Artificial heart valves

Modern biological artificial heart valves available for clinical use, with the exception of pulmonary autograft, are non-viable structures that lack the potential for growth and tissue reparation. This imposes significant limitations on their use, especially in children, for the correction of valve pathology. Tissue engineering has developed over the past 15 years. The goal of this scientific direction is to create in artificial conditions such structures as artificial heart valves with a thromboresistant surface and viable interstitium.

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How are artificial heart valves developed?

The scientific concept of tissue engineering is based on the idea of populating and growing living cells (fibroblasts, stem cells, etc.) in a synthetic or natural absorbable scaffold (matrix), which is a three-dimensional valve structure, as well as the use of signals that regulate 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 their structure and function. In this case, a new collagen-elastin framework or, more precisely, an extracellular matrix is formed on the original matrix as a result of the functioning of cells (fibroblasts, myofibroblasts, etc.). As a result, optimal artificial heart valves created by tissue engineering should be close to the native one in terms of anatomical structure and function, and also have biomechanical adaptability, the ability to reparation and growth.

Tissue engineering develops artificial heart valves using various sources of cell collection. Thus, xenogeneic or allogeneic cells can be used, although the former are associated with the risk of transmitting zoonoses to humans. It is possible to reduce antigenicity and prevent rejection reactions of the body by genetic modification of allogeneic cells. Tissue engineering requires a reliable source of cells. Such a source is autogenous cells taken directly from the patient and do not produce immune reactions during reimplantation. Effective artificial heart valves are produced on the basis of autologous cells obtained from blood vessels (arteries and veins). A method based on the use of fluorescence-activated cell sorting - FACS has been developed to obtain pure cell cultures. A mixed cell population obtained from a blood vessel is labeled with an acetylated, low-density lipoprotein marker, which is selectively absorbed on the surface of endotheliocytes. The endothelial cells can then be easily separated from the bulk of the cells obtained from the vessels, which will be a mixture of smooth muscle cells, myofibroblasts, and fibroblasts. The source of the cells, whether artery or vein, will affect the properties of the final construct. Thus, artificial heart valves with a matrix seeded with venous cells are superior in collagen formation and mechanical stability to constructs seeded with arterial cells. The choice of peripheral veins seems to be a more convenient source of cell collection.

Myofibroblasts can also be harvested from carotid arteries. However, vessel-derived cells have significantly different characteristics from natural interstitial cells. Autologous umbilical cord cells can be used as an alternative cell source.

Artificial Heart Valves Based on Stem Cells

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

Human red bone marrow stem cells are obtained by sternal puncture or iliac crest puncture. They are isolated from 10-15 ml of sternum aspirate, separated from other cells and cultured. Upon reaching the required number of cells (usually within 21-28 days), they are seeded (colonized) on matrices and 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). Subsequently, cell growth is stimulated through the cuptural medium (biological stimuli) or by creating physiological conditions for tissue growth during its isometric deformation in a reproduction apparatus with a pulsating flow - a bioreactor (mechanical stimuli). Fibroblasts are sensitive to mechanical stimuli that promote their growth and functional activity. The pulsating flow causes an increase in both radial and circumferential deformations, which leads to the orientation (elongation) of the populated cells in the direction of such stresses. This, in turn, leads to the formation of oriented fibrous structures of the valves. A constant flow causes only tangential stresses on the walls. The pulsating flow has a beneficial effect on cellular morphology, proliferation and the composition of the extracellular matrix. The nature of the nutrient medium flow, physicochemical conditions (pH, pO2 and pCO2) in the bioreactor also significantly affect collagen production. Thus, laminar flow, cyclic eddy currents increase collagen production, which leads to improved mechanical properties.

Another approach to growing tissue structures is to create embryonic conditions in a bioreactor instead of simulating physiological conditions of the human body. Tissue biovalves grown on the basis of stem cells have mobile and flexible flaps, functionally capable under the influence of high pressure and flow exceeding the physiological level. Histological and histochemical studies of the flaps of these structures showed the presence of active processes of matrix biodestruction and its replacement with viable tissue. The tissue is organized according to the layered type with the characteristics of extracellular matrix proteins similar to those of native tissue, the presence of collagen types I and III and glycosaminoglycans. However, the typical three-layer structure of the flaps - ventricular, spongy and fibrous layers - was not obtained. ASMA-positive cells expressing vimentin found in all fragments had characteristics similar to those of myofibroblasts. Electron microscopy revealed cellular elements with features characteristic of viable, secretory active myofibroblasts (actin/myosin filaments, collagen threads, elastin), and endothelial cells on the tissue surface.

Collagen types I, III, ASMA and vimentin were detected on the leaflets. The mechanical properties of the leaflets of tissue and native structures were comparable. Tissue artificial heart valves showed excellent performance over 20 weeks and resembled natural anatomical structures in their microstructure, biochemical profile and protein matrix formation.

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

Ideal artificial heart valves should have a porosity of at least 90%, as this is essential for cell growth, nutrient delivery, and removal of cellular metabolic products. In addition to biocompatibility and biodegradability, artificial heart valves should have a chemically favorable surface for cell seeding and match the mechanical properties of natural tissue. The level of matrix biodegradation should be controllable and proportional to the level of new tissue formation to ensure mechanical stability over 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 being designed to biodegrade after implantation, once the implanted cells begin to produce and organize their own extracellular matrix network. The formation of new matrix tissue can be regulated or stimulated by growth factors, cytokines or hormones.

