradable vascular device with buffering agent: pat. 7,803,182 USA. No. 10/856,459; Fil. 28.05.2004; Pub. Dat 28.09.2010), involving the incorporation of one or more structural components made of degradable biopolymers. This bothersome agent is released in the process of resorption of the polymer and allows you to keep the pH surrounding the implant tissues, which in turn can provide no reaction, Respecify the definition of inflammation, may affect the permeability of the structure.

The disadvantage of this method is the selection in the surrounding tissue acidic products resorption polymers (lactic and glycolic acid), which causes acidification of the surrounding tissues, causing chronic inflammation and thrombosis of the vascular implant. This circumstance requires additional introduction in the design bothersome agent, which complicates the production process and increases its cost. An important drawback of the method is the rejection of the use of autologous cells for forming the inner surface of the vascular implant that would avoid reaction nonspecific inflammation and blood clots.

Closest to the claimed is a method of forming a three-dimensional tissue-engineered constructs based on biocompatible and biodegradable polymers with the introduction of the copolymer in the frame of biologically active substances (growth factors and chemoattractants) (Engineering of strong, pliable tissues: pat. RE 42,575 USA. No. 11/529,691; Fil. 28.08.2006; Pub. Date 26.07.11.). Manufactured polymer frame pre-implanted in the subcutaneous tissue for the formation of cell layers of the future implant, after which the graft is implanted in the cardiovascular network. Synthetic porous matrix containing growth factors and chemoattractants in the process of decay helps to attract and organize autoclitic of different origin. It is this method we accept as a prototype.

The disadvantage of this method is the need for two-stage implantation, comprising the first stage of subcutaneous implantation of biodegradable design, and at the second stage - setting inhabited by autolycum implant in its final location. The result requires the patient to additional surgical procedures, increases the risk of complications and adverse outcomes. The disadvantage of the proposed method is the need for long-term (from 3 to 6 months) subcutaneous implantation for full settlement of the polymer matrix autolycum.

The technical result of the invention is to provide a bioresorbable vascular implant of small diameter, with enhanced biocompatible properties, through the use of polymer compositions based on polyhydroxybutyrate with oxovalerate (PHBV) and Epsilon-polycaprolactone with additional introduction in its composition of type IV collagen, human fibronectin and human fibroblast growth factor (bFGF).

Strukturoobrazujushchimi polymer in the manufacture of the proposed implant is Epsilon-polycaprolacton (PCL), which is a product of the oil industry and represents a cyclic monomer having high is some strength and elasticity. In addition, the PCL can bioresorbability in the animal or human body with medium term biodegradation under 3 years.

Polyhydroxybutyrate with oxovalerate (PHBV), entered into the composition of the polymer composition, refers to bacterial polymers of the class of polyhydroxyalkanoates (GAA) and has high biocompatibility to the tissues of the body, capable of biodegradation with the formation of toxic degradation products. It is known that the inclusion in the composition of the polymer composition of the copolymer microbial origin PHBV level oxovalerate from 8.5% to 37% allows to solve the problem of cytotoxicity when creating a hybrid vascular implant on the basis of the net polycaprolactone. Polymer composition based PHBV and PCL resorbed in the body with the formation of secure components hydroxybutiric acid, water and carbon dioxide, so you can use them in different ratios when creating bioresorbable structures for medical purposes.

In addition, the introduction in the composition PHBV can improve the adhesive properties of the implant, which is a critical factor in the manufacture of vascular grafts using cellular technologies.

The main task of selecting the ratio of these polymers in the composition - saving speed bioresorption in the body, sufficient for having evershine recreate his new vessel at the place of the resorbable implant, and considering features of the method of manufacturing a tubular structure of future vascular implant of small diameter.

Study of physico-mechanical characteristics, cytotoxicity in vitro and biodegradation bioresorbable polymer structures in vivo was conducted at the Department of experimental and clinical cardiology fsbi "research Institute for complex issues of cardiovascular diseases SB RAMS.

