Artificial heart valves

Modern, available for clinical use, biological artificial heart valves, with the exception of pulmonary autograft, are non-viable structures that lack the potential for growth and tissue repair. This imposes significant limitations on their use, especially in children in the correction of valvular pathology. Tissue engineering has been formed over the past 15 years. The purpose of this scientific direction is the creation in artificial conditions of such structures as artificial heart valves with a thrombo-resistant surface and viable interstitium.

[1], [2],

How are artificial heart valves developed?

The scientific concept of tissue engineering is based on the idea of colonization and growth of living cells (fibroblasts, stem cells, etc.) in a synthetic or natural resorbable framework, which is a three-dimensional valve structure, and the use of signals regulating gene expression, organization and productivity of transplanted cells during the period of formation of the extracellular matrix.

Such artificial heart valves are integrated with the patient's tissue for the final restoration and further maintenance of its structure and function. At the same time, a new collagenoelastine framework or, more precisely, an extracellular matrix is formed on the initial matrix as a result of the functioning of cells (fibroblasts, myofibroblasts, etc.). As a result, optimal artificial heart valves created by the method of tissue engineering should, by their anatomical structure and function, approach the native one, and also have biomechanical adaptability, ability to repair and grow.

Tissue engineering develops artificial heart valves using various sources of cell harvesting. Thus, xenogeneic or allogeneic cells can be used, although the former are associated with the risk of zoonotic transport to humans. To reduce antigenicity and prevent reactions of rejection of the organism is possible by genetic modification of allogeneic cells. Tissue engineering requires a reliable source of cell production. This source is autogenous cells taken directly from the patient and do not give immune responses during reimplantation. Effective artificial heart valves are produced on the basis of autologous cells derived from blood vessels (arteries and veins). To obtain pure cell cultures, a method based on the use of fluorescent activated cell sorting (FACS) has been developed. A mixed cell population derived from a blood vessel is labeled with an acetylated, low-density, lipoprotein marker that is selectively absorbed on the surface of the endotheliocytes. Endotheliocytes can subsequently be easily separated from the bulk of cells derived from the vessels, which will be represented by a mixture of smooth muscle cells, myofibroblasts and fibroblasts. A source of cells, be it an artery or a vein, will affect the properties of the final structure. Thus, artificial heart valves with a matrix sown with venous cells, in terms of the degree of collagen formation and mechanical stability, surpass the structures sown by arterial cells. The choice of peripheral veins seems to be a more convenient source of cell harvesting.

Myofibroblasts can also be taken from the carotid arteries. At the same time, the cells obtained from the vessels essentially differ from their natural interstitial cells. Autologous umbilical cord cells can be used as an alternative source of cells.

Artificial heart valves based on stem cells

The progress of tissue engineering in recent years is facilitated by stem cell research. The use of stem cells of red bone marrow has its advantages. In particular, the simplicity of biomaterial sampling and in vitro cultivation with subsequent differentiation into various types of mesenchymal cells allows to avoid the use of intact vessels. Stem cells are pluripotent sources of cell germs, have unique immunological characteristics that contribute to their stability in allogeneic conditions.

Human bone marrow stem cells are obtained by sternal puncture or puncture of the iliac crest. They are isolated from 10-15 ml of sternum aspirate, separated from other cells and cultured. After reaching the required number of cells (usually for 21-28 days), they are inoculated (colonized) on the matrix, cultured in a nutrient medium in a static position (for 7 days in a humidified incubator at 37 ° C in the presence of 5% CO2). Further stimulation of cell growth is carried out through a commercial medium (biological stimuli) or through the creation of physiological conditions for tissue growth with isometric deformation in a reproductive apparatus with a pulsating flow - a bioreactor (mechanical stimuli). Fibroblasts are sensitive to mechanical stimuli that promote their growth and functional activity. Pulsating flow causes an increase in both radial and circumferential deformations, which leads to orientation (elongation) of the populated cells in the direction of action of such stresses. This leads, in turn, to the formation of oriented fiber structures of the flaps. A constant flow causes only tangential stresses on the walls. The pulsating flow has a beneficial effect on cell morphology, proliferation and the composition of the extracellular matrix. The nature of the flow of the nutrient medium, the physico-chemical conditions (pH, pO2 and pCO2) in the bioreactor also significantly affect the production of collagen. So, laminar flow, cyclic eddy currents increase the production of collagen, which leads to improved mechanical properties.

