Study On Human Dermal Cell/Keratinocytes And An In Vivo Wound-healing Model

Wound healing is a constant ongoing hot topic puzzled the medical field, especially to plastic surgeons who are dealing with skin loss, extensive burns, and wound closure on a daily basis. Wound healing is a complex process involving multiple skin cell types including inflammation, re-epithelialization, tissue formation and tissue remodeling that operate in a well-organized manner both spatially and temporally, among them, dermal fibroblasts and keratinocytes are the most important functional cells. Fibroblasts follow certain signals by migrating into the wound, depositing extracellular matrix, organizing the substratum and contracting the wound. Abnormalities in cell migration can result in non-healing wounds or healed wounds with hypertrophic scars. Control of cell motility during wound healing is therefore a critical cellular response. On the other hand, cultured epidermis has been used as a component of skin substitutes to achieve a quick coverage of wounds when treating burns and wounds. In addition, keratinocyte cultures are used for gene therapy and pharmacologic strategies and for construction of skin equivalents. To achieve this, a reliable source of keratinocytes in culture is essential. Therefore, the focus of this thesis is to study the migration of dermal fibroblast and to seek a reliable approach to obtain keratinocytes for clinical applications. Furthermore, we have developed an in vivo human wound healing model on mice for potential cell therapy studies. The thesis thereby is composed by three independent sections, each of which is briefly described as below:Chapter oneHuman keratinocyte primary cultures are commonly established by tissue dissociation for clinical and research purposes. However, techniques for growing keratinocytes still often rely on feeder cell supports and culture medium that is not defined. Further, contamination by unwanted fibroblasts can be problematic. Consequently attempts to acquire purified adult progenitor keratinocytes from whole human skin are an ongoing line of investigation. Here, we developed an innovative skin explant method for growing primary keratinocytes that was rapid, simple, and reliably generated keratinocyte cultures free of fibroblast contamination. The process capitalized on the observation that fibroblasts migrate out of adult skin explants later than epidermal cells, allowing the early harvesting of keratinocytes by trypsinisation of cell outgrowths after3or4days. When grown subsequently in defined medium in the absence of feeder cells, the keratinocytes obtained from explant outgrowths had more rapid growth and colony forming characteristics than those obtained by epidermal cell dissociation. Moreover, whereas the latter could not be grown beyond passage3, the former could be passaged up to10times without differentiating. Antibody staining revealed that a high percentage of cells harvested from the outgrowths expressed K15while very few expressed the differentiation marker K10. Immunofluorescent antibody staining of cells at intervals during the outward migration of cells revealed that they expressed a number of markers associated with progenitor or stem cell like activity including p63, K15, and CD133as well as strong K6expression which is indicative of activated keratinocytes in wound healing epidermis. We consider that the migrating basal cells from the explants are responding in a manner that mimics epidermal cell responses to wounding, and that the activated keratinocytes that grow out therefore have greater growth potential than the dissociated cultures. Unexpectedly, by replenishing the explants with fresh medium after harvesting, further epidermal outgrowths could be obtained, offering the possibility of greatly increased keratinocyte yields for clinical applications.Chapter twoWounded skin instantly generates endogenous electric currents which direct epithelial cell migration and are likely to play an important role in epithelial wound healing. Migration of fibroblasts is critical in wound healing. Whether the physiological electric fields guide fibroblast migration appears unclear. We report here that mouse skin wounds generated and maintained endogenous electric fields for hours and days. In an electric field of50-100mV/mm, human dermal fibroblasts of both primary and cell-line cultures migrated directionally towards the anode, and with relatively decreased speed. This is in sharp contrast to epithelial cells, which migrate to the cathode with increased speed. Unlike epithelial cells, it took more than one hour for dermal fibroblasts to manifest detectable directional migration.We previously demonstrated that an EF activates Phosphatidylinositol-3-OH kinase (PI3-kinase) signaling, we herein tested the role of PI3-kinase in anodal-directed migration of fibroblasts and our current study showed that an applied electric field activated PI3kinase/Akt in dermal fibroblasts from wild type mouse while the cells from p110γ knock out mice were not affected. Our study clearly showed that observable migration of human dermal fibroblasts is time and voltage-dependent, i.e., in the presence of an electric field of physiologic voltage, observable migration toward the anode takes more than one hour to occur. Our study suggests that PI3K may play a role in the electrically-stimulated directional migration of human dermal fibroblast cells, as has been previously described in epithelial cells. Our study suggests that, in addition to the previously-described role of PI3K in the migration of epithelial cells in the presence of an electrical field, PI3K also plays that role in the electrically-stimulated directional migration of human dermal fibroblast cells.Chapter threeThe third chapter describes a preliminary study on a human skin wound healing model on mouse. One of the main components of wound healing study is the development of skin substitute. The majority of current researches are trying to incorporate living dermal and/or epidermal cells into dermal substitute to form a tissue engineered skin equivalent. However, it appears difficult to find the ideal model to test the efficacy of the skin equivalent achieved in which should be capable of accurately mimic the in vivo process of human skin wound healing. On the other hand, there is also a need to discover a cellular carrier for cell therapy. The current study, therefore, is aiming to set up an in vivo model of human skin wound healing that truthfully reflect the human wound healing process, and to establish a cell carrier system together to determine the best strategy of wound healing treatment. We have grafted nude mice with full thickness human skin obtained from excess skin after plastic surgery. The graft has survived after1week and graduately become matured around8weeks after surgery. Full thickness punch wounds are made in the human skin graft and immediately treated by Integra which inoculated with different type of dermal cells. The subsequent cellular responses were analyzed. We have observed all major features of human cutaneous replacement, those are re-epithelialization, dermal remodelling, and basal membrane reorganization could be accurately recapitulated in wounded skin treated with dermal skin equivalents. These results suggest that commercially available skin equivalents, such as Integra can be prepared with living cells (autologous or allogeneic cells) to study human skin wound healing in vivo, this model may be useful for addressing mechanistic questions and evaluating the efficacy of cell therapeutics for promoting wound healing.