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Study on Hair Follicle Dermal Cells Inducing Outer Root Sheath Cells to Form Hair Follicle-like Structure in Dermal Equivalent

Author: ZhuTangYou
Tutor: HaoFei
School: Third Military Medical University
Course: Dermatology and Venereology
Keywords: hair follicle hair follicle reconstruction dermal papilla dermal papilla cell dermal sheath dermal sheath cell outer root sheath cell extracellular matrix Matrigel tissue engineering dermal substitute skin substitute naked mouse transplantation development molecular mechanism
CLC: R751
Type: PhD thesis
Year: 2006
Downloads: 122
Quote: 0
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Abstract


Mature hair follicle is composed of epithelial cells known as outer root sheath and inner root sheath and dermal cells known as dermal papilla and dermal sheath.Hair follicle is pool of keratinocytes,melanocytes and mesenchymal dermal cells.Hair follicle has been target and tool for studying proliferation,migration and differentiation of adult stem cells and in tissue development and regeneration.The research of hair follicle reconstitution has important influence on the cosmetology, pharmacy and wound healing.The common defect of the tissue-engineered skin substitute at present is the lack of skin appendages and scar formation after implantation.Hair follicle might play an important role in promoting wound healing,reducing scar formation and improving healing quality. Specialized dermal cells of hair follicle might play an important role in the growth of epidermis and final structure of reconstructed dermis.Stem cells from outer root sheath cells of the bulge and specialized dermal cells may induce tissue-engineered skin substitute to form skin appendages.Therefor, the research of skin substitute constructed with hair follicle cells has become the focus.Researchers have constructed many kinds of hair follicles reconstituted models,such as cell transplantation, cell injection and conditional organ-type transplantation.And skin appendages such as hair follicle and hair have been successfully induced in definite location,such as under kidney envelope.These research models are impracticable,because of lacking scaffold and growth environment far from normal hair follicle.With development of tissue engineering,based on collagen gel scaffold, models close to normal growth environment of hair follicle have been established in skin substitute at the historic moment.But bilayered skin substitutes with various cells from hair follicle failed in produing any hair follicle-like structure in vitro.But in Gharzi’s research of rat, hair follicle and hair were induced successfully in vivo.In the research of Limat and Havlickova, based on typeⅠcollagen gel incorporated fibroblasts or dermal papilla cells,outer root sheath cells and dermal papilla cells were grown inside a three-dimensional network of Matrigel,the special extracellular matrix proteins containing plenty of basement membrane components.Organoid hair follicle-like structure,some cyst-like spheroids and spheroid epithelial cell aggregates,has been induced and the analysis of cell proliferation,differentiation and culture mediums was performed.The basic course of hair follicle morphogeneis is that the initial signal arises in the mesenchyme and instructs the overlying epithelium to thicken,forming a placode,from which signals induce the mesenchymal cells to form a dermal condensate conversely.The dermal condensate signals to placode promote follicular epithelium proliferatiation to form hair germ and grow down into the dermis and envelope the dermal condensate to form the dermal papilla, followed by concentric circle arrange of epithelium to form the hair follicle.In these signals,β-catenin plays a key role in hair follicle morphogenesis initially, while downstream secreted protein,Sonic hedgehogs(Shh),plays a major part in epithelial-mesenchymal signaling,and regulates proliferation and further downgrowth of the follicular epithelium and development of the dermal papilla.The relationship between Wnt signal expression and induced structure could be known through the investigation ofβ-catenin and Shh expressed by induced structure。In this experiment,tedious separation methods reported about dermal cells of hair follicle from human scalp were improved.Bilayered skin substitute reconstructed with dermal papilla cells and dermal sheath cells and outer root sheath cells respectively, and its ability on hair follicle induction was investigated and discussed.Dermal sheath cells and outer root sheath cells were mixed into Matrigel,then shifted onto composite chitosan dermal substitute reconstructed by dermal papilla cells and dermal sheath cells respectively.The double-layer system was transplanted under the skin of naked mouse.The histology, immunohistochemistry were investigated in vitro and in vivo.The protein expression ofβ-catenin and Shh were analyzed using Westem Blot with bilayered skin substitute reconstructed by foreskin resource and normal human hair follicle and naked mouce hair follicle as contrast.The main results are as follows:(1)Dermal papilla cells could be harvested purely and rapidly by transsecting the dermis-subcutis interface of scalp specimens,extracting the portions of hair follicle embedded in the adipose tissue.Dermal papilla were separated by two lmL plastic syringe with needle under dissecting microscope,captured by a pipettor, pre-incubated by collagenase.(2)Staining with alkaline phosphatase can distinguish dermal papilla cells from dermal sheath cells in lower passage respectively.(3)Bilayed skin substitute reconstructed by cells from hair follicle had closer arrange of epithelium cells and took outstanding cornification,but there was no certain hair follicle-like structure formation either in vitro or in vivo.This result is the same with most reports.(4)Double layer dermal substitute composed of dermal sheath cells and outer root sheath cells mixed into Matrigel layered on dermal substitute reconstructed by dermal papilla cells and dermal sheath cells induced organoid hair follicle-like structure either spheroid epithelial cells aggregates or cyst-like spheroids average diameter at 47±5μm and 38±2μm in the Matrigel respectively.After transplanted under the naked mouse’s skin,cyst-like spheroids enlarged with average diameter of 133±12μm and 131±9μm respectively, which completely cornified at inner and center part.Hair bulb-like structure and organoid hair follicle which grew in dermal substitute were seen. Hair follicle-like structure induced after transplantation was surrounded by basement membrane and positive cells with PAS staining and by connective tissue sheath with Ayoub Shklar(AS) staining.Keratin phenotype of all samples in vitro and in vivo was the same with normal hair follicle,which expressed CK6, CK14, AE1, AE3.(5)β-catenin was expressed in all samples in vitro and in vivo.β-catenin and Shh were expressed in double layer dermal substitute either in vitro or in vivo by Western Blot analysis,the expression was stronger in vivo than in vitro,but weaker than scalp sample.In conclusion,this experiment has established a high-efficient purified culturing method for dermal papilla cells of human hair follicle.Bilayered skin substitute was reconstructed by cells from human hair follicle.Preliminary exploration of the ability of skin appendages formation was carried out.Based on that,that tissue engineered skin substitute with appendages could be true if pluripotent stem cells were combined with hair follicle dermal cells with inductive ability has been brought up.This is the first time that organoid hair follicle-like structure was reconstructed successfully with dermal sheath cells and outer root sheath cells in vitro.Hair bulb,basement membrane and connective tissue sheath like structure were induced after transplanted onto nude mice subcutaneouly.Differentiation toward hair follicle of the induced structure was confirmed by immunohistochemistry. High expression ofβ-catenin may be respond for cyst-like spheroids formation by immunohistochemistry and Western Blot.This study demonstrated that environment and developing signals of hair follicle induction are very important on hair follicle reconstruction by tissue engineering and it has been further confirmed that human dermal sheath cells have an important effect on hair follicle development as dermal papilla cells.

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