Study On Restructuring Skin Equivalents With Human Hair Follicle Outer Root Sheath Cells And Fibroblasts

[Purpose] The bulge region, a portion of the outer root sheath, contains the hair follicle stem cells, which shows multipotency to differentiate into almost all epithelial cell types. We plan to restructure the skin equivalent with human outer root sheath cells, which isolated from hair follicles, and fibroblasts on the Integra matrix by a quickly, easy and efficient culture method.[Methods] Scalp tissue after face-lift surgery were dissected and incubated in 0.5% Dispase at 4°C overnight to remove dermal sheath of hair follicles. The middle and lower parts of the isolated hair follicles with outer root sheath were dissected and incubated in 0.05% trypsin-0.53Mm EDTA at 37P0PC for 5minutes to isolate outer root sheath cells. The outer root sheath cells were primary cultured and subcultured in Difined Keratinocytes–Serum Free Medium at 37°C and 5% COB2B for cell studies and to be used as the seed cells to restructure the skin equivalent. The immunofluorescence stain of CK15, CK19 and ?1 Integrin, which are regarded as markers of the epidermis stem cell, were used respectively to identify the characters of the isolated outer root sheath cells, while human keratinocytes and fibroblasts, which isolated from new burn foreskin were taken as the negative control. The immunofluorescence stain of Ki-67 was used to test the proliferation ability. The number of cultured cells was counted to draw the cell growth curve. The matrices were seeded with human fibroblasts, cultured in DMEM with 10% FBS to form a dermal equivalent. 24 h later, passage 1 outer root sheath cells were seeded into the dermal equivalent and cultured. 4 days later, the surface of the skin equivalent was lifted to the air–liquid interface. MTT assay and DAPI stain were used to monitor the cell proliferation in the cultured matrix among the matrices seeded with same density of outer root sheath cell alone, the matrices with same density of human fibroblasts alone and matrices with half density of outer rooter sheath cell and fibroblasts. One week after the air-liquid co-culture, matrices were transplanted into the symmetrical full-thickness defect wounds on athymic mice. At time points of 2 weeks and 4 weeks after transplantation, the grafts were harvested and examined by means of histology and immunohistochemistry. H&E stained sections were used to show the structure of new skin. Human anti-mouse HLA-A, CK MNF116 and Vimentin stains were used respectively to identify the origin of the cells in the regenerative tissues.[Results] The isolated outer root sheath cells showed high proliferation ability. The expression of the immunofluorescence antibodies of CK15, CK19 and ?1 Integrin were all positive in the primary cells. After passage 2, the expression of ?1 Integrin and CK15 decreased, while expression of CK19 was still positive. Compared with the matrices seeded with outer root sheath cells or human fibroblasts alone, the matrices seeded both with outer root sheath cells and fibroblasts show more cell density either in MTT assay or DAPI stain at the time point of day 1, day 7 and day 14. It indicated that outer root sheath cells cultured with fibroblasts in matrix have more proliferation ability. 2 weeks after transplantation, the positive stain of HLA-A could be seen in the whole layer of grafted skin. The human CK MNF116 was positive stained in the grafted epidermis, meanwhile human Vimentin in the new dermis. The regenerated epidermal tissue closely resembled that of normal human skin tissue comprised of a basal layer, five to ten granular layers and abundant corneum layer. In the new dermal layer, the matrix decreased 4 weeks after transplantation.[Conclusion]This study gives evidence that our isolation technique allows isolating and culturing of outer root sheath cells, which contain some bulge stem cells. The skin equivalent which we restructured with fibroblasts and outer root sheath cells in vivo is able to form histological structures similar to normal human dermis and epidermis within 2 weeks. We hope that this method may be the trend of tissue engineering skin in the future.