The Construction Of Tissue Engineering Skin With The Capability Of Hair Growth

BackgroundThe skin is the largest organ of the human body,which not only covers the whole body,but also has the function of protection,feeling,regulating body temperature,excretion and secretion,absorption,metabolism and immunity.As the skin is the body’s first barrier to the outside world,a variety of physical,chemical,biological factors can make the skin injured.The traditional repair method for skin injury is skin grafting or skin flap transplantation,which are faces problems such as donor shortage and immune rejection.Tissue engineering skin is a kind of artificial skin which is the mixture of the seed cells and extracellular matrix.Tissue engineering skin can repair wounds and rebuild skin function,thus has good prospects for development in the future.Though the tissue engineering skin shows great advantage in covering the wound,it still lacks skin appendages such as hair follicles.Hair follicles contain abundant hair follicle stem cells,which play an important role in skin repairing and reconstruction.Besides the basic functions of hair such as regulating body temperature,secretion and excretion,the hair also affects the appearance,and even psychological and social function of human.Not only does tissue engineering skin that lack of skin appendages delay the repair of skin defect,but also it weakens the beauty and lost basic functions of hair follicles.Therefore,it is urgent to construct tissue engineering skin with hair follicle units,which will accelerate skin repairing and reconstruct the function of skin.Objective1.To observe embryonic development of hair follicle and dynamic differentiation process of hair follicle stem cells,and to search for the seed cells with capability of hair follicle formation.2.To construct epidermis-dermis tissue engineering skin in vitro and to observe the outcome of its transplantation in vivo.3.To look for seed cells that have the capability of hair follicle formation.4.To construct epidermis-dermis tissue engineering skin with hair follicle.Methods1.The embryonic development of hair follicle of C57BL/6 fetal mice of different ages is observed by HE staining and the dynamic process of hair follicle stem cells is observed by immunofluorescence labeling.2.Epidermic cells and dermal fibroblasts derived from the foreskin of children are isolated and cultured as seed cells.SD rat tail collagen is prepared as matrix.The epidermis-dermis tissue engineering skin is constructed in vitro by air-liquid interface culture.The epidermis-dermis tissue engineering skin with human dermal papilla cell PO is constructed in vitro and the outcome is observed after transplantation.3.The hair is observed or not in the mixture of the fresh isolated mice dermal cells and fresh isolated epidermal cells of newborn mice after transplantion for three weeks.The hair is not observed or not in the mixture of 3D cultured newborn mice dermal cells and fresh isolated epidermal cells of newborn mice after transplantion for three weeks..The hair is observed or not in the mixture of 3D cultured dermal cells and fresh isolated epidermal cells of newborn mice after transplantion for three weeks.The hair is observed or not in the mixture of single dermis cell from 3D cultured cell and fresh isolated epidermal cells of newborn mice after transplantion for three weeks.4.A variety of seed cells in 3D culture are transplanted into the nude mice to verify the capability of hair follicle formation of different seed cells.5.The epidermis-dermis tissue engineering skin with seed cells in 3D culture is constructed by 3D printers.The epidermis-dermis tissue engineering skin is transplanted into nude mice to verify its hair-procucing capability.Results1.Embryonic development of hair follicle and dynamic process differentiation of hair follicle stem cells(1)At embryonic days 14.5,the epidermis form placodes,mesenchyme cells that under the placodes form a dermal condensate.At embryonic days 15.5,germ and peg is observed.And at embryonic days 16.5-17.5 hair papilla is observed.At embryonic days 18.5 to birth embryonic development of hair follicles continue till the end.