Construction Of Tissue Engineering Skin On ADM By Mix-seeding

Objective: In the past research about tissue engineering skin, fibroblasts and epidermal cells were all seeded separately. There is a problem that epidermal cells didn't easily adhere to ADM.what's more ,it can't used in clinical work because epidermis didn't stick to dermis tightly.According to some research,growth factors and extracellular matrix secreted by fibroblasts can promote to migration,proliferation and differentiation.The aim of this experiment is to investigate the feasibility of mix-seeding constructing tissue engineering skin in vitro.Methods: Fibroblasts and epidermal cells were cultured seperatedly from human foreskin. Fibroblasts were seeded on the dermal surface of acellular dermal matrix(ADM).7 days later, epidermal cells (5×105/cm2) were mixed with fibroblasts(0.2×105)and then seeded on epidermal surface of ADM. The culture medium was mixed liquor containing half of K-SFM and another half of the culture supernatants of fibroblasts.But in the control group,only epidermal cells (5×105/cm2) were seeded and cultured with K-SFM.After composite skin have been cultured for 1 week and 3week, samples were harvested respectively to observe the morphological alteration. Constructed 3-week composite skin was identified by immunohistochemitry and observed by transmission electron microscope .Results: Fibroblasts and epidermal cells obtained by two-step enzyme digestion are extraordinary much and vigorous.We can observe that fusional epidermal cells stick to ADM tightly in 1-week experiment group.As a control,only sporadic epidermal cells can be seen. After 3-week culture, there were 3-4 continuous layers of the epidermis in the experiment group. Not only the epidermal layer sticked tightly to the dermis,but also trochanterellus was formed.But in the control group,there were just 1-2 layers of the epidermis without sticking tightly to the dermis. Laminin(++) indicated that basement membrane was thoroughly formed in the experiment group.The same result can be seen by transmission electron microscope.Conclusion: perforated ADM is suitable for reconstruction of tissue engineering skin.The mix-seeding which contained epidermal cells and small fibroblasts would contribute to the adhesion and proliferation of epidermal cells on ADM,and promote to the complete formation of basement membrane.According to HE staining,immunohistochemitry and electronic microscope,constructed tissue engineering skin by this method has been verified to approach normal skin.