The Pilot Study Of Induction Of Tissue Engineered Hair Follicle Reformation

BackgroundHair plays an important role in biological necessity and performs a variety of beauty function.At present,autograft of hair follicles is the most important treatment for hair loss,especially for male pattern baldness.But there are some problems for this treatment,such as formation of scar in donor site,scantiness of donor site,low density of transplantations,so hair follicle tissue engineering provides a new minimally invasive treatment not restricted by the source for repairing hair loss.A hair follicle is composed of epithelial cells including matrix,out root sheath, inner root sheath and dermal cells including dermal papilla,dermal sheath cells. Dermal papilla cells are a kind of fibroblast cells,which can aggregate during growing in mediums containing FCS.According to the different phages of hair cycle, the shape of the dermal papilla change periodically.Dermal papilla plays an important role in the morphogenesis of hair follicle and hair cycle.Dermal sheath around the hair fllicle of the outermost layer are the dermis derived tissue,dermal sheath cells can be observed transforming to dermal papilla cells in the hair follicle regeneration process,It has been demonstrated that dermal sheath cells are the reserves cells of dermal papilla cells and the dermal sheath cells neighbor dermal papilla have the same capacity induction as dermal papilla cells.The human hair follicle bulge connectted with outer root sheath is an important niche for keratinocyte stem cells.It is widely accepted that a highly coordinated series of bidirectional epithelial-mesenchymal interactions(EMIs) is essential to hair follicle morphogenesis.Several lines of evidence have suggested that foreskin and scrotum epidermal is an important niche for keratinocyte stem cells.Although Japanese scholars has demonstrated that hair follicle would be reconstructed and hair fibers visible to the naked eye grow induced by mouse hair follicles dermis cells and human foreskin epidermal cells portfolio when transplanted into nude mice dorsal.There is no reported that human hair follicle cells and human dermis foreskin epidermal cells portfolio have been used for the experimental replication.Type I collagen scaffold composed of various glycoprotein molecules has been the most widely used in tissue engineering experiment in the present.Type I collagen scaffold,which is a kind of important component mesenchymal outside of cells,has many biological activity as an organizational support.It has been demonstrated that collagen could be participated in tissue repair as a kind of excellent biological materials because of its cellular compatibility and adjustable speed degradation.Rat tail collagen has been widely used in hair follicle biology studies as a scaffold for hair follicle cells in the dermis and epidermis cells composition organotypic culture. However,there has been no report that solid typeⅠcollagen could be used as a kind of scaffold for hair follicle seed cells culture in hair follicle engineering.DiI is a lipophilic carbon anthocyanin dye easily embedded within a biological membrane by lateral diffusion movement,so DiI can effectively label cytomembrane, and DiI can effectively label cytoplasm by living cells' pinocytosis.It has been demonstrated that DiI labeled cells have good morphology and DiI is no toxicity for living cells.There are some research reports show that DiI can be used as a fluorescent tracer for cultured nerve cells,then come DiI label of hepatocyte,bone marrow stromal stem cells and adipose derived stem cells,but so far there has been no labeled for cells derived from human hair follices.Objective1.To establish the method for isolating dermal papilla cells,dermal sheath cells, follicular epithelial cells and foreskin epidermal basal cells rapidly and efficiently.2.To explore appropriate scaffold materials for hair follicle tissue engineering, to establish the model for hair follicle cells organotypic culture.3.To explore the method of reconstructing hair follicles.Methods1.The isolation,culture and identification of dermis and epidermis cells derived from human hair follicle.Small scalp specimens were incubated in the presence of dispase at 37℃for 2 h,the hair shafts with ORS embedded in the dermal sheath (DS) were extracted under dissecting microscope,and the ORS tissue were inoculated onto