Utilization of the keratinocytes from epidermal lineage cells which differentiated from mouse embryonic sem cells, on primer wound healing

The skin plays a crucial role in protecting the body against the environment and integrity of the body?s internal milieu. The treatments of burns and skin injuries with less damage using autologous or allogenic skin grafts constitute from adult or embryonic stem cells in tissue engineering are at the forefront. Pluripotent embryonic stem cells are able to proliferate in suitable condition without differentiation, in addition to this, the cells can differentiate when the culture condition is changed. The aim of this project is to investigate the effects of keratinocytes which differentiated from mouse embryonic stem cells after transferring on the surgical wound model of the mouse during wound healing and secreted molecules which after wound healing.Keratinocyte like cells were obtained from embryoblasts which derived from embryonic stem cells on matrigel and culture media supplemented with BMP-4. Differentiated keratinocytes were fixed in 4% paraformaldehyde and distribution of cytokeratin-8 and cytokeratin-14 using with immunohistochemical technique, and the ultrastructural properties of the cells were analyzed after fixation with glutaraldehyde and osmic asid and embedded in an agar. The keratinocytes are labelled with Brd-U before transferring to the wound area. For creating experimental surgical wound model 60 Bulb C type mouse divided in 4 equal groups [experimental (n=15), control (n=15), sham (n=15) and healthy skin (n=15)]. In healthy skin group nothing is done however in control group only wound is made. In sham group, only mesh is transferred after the wound is made, while in experimental group, the mesh with keratinocytes which obtained from culture condition were transferred on to the wound area. From all groups, the samples were collected after the surgical process on the 3th, 5th and 7th day for both light and electron microscopic analyses. For the light microscope analyze, samples were fixed in 10% formalin and sections were taken after routine paraffin process, and some of the sections were stained either H-E or Masson Trichrom, also the rest of the sections were stained with indirect immunoperoxidase technique in order to determine distributions of cytokeratin-8, cytokeratin-14, EGF, IL-8, FGF-1, FGF-2, MCP-1 and collagen-1. For analyzing Brd-U labelled cells, the sections were also stained with anti-Brd-U. For electron microscope analyze, samples were fixed in glutaraldehyde and osmic asid, were then embedded in epon and half thin and thin sections were taken.The differentiated cells in culture condition were both positive for early and late term keratinocyte differentiation markers, cytokeratin-8 and cytokeratin-14 and similarity ultrastructurally with keratinocytes, were thought keratinocyte-like cells. In light microscopic analyze, on the 3th day in experimental group, on the wound area there was an epithelialization that consist of keratinocyte cells, while in control and sham groups near the wound area, only keratinocytes which were just started to proliferate were observed but there was no evidence about epithelialization. From the 3th day in experimental group in the wound area, Brd-U labelled transferred cells?s nucleuses were stained positively with anti-Brd-U stain.In indirect immunohistochemistry analyze, while cytokeratin-8 immunoreactivities were negative in control group on the 3th and 5th days, it was positive on the 7th day, in experimental group on the 3th and 5th days were positive, but on the 7th day it was negative. In the sham group, cytokeratin-8 immunoreactivities in all days were positive, while in healthy skin it was negative in all days. The cytokeratin-14 immunoreactivity in healthy skin on the 3th and 5th and 7th days stained strongly positive while in experimental, control and sham groups it was minimal or negative on the 3th day. In control and sham groups cytokeratin-14 immunoreactivity on the 5th and 7th days was moderate but in experimental group it was strongly positive. EGF immunoreactivity in healthy skin group was negative in all days while it was negative on the 3th day and positive on the 5th and 7th days in experimental, control and sham groups, but, immunoreactivity was especially detectable on the 7th day of experimental group. IL-8 immunoreactivity was negative in healthy skin group in all days, in experimental and control group in all days immunoreactivities were positive but it was more on the 3th day than the other days, in sham group on the 3th and 7th days it was minimal, on the 5th day it was moderate. FGF-1 immunoreactivity in all groups on the 3th, 5th and 7th days was negative. FGF-2 immunoreactivities in all groups on the 3th, 5th and 7th days were positive while FGF-2 immunoreactivity positive cells were more on the 7th day of experimental group than others. MCP-1 immunoreactivity in healthy skin group in all days was minimal in connective tissue while in experimental, control and sham groups on the 3th and 5th days was moderate where as on the 7th day in experimental group it was moderate and in control and sham group it was lessen. Collagen-1 immunoreactivity in healthy skin group was positive in dermis and there was no difference between the days , however, it was negative on the 3th day in experimental group, in control and sham group it was minimal and the immunoreactivity was gradually decreased on the 5th and 7th days, while in experimental group it was gradually increased. Electron microscope analyze of samples, in experimental group beginning from the 3th day it was observed that epithelialization started and keratinocytes were on the wound area.Keratinocytes which derived from mouse embryonic stem cells after transferring on the surgical wound area were evaluated healty because of positively stained with anti-Brd-U. Differentiated cells were demonstrated increased secretion of molecules that collagen-1, EGF and cytokeratin-14 secretion in late term and the cytokeratin-8, IL-8, FGF-2 and MCP-1 secretion in early term during wound healing on wound area. In conclusion differentiated cells are accelerate and influence positively to the wound healing stages.