Preliminary Study Of Artificial Skin Containing The Hair Follicles Subsidiary

OBJECT:To establish and investigate a preliminary an artificial skin with generated hair follicles based on the experiments that the artificial skin without generated hair follicles had been successfully established.METHOD:(1) Observed the characteristics of scalp tissue and micro-structure characteristics of scalp hair follicles by immunohistochemical and microscopic methods;(2)Isolated and gained the outer root sheath cells(ORSC),dermal papilla cells(DPC) and hair follicle dermal sheath cells(DSC) by using combination of enzyme digesting and micro-mechanism separating method,cultured and identified them respectively by immunohistochemical method;(3)Cultured keratinocytes and fibroblast observed and compared the growth characteristics of the cells from skin and from hair follicles;(4)Recognized the proliferation properties in different parts of hair follicle epithelial cells through observing their growth changing;(5) Observed and compared the influence of the supernatants of different generations of Papilla cells on the growth of outer root sheath cells by cell counting;(6) Fabricated two kinds of artificial dermis scaffold using bovine typeⅠcollagen and self-make rat tail collagen separately;(7) Planted hair follicle cells into the dermis scaffold and cultured them to establish a preliminary artificial skin with generated hair follicles.RESULTS:1) Human scalp contains a large number of hair follicles,sebaceous glands,sweat glands and other appendaqes,hair follicles are rich in the scalp,the dermal papilla locates in the subcutaneous tissue;2)Isolated and cultured the outer root sheath cells successfully,the formats of the classic "cobblestone"-like appearance of the cells could be observed.The outer root sheath cells have strong colony-forming ability,they could regenerate about 4 generations,the survival rate of the recovery of the second generation is beyond 85%; the survival rate of the recovery of the third generation is about 70%;3)The outer root sheath cells proliferated fast and had strong colony-forming abilities by observing their growing change.The ORSC from the "bulge" segments had stronger prolixferating abilities than the ORSC from the lower segments.The result of CK19 immunohistochemistry on the cell-climbing showed positive cells mainly from upper hair follicles;3)Isolated dermal papilla successfully by enzyme digesting method,and the number of the dermal papilla were larger,the dermal papilla cells grow fast and attachment rate is high.DPC had fibroblast-like morphology under microscope,they could regenerated more than 8 generations,DPC within 4 generations had the characteristics of growing together.Immunohistochemistry showed that DPC were anti-VIM-positive;4) Isolated and cultured hair follicle dermal sheath cells successfully and identified they were anti-VIM-positive by immunohistochemistry;5) The remaining source of cultured hair follicle cells besides outer root sheath cells had longer doubling time than the cells from epidermal and dermal,the hair follicle cells proliferated faster than the cells from epidermal and dermal especially after passage, there was a significant distinction between those two tissue-derived cells according to the growth shape of cells;6) The supernatants of different generations of dermal papilla cells(DPC) promoted the adherence and growth of the outer root sheath cells(ORSC).the lower generations of the DPC culture supernatant had more obvious growth-promoting role on ORSC;7) Made self-make rat tail collagen successfully,and fabricated dermal collagen sponge and collagen gel scaffold separately;8) Planted three groups of intermix-fluid of DPC,DSC,DPC+DPC into the dermal collagen sponge and collagen gel scaffold respectively,the cells died completely after culturing six days to nine days.CONCLUSIONS: 1.Isolated,cultured and identified hair follicle seed cells which could be used to establish artificial skin with generated hair follicles.They included outer root sheath cells(ORSC), dermal papilla cells(DPC) and dermal sheath cells(DSC).2.Got the growth characteristics of the different generations of outer root sheath cells (ORSC),dermal papilla cells(DPC) and dermal sheath cells(DSC),and verified the hypothesis that hair follicle stem cells exist mainly in the hair follicle "bulge".3.Found that the supernatants of the dermal papilla cells played an important role in promoting the growth and proliferation of ORSC;and verified the hypothesis that dermal papilla cells(DPC) may induce the regeneration of ORSC.4.)Fabricated dermal collagen sponge and collagen gel scaffold separately by using self-make rat tail collagen and commercial collagen.5) Planted hair follicle cells into the dermal collagen sponge and collagen gel scaffold respectively. Despite the expectiffg results could not be gotten,the establishing procedure of artificial skin with generated hair follicles and some relevant experiences were acquired. It is foundation for our future experiments.