The Application Research Of Autologous Skin Fibroblasts In Medical Cosmetology

Fibroblasts, the main cells in corium, can synthesize and secrete a great deal collagen. Those secreted from adults are mainly classified into two types, typeⅠand III collagen. The former takes up 70%～80% of the total, while the later takes up the rest. In the skin of adults, typeⅠCollagen concentrates to form big fibers paralleling to subcuticula,and the fibers intercross to form nets that possess a highly mechanical stability. TypeⅠCollagen is not only a important composition that maintain skin tension and endure tensile force, but also a material foundation that makes the skin satiated. Type III collagen,the immature and tenuous collagenous fibers,takes up 50% of the total composition in the embryo skin and down to 30% in adult skin where type III collagen exists only in epiderm and skin appendages and arranges in loose nets. The amount of type III collagen would increase significantly in wound healing. However , either physiological or pathologic factor would cause typeⅠcollagen decrease, while type III collagen increase oppositely, which would finally induce the decrease of collagen bundle with loosening as well as wrinkles on skin.Using culture in vitro and model of depressed repair in trauma, this experiment investigates the role of autologous skin fibroblasts in depressedcicatricle and wrinkle elimination from animals level and cells level.Main results were as follows:1. Culture in vitro,identification and biological character of fibroblasts(1) It has been shown that using digest isolation culture, fibroblasts of human skin grow slowly and do not coalesce until 7 d while that of rat skin grows fast and coalesce in 2～3 d. It is concluded that fibroblasts of human skin requires more culture time than that of rat.(2) Vimentin was detected by S-ABC in the cultured cells. The color of endochylema is buffy and nucelus is not stained,which certificate that this kind of cell is fibroblasts.(3) Determination of growth curve by MTT: cells fill the well in good condition in 5 d. Latency period,log phase and platform phase are respectively 0～1 d,1～5 d,and 5～7 d. Determination of recovery and growth curve of frozen fibroblasts by trypan blue and cytometry: cells are in good condition after recovery and the results are similar to that before recovery,proving their biological character stable.(4) Hydroxyproline (HYP) and collagen I consents of the fibroblasts nutrient fluid were detected by the hydroxyproline examination reagent box and ELISA methods. Results indicated that total collagen and typeⅠcollagen content in the fibroblasts nutrient fluid increased along with the cell multiplication.2. The research of animal's level(1) Wistar rats were treated to make skin depressed scar model by surgical excision of the skin and subcutaneous muscle. Autologous fibroblasts cultured in vitro for 21 d were injected back to the dermal layer of skin depressed scar model rats. The color in injection lateral scar was lighter significantly than that in control scar group after injected for 7 d.(2) HE staining and vimentin immunohistochemical staining confirmed that fibroblasts expression increased markedly in the dermal layer of injection group. Masson also found that the dermal layer of coarse collagen fibers in the injection group intertwined to nets,their number increased and color darkened.(3) Type I and III collagen immunohistochemical staining result also confirmed that injection cells group had more type I and III collagen positive cells in the dermal layer than control scar group. Furthermore,type I collagen positive expression was more obvious than type III collagen between these two types.The main conclusions of this study are as follows:1. By cold trypsin and collagen enzyme digestion,a new cultural method suitable to the human and the rat skin fibroblasts has been established.2. The recovery rats'fibroblasts after frozen have no significant difference of biological characteristic with pre-frozen fibroblasts. The recovery survival rate of frozen fibroblasts is about 85%. These results indicated that fibroblasts have strong reproductive activity as well as adaptability and it is stable and easy to cultivate.3. After re-injection back to the dermis layer of scar wounds ofmultiplication in vitro,autologous fibroblasts synthesis and secret typeⅠas well as type III collagen. Furthermore,the increase continues along with the time and type I collagen takes up the most part promoting the restoration of depressed scars.