Background: Tissue engineered (TE) skin offers promising alternative treatment in wound healing of acute and chronic skin injuries. Limited autografts from donor skin and risks of rejection with allograft have instigated the development of TE skin. Amniotic membrane (AM) has anti-fibroblastic, ati-angiogenic property and also reported to reduce scarring. We aim to produce TE skin substitute with the use of decellularized (DC) human AM matrix that can emulate dermis with human adipose derived epithelial like cells for re-epithelialization.

Methods: Human AM (n=6) were DC by treating with hypotonic buffer and then with sodium dodecyl sulphate and lastly with deoxyribonuclease I to wash away cell debris. The DC AM were characterized by histology, DNA quantification and electron microscopy. Human adipose derived stem cells (ADSC) were isolated from fat tissue (n=5) and EPCAM positive cells were selected using magnetic nanoparticles. These cells were further seeded on a decellularized AM for 2 and 4 weeks to generate TE skin substitute. For in vivo characterization, TE skin (AM + cells) and decellularized AM was orthotopically transplanted onto full thickness skin wounds (1.5cm x 1.5cm) in athymic nude mice and observed for four weeks.

Results: Complete decellularization of the AM was achieved in three DC cycles. We found that the isolated EpCAM+ cells from ADSC stained positive for different epithelial markers by immunofluoroscence staining and flow-cytometry analysis. Extensive epithelial like cellular in-growth forming well integrated TE skin was observed in histological findings in the in vitro TE skin. After transplantation, all animals were healthy and there was no sign of red swelling, necrosis and exudation in both, TE skin group and decellularized AM group. After four weeks, surfaces of both grafts were taken over by the skin and appeared pink-white, soft and normal in its texture, whereas the control group wound was with a scar. Histological analysis showed that in all three groups, keratinocytes were migrated around the wound boundaries, and acted like the barrier function of the epidermis by closing the wound. However in control group wound, dermal matrix in healed epidermis had fibrosis like scar.

Conclusion: Results from the animal studies further confirm the regeneration of TE skin in both epithelial layer and also in dermal layer. Use of autologous stem cells together with DC AM for tailor-made TE skin would be promising treatment for acute and chronic skin injuries.

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