Methods of cultivation of skin fibroblasts and keratinocytes in vitro prevention of scoliosis

Abstract

Introduction: Replacement of skin has long been the ultimate task for surgeons facing skinresurfacing challenges such as thermal burns and chronic ulceration. After data world statistics the thermal injury frequency in children varies from 3,4 to 36,0% and in adults from 5,6 to 10,0%, fatal outcomes are recorded in 4,9–14,5%.In Republic of Moldova during the period 2006–2013 frequency of thermal trauma ranged between 178-82 cases per 100,000 population, with a significant decrease in recent years; the general mortality decreased too, from 6,3 to 5,0% in adults and from 2,5% to 1,4% in children. It’s noted the risk of death depends on the total area of affected skin - for burns over 30% TBSA lethality reaches 31-54%, and it is not usually possible to cover the entire burns with autologous grafts, and another alternative cover is needed as tissue-engineered skin replacement: cultured autologous/allogeneic keratinocyte grafts, cultured autologous/allogeneic fibroblast grafts, autologous/allogeneic composites, acellular collagen matrices etc. The main objective of this study are studying and determining the optimal methods of in vitro cultivation of fibroblasts and keratinocytes for burned patients. Materials and methods: In the present study, we developed procedures for establishing confluent layers of cultured human fibroblasts on the surface of gelatinscaffold. The culture methods for propagation of keratinocytes obtained from human skin were developed too.Fibroblasts were isolated from normal human tissues and then cultured in nutritive medium that contained growth factors necessary to sustain cell growth and an antibiotic/antifungal mixture to prevent culture contamination. The cells’ growth and proliferation were evaluated by culture examination in phase-contrast microscope. In normal circumstances, fibroblasts appeared as spindle elongate cells with clear cytoplasm. Results: The study showed that by cultivation of isolated skin dermal cells in an adequate nutritive medium in a month can be obtained a confluent layer of fibroblasts that completely cover the culture dish. The final concentration of the cells in the culture was 5,0*104 cells/cm2. Also study demonstrated that gelatin scaffold is necessary to growth of fibroblastsby ensuring better cells attachment tothe flask surface. Keratinocytes are involved in the intricate mechanisms of initiation, maintenance, and completion of wound healing; also they stimulate fibroblasts to synthesize growth factors, which in turn will stimulate keratinocyte proliferation in a double paracrine manner. Conclusion: Cultured skin cells are a valuable material for the treatment, including burns and chronic wound. Fibroblasts are critical in supporting normal wound healing, involved in key processes such as breaking down the fibrin clot, creating new extra cellular matrix and collagen structures to support the other cells Associated with effective wound healing, as well as contracting the wound. It is necessary to rapidly grow optimal number of cells with desired potency, optimal harvest site identification based on desired therapeutic indication, cultivation, storage and transport of the cells for clinical application.