Petri dish.The specimens were transected at the interface between the dermis and subcutaneous tissue.The portions of DS and DP(linked with and enclosed by DS) embedded in the adipose tissue were pulled out and incubated with collagenase at 37℃for 6-8 h,and the DP and DSCs were isolated by repeated low-speed centrifugation and cultured respectively on petri dishes.The cultured ORS bulge cells were identified by immunohistochemistry with K19 antibody and DPCs and DSCs by immunohistochemistry withα-actin antibody.2.The separation and culture of dermal papilla cells and epithelial cells of rat vibrissa follicle.Dermal papilla cells of rate vibrissa hair follicles were isolated by microdissecting method,and cultured in DMED medium containing 15%FCS,the bioligical behavior in vitro was observed.Epithelial cells of rate vibrissa hair follicles were isolated by microdissecting and enzyme digesting method,and cultured in K-SFM medium,Dermal papillas were removed from the dermal sheathes under the microscope.The bioligical behavior in vitro was observed.3.The separation and culture of epidermal basal cells from the foreskin. Epidermal cells from the human foreskin were isolated by typeⅣcollagen rapid adhesion method,and cultured in K-SFM medium,the bioligical behavior in vitro was observed.4.Cell collagen gels preparation,mouse hair follicle dermal papilla cells(6×10~5) and outer root sheath cells(3×10~5) subcultured from several(＜4 generation) were gradient centrifugated and added into 0.5ml fetal bovine serum.4 ml of collagen from mice tendon(1.0-1.5g/L) was taken to small penicilin ampoule in ice-bath and 0.5ml 10×DMEM culture medium was added into and was mixed immediately.0.1 ml 1N NaOH was added dropwise to it until it changed to a pink colour.The mixture was then inoculated with cells and put into 35-mm culture dishes in a 37℃incubator for gel formation to take place.Gels were maintained in the incubator until use.5.Fluorescent reactive dye DiI labeled human dermal papilla cells and outer root sheath cells,epidermal cells from foreskin.Cells in 3- 4 generations were collected for the trial,washed with PBS and centrifugalized twice.Serum-free DMEM was added to make into cell suspension before 5μL DiI liquor at the density of 1×10~9/L was added,and then cells were incubated for 20 minutes at 37℃,1500 r/min centrifugalized for 5 minutes,washed with PBS and centrifugalized twice. DMEM medium containing 0.1 volume fraction FBS was added before coloration and morphologic change of cells were observed at hours 24,48 and 72 with fluorescent microscope.6.Cells- scaffold complex formation.The morphology and function of the human dermal papilla cells and outer root sheath cells,epidermal cells from foreskin were observed by inverted phase contrast microscope,scanning electron microscope when cocultured typeⅠcollagen scaffold with cells in vitro.7.Nude mice induced hair follicle reformation experimental.Mouse hair follicle cells in the collagen gel were transplanted in the subcutaneous tissue of dorsal part of the nude mice.After 4 weeks,The cultured tissue and local skin with cell transplantation were sectioned with routine paraffin,and stained by HE,and observed under optical microscope and fluorescence microscope.Cells- scaffold complex were transplanted in the subcutaneous tissue of dorsal part of the nude mice. Respectively,after 4,8,12 weeks,local skin with cell transplantation were sectioned with routine frozen sections and paraffin,and stained by HE,and observed under optical microscope and fluorescence microscope.Results1.Dermal papilla cells,dermal sheath cells and purified bulge-derived cells were isolated and cultured from the scalp hair follicles by using method of combination of microdissection and enzyme digestion,and other basic separation technology.Dermal papilla cells isolated by using enzyme digestion method. proliferated rapidly in vitro,the adhering rate was high and can reached 90%, Dermal papilla cells and dermal sheath cells grew in a aggregating way and were positive for immunohistochemical stain ofα-SMA.Bulge-derived cells cultured by tissue cultured method proliferated rapidly in vitro in the medium of K-SFM,like paving stones,and were positive for immunohistochemical stain of K-19.2.Microdissection method succeeded in isolating dermal papilla cells of rat vibrissa hair follicless.Cells proliferated rapidly in vitro,they grew in a aggregating way.ORS cells isolated by combination of microdissection and enzyme digestion. Cells proliferated rapidly in vitro in the medium of K-SFM,like paving stones. 