Abstract:

Objective To repair full-thichness skin defects by acellular amniotic membrane integrated with hair follicle stem cells. Methods Human hair follicle stem cells were isolated and cultured in vitro, and those of the fourth generation marked by lentivirus-mediated green fluorescent protein(pGC FU-GFP-Lentiviru) were grafted onto acellular amniotic membrane. Seven days after integration, cell proliferation was observed by light microscopy with HE staining. Wounds of full-thickness skin defects were prepared in 18 C57BL/6 nude mice, and experiment group (wounds of fullthickness skin defects were grafted with acellular amniotic membrane integrated with hair follicle stem cells), acellular amniotic membrane graft+stem cell injection group (wounds of full-thickness skin defects were grafted with acellular amniotic membrane, with injection of 5×10⁶ hair follicle stem cells under amniotic membrane) and acellular amniotic membrane graft group (wounds of full-thickness skin defects were grafted with acellular amniotic membrane) were divided, with 6 mice in each group. The areas and contraction rates of wounds in each group were measured 7 d, 14 d, 21 d and 28 d after grafting. The expression of green fluorescent protein (GFP) of wound tissues was detected by fluorescence microscopy, and the structure of regenerated skins was observed by light microscopy with HE staining. Results Seven days after acellular amniotic membrane integration with hair follicle stem cells, light microscopic observation with HE staining revealed that cells formed in lamellar shape and covered acellular amniotic membrane. The contraction rates of wounds of each time points in experiment group and acellular amniotic membrane graft+stem cell injection group were significantly lower than that in acellular amniotic membrane graft group (P<0.05). Twentyeight days after grafting, fluorescence microscopic observations revealed that the expression of GFP in cuticular layer of wounds in experiment group was positive, and light microscopic observations with HE staining unveiled that the cuticular layer of wounds in experiment group was significantly thickened, and hair follicle-like structure formed. Conclusion Acellular human amniotic membrane integrated with human hair follicle stem cells can be used in the repair of full-thichness skin defects in nude mice.

Key words: hair follicle stem cell, amniotic membrane, skin defect