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Donor artificial heart valves

Donor artificial heart valves obtained from humans or animals and depleted of cellular antigens by decellularization to reduce their immunogenicity can be used as matrices. The preserved proteins of the extracellular matrix are the basis for subsequent adhesion of seeded cells. The following methods of removing cellular elements (acellularization) exist: freezing, treatment with trypsin/EDTA, detergents - sodium dodecyl sulfate, sodium deoxycolate, Triton X-100, MEGA 10, TnBR CHAPS, Tween 20, as well as multi-stage enzymatic treatment methods. In this case, cell membranes, nucleic acids, lipids, cytoplasmic structures and soluble matrix molecules are removed while preserving collagen and elastin. However, an ideal method has not yet been found. Only sodium dodecyl sulfate (0.03-1%) or sodium deoxycolate (0.5-2%) resulted in complete cell removal after 24 h of treatment.

Histological examination of removed decellularized biovalves (allograft and xenograft) in an animal experiment (dog and pig) showed partial endothelialization and ingrowth of recipient myofibroblasts into the base, with no signs of calcification. Moderate inflammatory infiltration was noted. However, early failure developed during clinical trials of the decellularized SynerGraftTM valve. A pronounced inflammatory reaction was detected in the bioprosthesis matrix, which was initially nonspecific and accompanied by a lymphocytic reaction. Dysfunction and degeneration of the bioprosthesis developed over the course of one year. No cell colonization of the matrix was noted, but calcification of the valves and preimplantation cell remnants were detected.

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

It should be noted that collagen is also one of the potential biological materials for the production of matrices capable of biodegradation. It can be used in the form of foam, gel or plates, sponges and as a fiber-based blank. However, the use of collagen is associated with a number of technological difficulties. In particular, it is difficult to obtain from a patient. Therefore, at present, most collagen matrices are of animal origin. Slow biodegradation of animal collagen may carry an increased risk of infection with zoonoses, cause immunological and inflammatory reactions.

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

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Artificial heart valves made of synthetic materials

Artificial heart valves are also made of synthetic materials. Several attempts to manufacture valve matrices were based on the use of polyglactin, polyglycolic acid (PGA), polylactic acid (PLA), PGA and PLA copolymer (PLGA) and polyhydroxyalkanoates (PHA). Highly porous synthetic material can be obtained from braided or non-braided fiber and using salt leaching technology. A promising composite material (PGA/P4HB) for the manufacture of matrices is obtained from non-braided loops of polyglycolic acid (PGA) coated with poly-4-hydroxybutyrate (P4HB). Artificial heart valves manufactured from this material are sterilized with ethylene oxide. However, the significant initial rigidity 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 a search for other materials.

The use of autologous myofibroblast tissue culture plates cultured on a scaffold to form supporting matrices by stimulating the production of these cells has made it possible to obtain valve samples with active viable cells surrounded by an extracellular matrix. However, the mechanical properties of the tissues of these valves are still insufficient for their implantation.

The required level of proliferation and tissue regeneration of the valve being created may not be achieved by combining cells and matrix alone. Cell gene expression and tissue formation may be regulated or stimulated by adding growth factors, cytokines or hormones, mitogenic factors or adhesion factors to matrices and scaffolds. The possibility of introducing these regulators into matrix biomaterials is being studied. Overall, there is a significant lack of research on the regulation of tissue valve formation by biochemical stimuli.

The acellular porcine xenogeneic lung bioprosthesis Matrix P consists of decellularized tissue processed by a special patented procedure of AutoTissue GmbH, including treatment with antibiotics, sodium deoxycholate and alcohol. This processing method, approved by the International Organization for Standardization, eliminates all living cells and post-cellular structures (fibroblasts, endothelial cells, bacteria, viruses, fungi, mycoplasma), preserves the architecture of the extracellular matrix, reduces the level of DNA and RNA in the tissues to a minimum, which reduces to zero the probability of transmission of the porcine endogenous retrovirus (PERV) to humans. The Matrix P bioprosthesis consists exclusively of collagen and elastin with preserved structural integration.

In sheep experiments, minimal reaction from surrounding tissues was recorded 11 months after implantation of the Matrix P bioprosthesis with good survival rates, which was particularly evident in the shiny inner surface of its endocardium. Inflammatory reactions, thickening and shortening of the valve leaflets were virtually absent. Low tissue calcium levels in the Matrix P bioprosthesis were also recorded, the difference being statistically significant compared to those treated with glutaraldehyde.

The Matrix P artificial heart valve adapts to the individual patient's conditions within a few months after its implantation. The examination at the end of the control period revealed an intact extracellular matrix and confluent endothelium. The Matrix R xenograft implanted in 50 patients with congenital defects during the Ross procedure between 2002 and 2004 demonstrated superior performance and lower transvalvular pressure gradients compared to cryopreserved and decellularized SynerGraftMT allografts and glutaraldehyde-treated scaffoldless bioprostheses. Artificial heart valves Matrix P are intended for pulmonary valve replacement during reconstruction of the right ventricular outflow tract in surgery for congenital and acquired defects and during pulmonary valve replacement during the Ross procedure. They are 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 tissue-engineered valves will depend on advances in valve cell biology (including issues of gene expression and regulation), studies of embryogenic and age-related valve development (including angiogenic and neurogenic factors), precise knowledge of the biomechanics of each valve, identification of adequate cells for seeding, and development of optimal matrices. Further development of more advanced tissue valves will require a thorough understanding of the relationship between the mechanical and structural characteristics of native valves and the stimuli (biological and mechanical) to recreate these characteristics in vitro.

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