ABSTRACT
Objective: the organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts [DFs] on epidermal differentiation of adipose-derived stem cells [ASCs] using a three-dimensional [3D] organotypic co-culture technique
Materials and Methods: in this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone [PCL] matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction [RT-PCR] and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co-culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy [SEM]
Results: the early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups compared to the control ones [P<0.05]. We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes
Conclusion: the 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration
ABSTRACT
Tissue engineering is a new field of which the main purpose is to regenerate and repair the damaged tissues. Scaffolds serve as three dimensional matrices for neo-organogenesis and their substance can be biologic or synthetic. Natural polymers have good interactions with the cells and synthetic biomaterials are also highly useful in biomedical application because of their biocompatible properties. In addition to scaffold substance, surface properties of biomaterials have an important role in tissue engineering. In this study, we examined whether substrate substance is important for wound healing or its surface topography. Therefore, we fabricated two matrices, electrospun polycaprolactone (PCL) nanofibers and collagen/chitosan film, and implanted them to the same rat models. After 2 weeks, the sizes of healing wounds were measured and their cellular structures were evaluated by histochemistry and mmunohistochemistry. Histological staining showed a good level of cellularization and epidermis-dermis formation in PCL implant while no determinable epithelium was observed after 2 weeks in collagen-chitosan graft. Immunohistochemical study demonstrated the highly expressed pancytokeratin in PCL graft while its expression was weak in underdeveloped epidermis of collagen-chitosan implantation. In conclusion, this study suggested that PCL nanofibers with high surface area had a more ideal property than natural collagen-chitosan film, therefore the structure and topography of a matrix seemed to be more important in wound healing than its material substance.