It is revealed that the optimal are the following ratio of the polymers in the solvent: 3%-8% PGBV and 10%-14% PCL. Equity ratio PHBV:PCL in solution by dry weight of the polymers relative to each other will be as follows: 23,1:76,9 (if the final concentration in solution of 3% PGBV and 10% PCL) to 36.4:63,6 (if the final concentration in solution of 8% PGBV and 14% PCL) with intermediate variations. Received non-toxic biocompatible biodegradable copolymer composition of different composition, have adequate speed bioresorption and physico-mechanical properties to create vascular prostheses of small diameter, suitable to play the role as a cell-free prosthesis, as well as a matrix to create a hybrid vascular graft using the methods of cell technologies and tissue engineering.

The main characteristics of polymer structures explains the data the study is any, where in the table 1 presents the rate of biodegradation of the polymer structures in the subcutaneous implantation to Wistar rats for 8 months.

Table 1
Polymer design1 month.2 months.3 monthsto 4 months.5 months.6 months.7 months.8 months.
5% PGBV + 10% PCL--±+++++
7,5% PGBV + 10% PCL±++++++++

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

Analysis of cytotoxicity obtained matrices based on the different composition of polim the nuclear biological chemical (NBC composition was carried out on the basis of a state planted on matrices multipotent mesenchymal stromal cells (MMSC), cultured on matrices within 7 days. It was estimated by the relative number of cells in early apoptosis, late apoptosis and necrosis.

Table 2
The relative number MMSC viable, apoptosis and necrosis
no series of testsMatrixLive MMSC, %Early apoptosis MSC, %Late apoptosis MSC, %Necrosis MSC, %
15% PGBV + 10% PCL73,525,11,30,2
27,5% PGBV + 10% PCL72,824,91,70,5
310% PCL (sample for comparison)43,754,41,30,5

Thus, our studies demonstrate the suitability of bioresorbable TRU is striated polymer structures based on PHBV and PCL to create a vascular implant of small diameter and confirm the prospects of their use in cardiovascular surgery.

Additionally, the composition of the polymer composition is injected protein components, optimizing cell adhesion and promotes recreation neointima vascular implant based on autologous cells, thus avoiding immunological conflict, improve hemocompatibility and long-term patency of vascular implants of small diameter. Collagen type IV and fibronectin, are introduced into the composition, form similar basal membrane during the formation of the inner surface of the tubular structure, and growth factor bFGF is designed to activate chemotaxis own fibroblast-like cells in the wall of the implant, to form walls of a vessel.

Fabrication of bioresorbable tubular polymeric implant small diameter carried out by the method of two-phase electrospinning that allows you to create the most porous tubular structure due to the chaotic distribution of polymeric filaments to the filament winding collector with a diameter of 2-6 mm optimal for settlement implant cells is the pore size of 30-150 μm as in the use of cellular technologies in vitro, and through the natural blood flow in vivo.

The formation of filaments of polymeric compositions with the introduced protein substrates occurs in a strong electric field that occurs between the two electrodes PR is the Fig seragnoli. When the solution of polymers from a syringe through a needle of the polymer solidifies and forms a fiber, which is wound on the pin header of a selected diameter, situated in this chaotic and forming porosity.

The advantage of this method of execution tubular frame in addition to the formation of porosity is low temperature, allowing to combine polymers with heat-sensitive protein components with retention of bioactivity of the latter. Sealing of protein substrates in polymer fiber ensures the safety of their functions as in the sterilization of the tubular polymeric structure, and in the process of long term bioresorbable after implantation in the body.

The process of electrospinning is divided into 2 phases, due to the necessity of introducing into the solution polymers of the three biologically active substances multidirectional action and their subsequent localization in the inner and outer layers of the tubular polymeric structure.

The method is as follows. The polymer composition is made by mixing dry matter Epsilon-polycaprolactone (polycaprolactone, PCL) molecular weight of 80,000 KDa and polyhydroxybutyrate with oxovalerate (PHBV) molecular weight 2307 KDa with the inclusion of oxovalerate from 8.5% to 37%. equity ratio PHBV:PCL solution su is th mass - 23,1-36,4:76,9-63,6. As a solvent of the polymer is chloroform, the amount of which will depend on the desired final volume of the solution (for example, from 10 ml to 100 ml). Mixing the polymers in a solvent is carried out at room temperature on a magnetic stirrer until complete dissolution of the polymers.

