In this study, it was aimed to present an approach for the development of multi-layered tissue engineering constructs by using cell sheet engineering. Briefly, MC3T3-E1 mouse pre-osteoblast cells were cultured in temperature-responsive plates (Nunc Upcell®) in the presence of osteogenic medium and the resulting cell sheets were laminated with electrospun poly(L-lactic acid) (PLLA) membranes to obtain viable three-dimensional, thick constructs. The constructs prepared without PLLA membranes were used as control. The cell viability and death in the resulting structures were investigated by microscopic and colorimetric methods. The in vitro performance of the structures was discussed comparatively. Alkaline phosphatase (ALP) activity, collagen and sulfated glycosaminoglycan (sGAG) content values were calculated. The presented approach shows potential for engineering applications of complex tissues with at least two or more microenvironments such as osteochondral, corneal or vascular tissues.
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