Study On The Reconstruction Of Tissue-Engineered Skin With Appendages Using Dermal Papillae Cells And Keratinocytes

Research 1.Objective To found an efficient method to isolate clear dermal papillaes without operation under microscope. Method The scalp specimens were transected at the dermis-subcutis interface after incubated with dispase solution. The subcutaneous tissue was cut continuously into a mash and mixed with collagenase I solution and incubated at 37℃for 2-2.5 h. Dermal papillaes were obtained by centrifuge repeatedly. Results Clear dermal papillaes were harvested using new two steps digestive treatment without complicated operation under dissecting microscopes.Conclusion New two steps digestive treatment is an efficient method for isolating and culturing of dermal papillae cells and needs less labour.Research 2.Objective The differences of biological characteristics of dermal papillae cells cultured in keratinocyte medium and normal medium were compared and we determined whether it is feasible for the reconstruction of tissue-engineered hair follicle using dermal papillae cells cultured in keratinocyte medium. Method Dermal papillae cells were cultured in keratinocyte medium and normal medium in two group, the time of dermal papillae adherence and cell outgrowth were recorded and the rate of dermal papillae adherence were determined after 5 days. As well as, we observed the differences of cell morphology under inverted phase contrast microscope. The maximum generations were determined in two groups. In third-generation cells, the number of aggregates in every dish and the proliferation by MTT were compared between those two groups. Meanwhile, the expression of a-SMA and ALP were detected by immunofluorescence and specific stain in two groups. Results Dermal papillaes of keratinocyte medium group had a higher rate of adherence and quickly outgrowth. Cells were bigger in NM than KM. There are more aggregates in third generation cells in NM than KM. In addition, cells could form cell sheets which were multi-layers only in KM group. Mostly 7 and 15 generations could been subcultured in NM and KM respectively. The result of MTT indicated cells proliferated more actively in KM than NM. The positive of a-SMA were detected in both groups third passage cells. Ocassionally a little few cells expressed ALP in the sixth-generation cells of NM group. However, the cells still expressed ALP in the fifteenth-generation cells of KM group. Conclusion Dermal papillae cells proliferated actively,'aggregated obviously and could been subcultured more generations in KM group. Therefore, culturing dermal papillae cells with keratinocyte medium is feasible for the reconstruction of tissue-engineering hair follicle.Research 3.Objective To reconstruct tissue-engineered skin substitutes with skin appendages using human dermal papillae cells and keratinocytes together. Method The dermis substitute was reconstructed by mixing both keratinocytes and dermal papillae cells into collagen gel. Next, keratinocytes were seeded onto the surface of dermal substitutes. The composite skin substitutes were cultured at the air-liquid interface for 3-5 days. A lxlcm full-thickness skin defect was maken and was covered with the skin substitute, to observe the healing and contraction of wound. At 4,6,8 weeks after transplantion, samples were obtained and histological study was carried out. Results The skin substitute which was reconstructed with dermal papillae cells and keratinocytes could contribute to wound healing and basement membrane forming and reduce scar contraction. Hair follicles and sebaceous glands formed in the dermis of skin substitutes, at 6 weeks after transplantation. Conclusion The reconstruction of tissue-engineered skin substitutes with appendage needs to seed dermal papillae cells and keratinocytes together in the dermis layer of skin substitutes.