3.Epidermal basal cells from the foreskin isolated by using typeⅣcollagen attachment method grew in a typical cloning of growth way in vitro,and cells growth cycle is long.4.Cells just inoculated in collagen gel are round shape,uniform distributed within the collagen gel.Afeter 24 h,cells extended,adhesion to scaffold.After 2 d, some cells extended and deformated in triangle-shap or fusiform-shape.5.Fluorescent microscope showed all the cells was with red fluorescence after labeling and cells with plenty kytoplasm were fusiform shape or round shape, keeping good normal shape,DiI positive rate was 100%.In the early period the cells were fluorescent ring shape,and 48 hours later fluorescent granules increased and fluorescence enhanced.No fluorescence was found in cell nucleus.6.Dermal papilla cells,bulge-derived cells and epidermal basal cells from foreskin inoculated in scaffold material and incubated for 2 h in sterile box.Cells began to attach on the scaffolds and gradually increased,after 2-3 days,cells in the material gradually extended processes,we can see that a large number of cells attached to the pore and the surrounding material,cells were polygonal, spindle-shape or round-shape,some cells attach to collagen sponge or pore walls, with cluster-like growth,after 3 d,cells were flat polygonal or round,extending in the material surface,after 1 week,cells were round or spindle shape,significantly increased,distributing in aggregated way,there was a large number of stromal surrounding the cells.7.Nude mice induced experimental.There was no hair fiber formation in the dorsal skin of nude mice.paraffin sections were observed under an optical microscope.Follicle-like structures formation were seen in the cells collagen gel experimental group,cell - typeⅠcollagen scaffold protein complexes(dermal papilla cells and bulge-derived ceils ) experimental group and cell - typeⅠcollagen scaffold protein complexes(dermal papilla cells and foreskin epidermal cells ) experimental group in 4 week,8 week and 12 week.There was no follicle-like sturcture formation in blank control group.De novo hair follicle-like structures were with red fluorescence under fluorescence microscope in the experimental group of DiI labeled dermal papilla cells combined with bulge-derived cells and in the experimental group of DiI labeled dermal papilla cells combined with epidermal cells from foreskin.There was no red fluorescent display in unlabeled control group. Conclusion1.Three cells derived from human hair follicle could be isolated and cultured from the same sample at the same time by using the method of combination of microdissection and enzyme digestion and other basic separation technology,and the sequencing of the isolated steps was improved,so the sample was to be maxmize used.Adhering rate and integration time of dermal papilla cells isolated by this method is no significant difference with that of dermal papilla cells isolated by enzyme digestion.The problems of immmunogenicity in experimental induction of hair follicle would be avoid because of all hair follicle cells derived from the same samples.2.Epidermal cells from foreskin rapid isolated by using typeⅣcollagen attachment method grew in a typical cloning of growth way in vitro,and cells growth cycle is long.It show that epidermal cells from foreskin isolated by this method may contain more undifferertiated epidermal stem cells.3.DiI positive rate was 100%.Cells in typeⅠcollagen scaffold cells were polygonal,spindle-shape or round-shape,in a gathering distuibution.There was a large number of stromal surrounding the cells.It shew that cells were able to attach, grow and proliferate well on the scaffolds.4.Nude mice induced experimental tissue slices were observed under an optical microscope.The de nove follicle-like structures shew red fluorescent induced by the experimental group of DiI labeled human dermal papilla cells and bulge-derived cells and by the group of DiI labeled human dermal papilla cells and epidermal cells from foreskin,while the de nove follicle-like structures induced by the control group of DiI unlabelled cells were no red fluorescent display.This phenomenon shew that reformation of the de nove follicle-like structures were induced by cytoskeletal complexes...