Electrospinning is a versatile technique for fabrication of made-on-purpose biomimetic scaffolds. In this study, optimized electrospun fibrous membranes were produced by simultaneous electrospinning of polycaprolactone (PCL) and polyvinylpyrrolidone (PVP), followed by the selective removal of PVP from the PCL/PVP mesh. After aminolysis, a blend of collagen/chitosan was grafted on the surface. Physicochemical characterizations as well as in vitro evaluations were conducted using different methods. Successful cell infiltration into samples was observed. It seems that the positive trend of cell ingress originates from the proper pore size obtained after removal of pvp (from 4.46 μm before immersion in water to 33.55 μm after immersion in water for 24 h). Furthermore, grafting the surface with the collagen/chitosan blend rendered the scaffolds more biocompatible with improved attachment and spreading of keratinocyte cell lines (HaCaT). Viability evaluation through MTT assay for HDF cells did not reveal any cytotoxic effects. Antibacterial assay with Staphylococcus aureus as Gram-positive and Escherichia coli as Gram-negative species corroborated the bactericidal effects of chitosan utilized in the composition of the coated blend. The results of in vitro studies along with physicochemical characterizations reflect the great potentials of the produced samples as scaffolds for application in skin tissue engineering.

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Background: Off-the-shelf supply of viable engineered tissue is critical for effective and fast treatment of life-threatening injuries such as deep burns. An expanded keratinocyte sheet on the human amniotic membrane (KC sheet-HAM) is a beneficial tissue-engineering product for wound healing. To access an on-hand supply for the widespread application and overcome the time-consuming process, it is necessary to develop a cryopreservation protocol that guarantees the higher recovery of viable keratinocyte sheets after freeze-thawing. This research aimed to compare the recovery rate of KC sheet-HAM after cryopreservation by dimethyl-sulfoxide (DMSO) and glycerol. Methods: Amniotic membrane was decellularized with trypsin, and keratinocytes were cultured on it to form a multilayer, flexible, easy-to-handle KC sheet-HAM. The effects of 2 different cryoprotectants were investigated by histological analysis, live-dead staining, and proliferative capacity assessments before and after cryopreservation. Results: KCs well adhered and proliferated on the decellularized amniotic membrane and successfully represented 3 to 4 stratified layers of epithelialization after 2 to 3 weeks culture period; making it easy to cut, transfer, and cryopreserve. However, viability and proliferation assay indicated that both DMSO and glycerol cryosolutions have detrimental effects on KCs, and KCs-sheet HAM could not recover to the control level after 8 days of culture post-cryo. The KC sheet lost its stratified multilayer nature on AM, and sheet layers were reduced in both cryo-groups compared to the control. Conclusion: Expanding keratinocytes on the decellularized amniotic membrane as a multilayer sheet made a viable easy-to-handle sheet, nonetheless cryopreservation reduced viability and affected histological structure after thawing. Although some viable cells were detectable, our research highlighted the need for a better cryoprotectant protocol other than DMSO and glycerol, specific for the successful banking of viable tissue constructs.