Another approach in growing tissue structures is to create embryonic conditions in the bioreactor instead of modeling the physiological conditions of the human body. Cultured on the basis of stem cells, tissue bioclaps have movable and plastic valves that are functionally well-behaved when exposed to high pressure and a flow exceeding the physiological level. Histological and histochemical studies of the leaflets of these structures showed the presence in them of actively proceeding biodegradation processes of the matrix and its replacement by viable tissue. The tissue is organized in a laminated type with the characteristics of extracellular matrix proteins, similar to the characteristics of native tissue by the presence of type I and III collagen and glycosaminoglycans. However, a typical three-layered structure of the valves - ventricular, spongy and fibrous layers - was not obtained. Discovered in all fragments, ASMA-positive cells expressing vimentin had characteristics similar to the characteristics of myofibroblasts. Electron microscopy revealed cellular elements with characteristics characteristic of viable, secretionally active myofibroblasts (actin / myosin filaments, collagen filaments, elastin), and endothelial cells on the surface of the tissue.

Collars of I, III types, ASMA and vimentin were found on the valves. The mechanical properties of the wings of the tissue and native structures were comparable. Tissue artificial heart valves showed excellent performance for 20 weeks and resembled natural anatomical structures for their microstructure, biochemical profile and the formation of a protein matrix.

All artificial heart valves, obtained by the method of tissue engineering, were implanted into the pulmonary position by the animal, since their mechanical characteristics do not correspond to the loads in the aortic position. The tissue valves implanted from animals are structurally similar in their structure to native ones, which indicates their further development and rearrangement under in vivo conditions. Whether the process of tissue restructuring and maturation will continue in physiological conditions after artificial heart valves are implanted, as observed in animal experiments, further studies will show.

Ideal artificial heart valves should have a porosity of at least 90%, as it is essential for cell growth, nutrient delivery and removal of cell metabolism products. In addition to biocompatibility and biodegradability, artificial heart valves should have a surface chemically favorable for sowing cells and correspond to mechanical properties of natural tissue. The level of biodegradation of the matrix must be controlled and proportional to the level of formation of the new tissue to ensure a guarantee of mechanical stability for a certain time.

Currently, synthetic and biological matrices are being developed. The most common biological materials for creating matrices are donor anatomical structures, collagen and fibrin. Polymer artificial heart valves are designed to biodegrade after implantation as soon as the implanted cells begin to produce and organize their own extracellular matrix network. The formation of a new matrix tissue can be regulated or stimulated by growth factors, cytokines or hormones.

[3], [4], [5], [6], [7]

Donor artificial heart valves

Donated artificial heart valves derived from humans or animals and devoid of cell antigens by decellularization to reduce their immunogenicity can be used as matrices. The preserved proteins of the extracellular matrix are the basis for the subsequent adhesion of the cells that are sown. There are the following ways to remove cellular elements (cellulization): freezing, processing of trypsin / EDTA, detergents - sodium dodecyl sulfate, sodium deoxychlorate, Triton X-100, MEGA 10, TnBR CHAPS, Tween 20, and multistage methods of enzymatic treatment. At the same time, cell membranes, nucleic acids, lipids, cytoplasmic structures and soluble matrix molecules are retained, while collagen and elastin are retained. However, an ideal method has not yet been found. Only dodecyl sodium sulfate (0.03-1%) or sodium deoxycolate (0.5-2%) resulted in complete cell removal after 24 hours treatment.

Histological examination of remote decellularized bioclaps (allograft and xenograft) in an animal experiment (dog and pig) showed that partial endothelialization and ingrowth of the recipient's myofibroblasts into the base occur, and there are no signs of calcification. Moderately pronounced inflammatory infiltration was noted. However, in clinical trials of the decellularized SynerGraftTM valve, early insufficiency developed. In the matrix of the bioprosthesis, a pronounced inflammatory reaction was determined, which at first was non-specific and was accompanied by a lymphocytic reaction. Dysfunction and degeneration of the bioprosthesis developed within one year. Cell colonization was not observed in cells, however, calcification of valves and preimplantation cell debris were detected.

The cell-free cells, which were seeded with endothelial cells and cultured under in vitro and in vivo conditions, formed an integral layer on the surface of the valves, and the sested interstitial cells of the native structure showed their ability to differentiate. However, it was not possible to achieve the necessary physiological level of colonization of cells on the matrix under dynamic bioreactor conditions, and implanted artificial heart valves were accompanied by a fairly rapid (three months) thickening due to accelerated cell proliferation and the formation of an extracellular matrix. Thus, at this stage, the use of donor cell-free matrices for their colonization by cells has a number of unresolved problems, 8 including the immunological and infectious nature, the work on decellularized bioprostheses continues.

