Exploration And Research On The Reconstruction Of Hair Follicle By In Vivo And Vitro

Objective:Isolating and culturing seed cells from source of hair follicles in vitro. In vivo and vitro inducing them to reconstruct hair follicles. To explore the biological characteristics of the hair follicle and the the role of seed cells play in the reconstruction of hair follicle.Methods:(1) Fibroblast (Fb) was isolated and culrured by enzyme digestion method combined with tissue cultured by ourselves from newborn foreskin. (2) Human hair follicle outer root sheath cell (HHFORSC) and Human hair dermal papilla cells (HHDPC) purchased from the ScienCell Company were recoveried, cultured and passaged. (3) DPC, ORSC and fibriblast cultured with Mitomycin primarily were injected into alginate 3D cell scaffold in different cell's combination, order and rate to fabricate the three-dimensional model of hair follicles in vitro. After culturing for 8 weeks, the situation of hair follicle formation was observed by light microscope. Meanwhile, the three-dimensional model of hair follicles were transplanted into the subcutaneous tissue of bal/bcl nude mice.After 8 weeks, the bal/bcl nude mice were harvested and the transplant sites were observed by HE staining, immunohistochemistry and electron microscopy separately.Results:(1) Fb was obstained sucessfully by ourselves (2) Fb which was isolated and cultured and HHORSC, DPC which were recoveryed, cultured and passaged grew normally (3) There were not follicle-like structures and substances of keratosis be observed except scatting hair follicle cells in the scaffold in vitro by light microscopy; (4) Slices from the transplant site showed cells aggregated and formed cell ring, appeared to hair follicles-like structure by HE staining. CK14, CK15,β1 integrin and vimentin staining of the aggregated cell showed positive. Hair follicle cells and red blood cells attaching into the scaffold were observed by electron microscopy. (5) Slices from the transplant site showed that there were more obvious and much hair follicles-like structure by HE staining when the cell combination and order is injecting fibroblast cultured primarily with Mitomycin, after 1 week culturing, add the suspension mixture of DPC and ORSC, the suspension mixture rate of DPC:ORSC is 1:5. The result of MTT showed that lOng/ml BMP-4 can significantly promote the DPC and ORSC's proliferation; (7) Slices from the transplant site showed cells aggregated and formed cell ring, they appeared to hair follicles-like structure when seed cell were cultured with KGM in vitro, but cell were not formed cell ring and appeared to no hair follicles-like structure when cell were cultured with MSCM.Conclusions:(1) fibroblast obtained by ourselves and HHORSC, DPC purchased keep the normal growth character; There were not follicle-like structures formed in the three-dimensional model in vitro, but hair follicles-like structure be gotten in vivo. DPC keep the inducing function in hair follicle reconstruction, ORSC keep the activity of hair follicle stem cell; (3) We explored the best combination, order and rate of cells which could induce hair follicle resconstrution. The best combination and order is injecting fibroblast cultured primarily with Mitomycin, after 1 week culturing, add the suspension mixture of DPC and ORSC, the suspension mixture rate of DPC: ORSC is 1:5. (4)In addition, we confirmed that 10ng/ml BMP-4 can significantly promote the DPC and ORSC's proliferation. (5) KGM medium is better than MSCM when seed cells are cultured in the scaffold in vitro. Our research could guide the direction of reconstruction of the hair follicle.