OBJECTIVE: To investigate the skin regeneration using cultured human keratinocytes with collagen sponge transplanted into thickness wound of nude mice. METHODS: Human foreskin from foreskin ectomy procedures was detached with 0.5% Dispase II. Epidermis sheets were separated from dermis and digested with 0.05% Trypsin into single cell suspension. Keratinocytes were cultured and seeded into collagen sponge during logarithmic growth phase. After 3 days, the keratinocytes-collagen sponge were grafted on full thickness wound of nude mice, compared with simple collagen sponge without keratinocytes. The histological, immunohistochemical examination and electron microscopy were detected. RESULTS: After the epidermal substitute was grafted onto wound, the human keratinocytes were able to further proliferate and differentiate and develop into new epithelia. Compared with the control group, the wound healed earlier and contracted less, epithelia matured earlier, and the collagen fiber was less beneath epithelia. CONCLUSION: Keratinocytes can grow on collagen sponge and migrate onto wound to develop into stratified epithelia and inhibit wound contract. The keratinocyte graft can be used to repair skin defect.
Objective To investigate the feasibility of human amniotic membrane-living skin equivalent (AM-LSE) in repairing the skin defect. Methods A 5-year-old boy with giant nevus at neck, shoulder, and back was admitted in July 2016. Normal skin tissue of the patient was harvested and keratinocytes and dermal fibroblasts were separated and expanded in vitro. Human AM was donated from a normal delivery and de-epithelialized for constructing an LSE as a matrix. Keratinocytes were seeded on the epithelial side of the AM which was previously seeded with fibroblasts on the stromal side and then the complex was lifted for air-liquid surface cultivation for 10 days and observed under naked eyes and sampled for histological study. The nevus was excised to deep fascia and the skin defect in size of 20 cm×15 cm was covered with artificial skin of collagen sponge for 2 weeks to enhance granulation tissue formation, and then the AM-LSE grafts of stamp size were grafted on. The dressing was changed until the wound healed. Results After 10 days of air-liquid surface cultivation, the AM-LSE developed a multilayered and differentiated epidermis with the fibroblasts-populated amnion as the dermal matrix. The LSE stamps survived and expanded to cover the whole wound. The grafted area showed normal skin color and soft contexture at 6 months after operation, and histological study showed well developed epidermis with compactly aligned basal cells, stratified and well differentiated squamous, granular layers and stratum corneum and well vascularized dermal compartment without inflammatory cells infiltration. Conclusion The cultivated AM-LSE with autologous cells can repair skin defect and survive for a long term without rejection.