(2)The dynamic differentiation process of hair follicle stem cells is observed as the embryonic develepment of hair follicle.But none of valuable new discovery is not observed.2.Construction of epidermis-dermis tissue engineering skin(1)The epidermis layer and dermis layer is observed in epidermis-dermis tissue engineering skin and multilayer of the epidermis is observed by air-liquid interface culture for five days.Epidermic cells and dermal fibroblasts is in good condition in epidermis-dermis tissue engineering skin.After transplantation to skin wound animal model for three weeks,the TES closely connected with the surrounding tissue and the skin wound healed completely with no hair was observed.The epidermis layer and dermis layer structure is observed and hair follicle structure is not seen in histological observation.(2)Hair is not observed in tissue engineering skin constructed by cultured human dermal papilla cell PO and fresh isolated human hair follicle stem cell.HE staining showed that no Hair follicle structure exist in TES.3.Looking for seed cells with capability of hair follicle formation(1)The pigmented hair is observed in the mixture of the fresh isolated mice dermal cells and fresh isolated epidermal cells of newborn mice after transplantion for three weeks.(2)The hair is not observed in the mixture of 3D cultured newborn mice dermal cells and fresh isolated epidermal cells of newborn mice after transplantion for three weeks.Hair follicle structure is not seen in histological observation.The pigmented hair is observed in the mixture of 3D cultured dermal cells and fresh isolated epidermal cells of newborn mice after transplantion for three weeks.The hair is not observed in the mixture of single dermis cell from 3D cultured cell and fresh isolated epidermal cells of newborn mice after transplantion for three weeks.(3)The hair is observed in 3D structure of P1 cultured beard dermal papilla cells from adult mice and fresh isolated epidermis cell from newborn mice after transplantion for three weeks.Hair follicle structure is observed in histological examination.(4)The unpigmented hair is observed in 3D structure of PO cultured scalp dermal papilla cells from adult human and fresh isolated epidermis cell from juvenile after transplantion for three weeks.(5)The unpigmented hair is observed in 3D structure of PO cultured dermal fibroblasts from juvenile and fresh isolated epidermis cell from juvenile after transplantion for three weeks.DNA of human is tested at hair detected by multiplex amplifying mtDNA-HVI,HVII and cytb regions.(6)The pigmented hair is observed in the mixture of hESC-DP which is differenciated from NC cells and fresh and uncultured epidermis cells from newborn mice after transplantion for three weeks.The pigmented hair is observed in the mixture of hESC-DP from NC cells fresh isolated epidermis cell from child after transplantion for three weeks.4.Construction of epidermis-dermis tissue engineering skin with hair follicle(1)The hair is observed in the epidermis-dermis tissue engineering skin constructed by 3D printers after transplanted for three weeks.(2)The pigmented hair is observed in tissue engineering skin contained hESC-DP from NC cells intermediate and fresh and uncultured epidermis cell from newborn mice after transplantion for three weeks.Conclusion1.To observe embryonic development of hair follicle of C57BL/6 fetal mice and dynamic differented process of hair follicle stem cells.E14.5-E17.5 is the key time point to generate hair follicles.It further confirmed the embryonic development of hair follicles is the result of the interaction between epidermal cells and dermal cells.2.Construction of epidermis-dermis tissue engineering skin.(1)Tissue engineering skin constructed in vitro after air-liquid culture for five days show bilateral structure of epidermis and dermis.Tissue engineering skin transplanted into nude mice can accelerate skin wound healing.(2)Tissue engineering skin contained single cell from cultured human dermal papilla cells do not show the capability of hair follicle structure formation.3.Looking for the seed cells which have the capability of hair follicle formation(1)The hair inducing capability of cells can be better preserved after 3D culture.(2)3D structure contained epidermal cells and fibroblasts derived from juvenile foreskin has the capability of unpigmented hair inducing formation.(3)Dermal papilla cell induced from human neural crest cells has the capability of inducing pigmented hair follicle formation.4.Construction of epidermis-dermis tissue engineering skin with hair follicle(1)Tissue engineering skin contained 3D struction of seed cells by using 3 d printers regenerate unpigmented hair after transplantation for 3 weeks.(2)Tissue engineering skin contained dermal papilla cell induced from human neural crest cells and uncultured epidermic cells derived from juvenile foreskin regenerate unpigmented hair after transplantation for three weeks.