Establishment Of 3D Artificial Skin And Study On Wound Healing Effects Of Rhamnolipids

Skin is the largest organ of the human body, which plays a vital role in maintaining homeostasis and as a barrier between human and environment. For the often occurred burns and chronic wounds, autologous skin graft cannot meet the therapy requirement. So skin equivalents were paid more and more attention. Among them, three-dimensional artificial skin consisting of epidermis and dermis is most approached to real skin, showing high value than other artificial skins. In this thesis, primary human skin fibroblasts and keratinocytes will be firstly isolated and cultured. Then 3D artificial skin will be established to obtain better skin and reduce the cost. Finally, rhamnolipids, excellent biosurfactants possessing outstanding surface/interfacial activity and low toxicity, will be investigated for their effect on wound healing and anti-scar in the in vitro skin model.Firstly, human skin fibroblasts and keratinocytes were isolated from children’s foreskin. The results showed that primary fibroblast feeder layer was superior to mouse fibroblast cell line (NIH3T3) feeder layer in culturing primary keratinocytes. Density of feeder cells had great influence on the proliferation of keratinocytes. When density of feeder cells and keratinocyte is 1:1, keratinocytes grow fastest. Moreover, domestic trypsin cannot digest keratinocytes well and exert injury on keratinocytes, while TrypLETE can easily digest keratinocytes to single cells with little cell damage.Secondly, keratinocytes were seeded on fibroblasts embedded collagen to establish 3D artificial skin. The effect of calcium content in keratinocytes proliferation medium —EPil on the formation of skin epidermis was analyzed. The result showed that the medium without calcium or low calcium was more beneficial to the maturation of epidermis. Then bovine and rat tail collagen were used to construct dermis. The morphology of skin equivalents was analyzed by HE staining, Sirius red staining and immunofluorescence analysis. The results showed that the area of skin constructed by bovine collagen is reduced by 21%, while that of skin constructed by rat tail collagen decreased about a half. The water contact angle of skin constructed by bovine collagen is similar to that of human skin which is much higher than skin constructed by rat tail collagen. Moreover, intact skin structure can be seen by HE staining and immunofluorescence analysis in both skin equivalents, but the skin constructed by bovine collagen have more multilayer keratinocytes.Finally, the wound healing and anti-scar effects of rhamnolipids were analyzed by in vitro skin model. In 2D wound scratch assay, rhamnolipids promoted the migration of fibroblasts and keratinocytes, indicating the effect in accelerating wound healing. In addition, rhamnolipids inhibited the collagen contraction by myofibroblasts and down-regulated collagen synthesis and rearrangement, indicating the potential anti-scar effect.In summary,3D artificial skin was established using primary fibroblasts and keratinocytes, while bovine tail collagen was used as dermis scaffold. The 3D artificial skin was similar to real skin in hydrophobicity and structure integrity. In addition, the effects and mechanism of rhamnolipids on wound healing and anti-scar were confirmed by in vitro skin model.