To create a bioresorbable tubular polymeric structure use the following modes: voltage - 10-50 kV, the feed rate of solution polymers - 1-10 ml/HR, the distance between needle and collector - 1-20 cm, the rotation speed of the collector - 10-300 rpm

The first stage of electrospinning is that to a ready solution polymers add the type IV collagen at a concentration of 100 μg per 1 ml of solution polymers and human fibronectin at a concentration of 10 μg per 1 ml of the composition. Thread polymeric composition containing feeder components of the wind collector selected diameter for 10-15 minutes until the formation of the wall thickness of the implant 75-125 microns, thus forming its inner surface.

The formation of the outer wall bioresorbable implants carry out the second stage of electrospinning, for which the solution of polymer is injected human fibroblast growth factor (bFGF) at a concentration of 0.01 μg per 1 ml of solution polymers and continue winding the thread in the specified mode of electrospinning to f is Mirovaya wall of the tubular design 300-500 microns.

Below are the results of physical-mechanical tests of the samples made from the polymer compositions on the basis of PHBV and PCL entered in the composition of protein components, optimizing cellular adhesion.

Table 3.
no series of testsThe synthesis matrix (PHBV+PCL)The diameter of the matrix mmσ, MPa□, %Emod, N/mm2
15% PGBV + 10% PCL 30 min2,02,0±0,28284,7±11,534,62±0,81
27,5% PGBV + 10% PCL 30 min3,01,75±0,09369,1±51,434,18±0,42
35% PGBV + 10% PCL 60 min4,01,4±0,05287,9±21,452,31±0,23
Note: σ - ultimate tensile stress tensile; the - elongation; Emod - the modulus of elasticity.

The following is an example of the method.

Example 1. The manufacture of polymeric skeleton of the vascular implant with a diameter of 4 mm on the basis of 3% PGBV and 10% PCL. Why perform a portion of the dry polymer at the rate of 0.3 g PHBV (including oxovalerate 37%) and 1.0 g of PCL, the solvent is injected 10 ml of chloroform. Mixing of the ingredients is carried out on a magnetic stirrer until complete dissolution of the polymers. The process of electrospinning carried out with the following parameters: voltage of 25 kV, a flow rate of solution of polymer 1 ml/h, the distance between needle and collector - 15 cm, the rotation speed of the collector - 150 rpm

To implement the first phase of electrospinning to 2.5 ml of a solution of polymers (¼ of the total volume of solution polymers) add a solution of type IV collagen at a concentration of 100 μg/ml, and the solution of human fibronectin at a concentration of 10 μg/ml of composition filled in the first sterile syringe and start the process of making the polymer matrix by the method of two-phase electrospinning. Forming nanowires within 7.5 minutes randomly wound on the pin with a diameter of 4 mm to a complete emptying of the syringe, after which the feeding device off and remove the syringe.

The second stage of electrospinning carried out using compiled by the VA, made from the remaining 7.5 ml polymer composition (¾ of the total volume of solution polymers) and added the solution of human growth factor bFGF at a concentration of 0.01 μg/ml Obtained by the composition of the fill of the second sterile syringe and without changing the settings electrospinning, continue to form nanowires, which for 22.5 minutes randomly popping up on the first (inner) layer of the wall of the implant to the complete devastation of the second syringe.

Thus, the internal 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 appropriate components required for the formation of a proper vessel in the implantation site.

1. A method of manufacturing a bioresorbable hybrid vascular implant of small diameter, including the use of biodegradable polymeric composition obtained by mixing chloroform polyepoxysuccinic molecular weight 2307 KDa with the inclusion of oxovalerate from 8.5% to 37% and Epsilon-polycaprolactone molecular weight of 80,000 KDa and executed by a method of two-phase electrospinning, where the pore size between randomly arranged filaments is 30-150 μm, wherein the ratio of polymers in the dry mix composition PHBV:PCL of $ 23.1-3,4:76,9-63,6, in the first phase of electrospinning to the polymer solution add the type IV collagen at a concentration of 100 μg per 1 ml human fibronectin at a concentration of 10 μg per 1 ml of the composition, and the second phase electrospinning carried out using a polymer composition, supplemented with 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 ranges from 2 to 6 mm, the thickness of the inner wall is 75-125 microns and the total thickness of the wall of the implant 300 to 500 μm.



 

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