It should be noted that collagen is also one of the potential biological materials for the manufacture of matrices capable of biodegradation. It can be used in the form of foam, gel or plates, sponges and as a preform on a fiber basis. However, the use of collagen is associated with a number of technological difficulties. In particular, it is difficult to obtain from the patient. Therefore, at the present time, most collagen matrices are of animal origin. The delayed biodegradation of animal collagen can carry an increased risk of zoonotic infection, cause immunological and inflammatory responses.

Fibrin is another biological material with controlled characteristics of biodegradation. Since fibrin gels can be made from the patient's blood for the subsequent manufacture of an autologous matrix, implantation of such a structure will not cause its toxic degradation and inflammatory response. However, fibrin has such drawbacks as diffusion and leaching into the environment and low mechanical characteristics.

[8], [9], [10], [11], [12]

Artificial heart valves made of synthetic materials

Artificial heart valves are also made of synthetic materials. Several attempts to fabricate the valve arrays were based on the use of polyglactin, polyglycolic acid (PGA), polylactic acid (PLA), a copolymer of PGA and PLA (PLGA), and polyhydroxyalkanoates (PHA). The highly porous synthetic material can be obtained from woven or non-woven fibers and using salt leaching technology. A promising composite material (PGA / P4HB) for the manufacture of matrices was obtained from uncoated polyglycolic acid (PGA) loops coated with poly-4-hydroxybutyrate (P4HB). The manufactured artificial heart valves from this material are sterilized with ethylene oxide. However, the considerable initial stiffness and thickness of the loops of these polymers, their rapid and uncontrolled degradation, accompanied by the release of acidic cytotoxic products, require further research and the search for other materials.

The use of tissue culture plates of autologous myofibroblasts cultured on a framework to form support matrices by stimulating the production of these cells allowed the production of valve samples with active viable cells surrounded by an extracellular matrix. However, the mechanical properties of the tissues of these valves are not sufficient for their implantation.

The necessary level of proliferation and regeneration of the tissue of the created valve can not be achieved by only combining the cells and the matrix. Expression of the cell gene and tissue formation can be regulated or stimulated by the addition of growth factors, cytokines or hormones, mitogenic factors or adhesion factors in matrices and matrices. The possibility of introducing these regulators into the biomaterials of the matrix is being studied. In general, there is a significant lack of research on the regulation of the process of tissue valve formation by biochemical stimuli.

The cell-free porcine xenogeneic pulmonary bioprosthesis Matrix P consists of a decellularized tissue treated with a special patented AutoTissue GmbH procedure involving treatment with antibiotics, sodium deoxycholate and alcohol. This treatment method, approved by the International Organization for Standardization, eliminates all living cells and postcellular structures (fibroblasts, endotheliocytes, bacteria, viruses, fungi, mycoplasmas), preserves the architecture of the extracellular matrix, reduces the level of DNA and RNA in tissues to min mA, which reduces to zero the probability of transmission of porcine endogenous retrovirus (PERV) person. The Matrix P bioprosthesis consists exclusively of collagen and elastin with preserved structural integration.

During experiments on sheep, a minimal reaction from surrounding tissues was recorded 11 months after the implantation of the Matrix R bioprosthesis with good indices of its survival, which, in particular, manifested itself in the shiny inner surface of its endocardium. In fact, there were no inflammatory reactions, thickening and shortening of valve flaps. A low calcium level of the Matrix P bioprosthesis tissue was also recorded, the difference was statistically significant compared to the treated glutaraldehyde.

Artificial heart valves Matrix P adapts to the individual conditions of the patient for several months after its implantation. In the study, after the expiration of the control period, an intact extracellular matrix and a drain endothelium were identified. The x-ray Matrix R implanted in a Ross operation performed in 50 patients with congenital malformations between 2002 and 2004 showed excellent performance and lower transvalvescent pressure gradients compared to cryopreserved and decellularized allografts of SynerGraftMT, as well as frameless glutaraldehyde-treated bioprostheses. Artificial heart valves Matrix P is designed for prosthetics of the pulmonary artery valve in the reconstruction of the right ventricular outflow tract in the congenital and acquired defects and during prosthetic repair of the pulmonary valve during the Ross procedure is available in 4 sizes (by internal diameter): for newborns (15-17 mm ), for children (18-21 mm), intermediate (22-24 mm) and adult (25-28 mm).

Progress in the development of valves based on tissue engineering will depend on the success of valvular cell biology (including gene expression and regulation), the study of embryogenic and age development of valves (including angiogenic and neurogenic factors), accurate knowledge of the biomechanics of each valve, identification of adequate cells for colonization, development of optimal matrices. To further develop better fabric valves, it is necessary to fully understand the relationship between the mechanical and structural characteristics of native valves and stimuli (biological and mechanical) in order to recreate these characteristics in vitro.

[13], [14], [15], [16]