| ObjectiveTo investigate the characteristics of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and collagen-chitosan scaffolds. To explore the potential epidermal differentiation of hUC-MSCs and the formation of epidermis substitutes in a three-dimensional (3D) microenvironment, which was fabricated by hUC-MSCs embedded into collagen-chitosan scaffolds combined with an air-liquid interface (ALI) culture system. To observed the effect of epidermis substitutes for repairing full-thickness wounds in rats.Methods1. Get healthy full-term cesarean umbilical cord and isolate hUC-MSCs by explant culture. Observe the morphology and the distribution of hUC-MSCs by inverted microscope and fluorescence microscope. Identify the surface markers of hUC-MSCs by flow cytometry and the multi-differentiation potential of hUC-MSCs by hUC-MSCs differentiation into adipocytes and osteocytes.2. Collagen and chitosan was mixed, freeze-dried and cross-linked to form the scaffolds. The microstructure of the scaffolds was observed by scanning electron microscopy (SEM). The biocompatibility of hUC-MSCs with CCSs was proved by detecting cell proliferation situation in collagen-chitosan scaffolds by Methyl thiazolyl blue tetrazolium bromide (MTT) assay and Live/Dead assay.3. hUC-MSCs were grown on the collagen-chitosan scaffolds with viable UC-MSCs and cultured in L-DMEM medium. The cells were exposed to air by lowering the medium level to the lower part of the collagen-chitosan scaffolds after 2 days in the immersed culture. The medium was changed once each day. hUC-MSCs were cultured in this condition for 7 to 14 days to differentiating hUC-MSCs into epidermal lineage. Cytokeratin 19 (CK19) and involucrin expression in hUC-MSCs were measured by immunohistochemistry and western blotting assay.4. Forty-five rats were randomly divided into 3 groups, the control group, the collagen-chitosan scaffolds group, and the epidermal substitute group. Rats’full-thickness skin wounds were prepared on the backs, followed by covering with collagen-chitosan scaffolds or epidermal substitutes. Wound condition was observed on 3,7,14,21 days after operation. Wound healing rate was determined on 3,7,14,21 days. Hematoxylin-eosin (H&E) staining was performed on 3,7, 14,21 days after operation.ResultsThe isolated UC-MSCs by explant culture were elongated and spindle-shaped after passage 3, and they could differentiate into adipocytes and osteoblasts. Flow cytometry revealed that UC-MSCs were positive for CD90, CD105, and CD73, whereas they were negative for CD34, CD45, HLA-DR, and CD lla. Using fluorescence microscope, we observed that hUC-MSCs were spindle-shaped and evenly distributed in the scaffolds. MTT assay and Live/Dead assay indicated that the collagen-chitosan scaffold has good biocompatibility with hUC-MSCs. Immunohistochemistry and western blotting assay both showed that the hUC-MSCs on the surface of the collagen-chitosan scaffolds were positive for the epidermal markers CK19 and involucrin at day 7 and day 14. In addition, H&E staining indicated that multi-layered epidermis substitutes were established. The constructed epidermis substitutes were applied to treat full-thickness wounds in rats and promoted wound healing.ConclusionIn this study, manipulating the 3D microenvironment is a novel method for inducing the epidermal differentiation of hUC-MSCs to engineer epidermal substitutes, which provides an alternative strategy for skin tissue engineering. |
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