Tissue-engineered small-diameter vascular graft and method for making it

FIELD: medicine.

SUBSTANCE: invention refers to medicine and tissue engineering, namely to cardiovascular surgery and may be used in coronary artery bypass surgery, as well as in surgical reconstruction of peripheral vessels. What is described is a method for making a porous tubular matrix of a vascular graft of a biodegradable polymer by two-phase electric spinning, with biologically active molecules stimulating the vascular regeneration being incorporated into a matrix wall matrix incorporated biologically active molecules.

EFFECT: creating the tissue-engineered high-patency and durability small-diameter vascular graft for biological re-modelling of the damaged vessels in vivo.

2 cl, 1 ex

 

The present invention relates to the field of medicine and tissue engineering, namely cardiovascular surgery and can be used in coronary artery bypass surgery, and surgical reconstruction of peripheral vessels.

Now for the surgical treatment of cardiovascular diseases associated with atherosclerotic occlusion of peripheral vessels and coronary arteries, use of autologous arteries and veins or vessels made of Xenomania. At the same time the effective functioning of biological shunts average of 5 years, resulting in the need for revascularization procedures (Bokeria L.A., a High percentage of reoperations in patients with coronary heart disease - modern state of the problem / Bokeria L.A., Berishvili I.I., Solnyshkov etc. // Bulletin of the Bakulev them. Bakulev RAMS. - 2009. No. 10(3). - P.5-27). The use of synthetic materials for the manufacture of vascular prostheses, such as polytetrafluoroethylene (PTFE) or Dacron, allows to solve the problem, however, when the diameter of the grafts less than 6 mm is the rapid formation of blood clots in the lumen of the prosthesis.

Alternative to the use of autologous and xenogenic veins and arteries, as well as synthetic blood vessels for cardiovascular surgery may be deninger the e grafts. The main idea of tissue engineering is the creation of a complete vascular graft for use in cardiovascular surgery, which has led to attempts to create absorbable grafts with cells derived from the patient's body.

Known tissue-engineered blood vessel, consisting of a biocompatible, biodegradable matrix, covered with AutoClean one or a few species, obtained from bone marrow or peripheral blood of the patient (application USA 20090275129 A1, IPC C12N 5/08, C12N 5/06, publ. 05.11.2009). Biocompatible matrix has a porous structure and is made of natural or synthetic biodegradable polymers. Cells obtained from the patient for the settlement of the vascular graft, cultivated in sterile conditions to increase the weight, and then "thrown" on the matrix. For further cell proliferation and formation of extracellular matrix the matrix is placed in a bioreactor.

The disadvantage of this timeintensive blood vessel is the complexity of the fence of sufficient cellular material from the patient, and the duration of the process of cultivation and planting of cells on the matrix to form a complete vessel prior to its implantation.

Closest to the claimed technical solution is tissue-engineered vascular graft of small diameter, is the output for implantation in the blood stream of the patient (application USA 2010/0221304 "Bionanocomposite Materials and Methods For Producing and Using the Same", Saul. 26.02.2010 g , publ. 02.09.2010,, IPC - A61F 2/06, A61L 27/34, A61F 2/82). The graft is made by a method of double electrospinning and consists of two biocompatible bionanocomposite materials, the core of which includes polycaprolactone (PCL), and in the outer layer of the wall matrix incorporate angiogenic growth factors such as transforming growth factor (TGF-b) and fibroblast growth factor (FGF-b).

A disadvantage of the known technical solution is that the core structure of the fiber, in which PCL combined with natural polymers (collagen, chitosan, elastin), or synthetic polymers with short term biodegradation (PLA, PLGA, PGA, PDLLA), will lead to early loss of strength of the graft after implantation in the bloodstream, making the product unsuitable for long operation. In addition, the joint use of TGF-beta and bFGF may trigger the active elements formation of connective tissue, as TGF-beta stimulates the expression of extracellular matrix components such as elastin, collagen, fibronectin, proteoglycans, leading to hyperplasia neointima and obstruction of the graft, especially in small diameter grafts.

The technical result of the invention is the creation of tissue-engineered vascular graft of small diameter for bioremediative damaged blood vessels in vivo with high PR is a need, biogeosociology and durability.

The problem is solved by manufacturing a porous tubular matrix of the vascular graft of the biodegradable polymer by the method of two-phase electrospinning in the wall matrix incorporated biologically active molecules, stimulating the regeneration of the vessel wall in the body.

As a material for the manufacture of a matrix of the vascular graft using a synthetic polymer with a long period of biodegradation - polycaprolacton (poly(e-caprolactone (PCL)), which is well known as strong enough and elastic polymer. In addition, this polymer is biocompatible and bioresistance, and the rate of degradation of PCL fibers obtained by the method of electrospinning in the body ranging from three months to one year. This rate of degradation of PCL promotes long enough to maintain the required mechanical properties of the graft to complete the formation of the native vessel, and the processes of hydrolysis of the polymer and the regeneration vessel coordinated in time and parallel. As a result of biodegradation are formed of non-toxic substances: water and hexanoic acid. Declared vascular graft consists only of PCL, which is a very durable, flexible polymer with a long term degradation, thereby about the capable of withstanding the pressure of the blood stream for a long time, before the formation of the native tissue of the vessel.

Method electrospinning for manufacturing matrix allows to obtain micro - and nantoka fiber and porous structure of the solutions and polymer melts of different composition. The principle of the method consists in the formation of fibers in a strong electric field that occurs between two electrodes of opposite seragnoli, with one electrode placed in the solution or melt polymer material, the second place on the receiving metal collector. Vascular grafts are made at the facility for electrospinning, while the polymer solution is placed in a syringe, the plunger of which slowly pressure pump at a given speed. The syringe attached to the needle with a blunt end, which is supplied with the electrical potential. The polymer at the exit of the syringe solidifies, forming the fiber. Polymer filaments are collected on a rotating collector, connects the second electrode, forming a porous material. The pore size of not more than 20 μm in order to prevent bleeding through the wall of the prosthesis.

For making a vascular graft using the following parameters electrospinning: voltage - 10-50 kV, a flow rate of polymer solution - 1-10 ml/h, the distance between needle and collector - 1-20 cm, the rotation speed of the collector - 10-300 rpm

In the process of electrospinning in the polymer of the second fiber incorporate such biological molecules, as vascular endothelial growth factor (vascular endothelial growth factor (VEGF), fibroblast growth factor (fibroblast growth factor beta (b-FGF)), factor stromal cells (stromal derived factor-1 alpha (SDF-1α)and molecules of heparin. The introduction of VEGF in the structure of the graft, contributes to its faster endothelialization, because this growth factor plays an important role in the regulation of migration and proliferation of endothelial cells. In addition as inducer of proliferation of endothelial cells and fibroblasts used bFGF. In turn, SDF-1α activates the directional migration of autologous stem cells into the injury site, contributing to the regeneration of the vessel wall. The incorporation of molecules of heparin into the wall of the matrix provides a reduced risk of blood clots in the lumen of the conduit.

The incorporation of these growth factors and heparin into the wall of the graft, is carried out by mixing a solution of the biodegradable polymer with a solution of biological molecules in phosphate-buffered saline in a ratio of 20:1, then vupolnyaut electrospinning. Because each type of biomolecules has a wide range of effects on cells, the proposed vascular graft may be composed of or one type of molecules, or combinations thereof.

Declared vascular graft, contains a combination of growth factors: VEGF, bFGF and SDF-1a that JV is contributes to the optimal formation of the wall of the graft and the endothelial layer.

In the process of biodegradation of the polymer incorporated molecules out into the surrounding tissue and carry out their biological functions, stimulating and regulating the formation of a new vessel. Additionally, the molecules are "sealed" in polymer fiber and have no contact with the external environment, which ensures the preservation of their functions sufficiently long period, which allows for the sterilization of these grafts before implantation. Using polycaprolactone for the manufacture of the conduit, prevents immune and allergic reactions on the part of the body after implantation. Due to the low rate of biodegradation of the polymer is ensured continuous delivery of bioactive molecules into the surrounding tissue.

The invention is illustrated by drawings, where figure 1 shows the structure of a vascular graft, a vascular graft in the form of a hollow tube, b - porous structure of the wall of the graft, formed by the fibers of the biopolymer in the process of electrospinning, biomolecules incorporated in the polymer fiber.

The study of the functioning of the vascular PCL grafts carried out on the basis of The Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA.

Example 1.

Vascular grafts (internal diameter 2 mm, thickness 100 μm) of the biodegradable polymer polycaprolactone (poly(caprolactone), PCL,) (M=80.000), were manufacturing the mould is prepared since the method electrospinning and implanted five male Wistar rats (400-450 g). PCL-graft implanted in the abdominal aorta between the renal artery and the bifurcation of the aorta. After removing the clamps the blood flow through the graft was evaluated using Doppler. After 6 weeks, the animals were taken from experiment, and conducted an evaluation of the anastomosis and graft on histological preparations with staining hematoxylin-eosin, Mallory and van Gison.

Histological examination in the lumen of the graft and anastomosis was identified by a continuous layer neointima. The inner surface of the graft was covered with endothelial cells, most of which had increased hyperchromic nuclei and reduced nuclear-cytoplasmic index compared to endothelial cells of the native aorta. The graft was infiltrated cells with morphological features of myofibroblasts and macrophages. The accumulation zones of the collagen rich glycosaminoglycans, laminin and fibronectin, were vyyavleny throughout the thickness and length of the graft. When macroscopic evaluation of an implanted conduit in perivascular tissue was not detected signs of bleeding.

Thus, the study showed the formation of structures on the PCL-graft characteristic of a blood vessel that makes the data of polymer grafts promising for use in cardiovascular surgery as tone the engineering of the vascular conduit.

1. Tissue-engineered vascular graft of small diameter, made of biodegradable polymer, polycaprolactone (PCL), by the method of two-phase electrospinning, while the porous structure of the walls of the matrix contains the incorporated fibroblast growth factor (FGF-b), characterized in that the wall thickness of the matrix incorporate vascular endothelial growth factor (VEGF) and factor stromal cells (SDF-1α), and molecules of heparin.

2. A method of manufacturing a tissue-engineered vascular graft according to claim 1, characterized in that the incorporation of biological molecules in the wall of the matrix is carried out by mixing a solution of polycaprolactone (PCL) with a solution of biological molecules in phosphate-buffered saline in a ratio of 20:1.



 

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