Application Of Silk Fibroin Nanofibers And Human Keratinocytes In Skin Tissue Engineering

With the development on treatment of skin damage, skin tissue engineering isbecoming a hot topic in recent years, which has a broad prospect for clinical application.Facing of large area of burns or damages, in order to promote the regeneration andrepair of skin tissue, it is urgent to obtain sufficient epidermal coverage. Silkfibroin, in consideration of different properties of other biological materials, can beprocessed into multiple material formats with controllable structure and property whichprovides good biological materials for the application in skin tissue engineering. Humankeratinocytes are derived from the outmost layer of skin epidermis. Culture techniquesof epidermal cells in vitro have laid a solid foundation for the study of the mechanismof epidermal differentiation. This study aims to use silk fibroin nanofibers andkeratinocytes to construct the tissue-engineered skin in vitro, so as to explore its role inthe repair of damaged skin tissue. This experiment adopted human keratinocytesextracted from surgical operation of abandoned children finger skin. Taking advantagesof the controllable structure and property of silk fibroin, we inspected the scaffoldsthrough three-dimensional culture system with keratinocytes in vitro. Furthermore, thecompatibility of silk fibroin with keratinocytes and its role in the repair of skin damagewere analyzed to lay a foundation for further study and application.Part Ι Culture and identification of human keratinocytes and the comparativestudy on silk fibroin nanofibersObjective: To isolate, culture and identify the kerotinocytes extracted fromabandoned children finger skin and study on the compatibility with silk fibroinnanofibers.Methods: The keratinocytes were isolated from the children skin by two stepcombined dissociation using dispase and trypsin.The cells were cultured and passaged in serum-free human epidermal cells medium. We detected the specific markers andproteins of keratinocytes by RT-PCR and immunofluorescence. The keratinocytes werethen seeded on silk fibroin nanofibers. The cells were cultured and observed underinverted microscope at different time point.We also detected the adhesion andproliferation ability of cells on the materials by scanning electron microcopy.Results: Human keratinocytes showed a typical epithelial-like growth withobvious boundary and clear nucleus. RT-PCR results showed that the cells isolated andcultured expressed keratinocytes marker genes CK5， CK14, β1-intergin andimmunofluorescence staining results revealed the expression of cytokeratin CK5andCK14. After seeding keratinocytes on silk fibroin nanofibers, the material could guidethe adhesion and growth of cells so as to spread and proliferate effectively. The secretedextracellular matrixes indicated that human keratinocytes in vitro were grown inexcellent condition and have good compatibility with silk fibroin nanofibers.Conclusion: We successfully established the system to extract and culture humankeratinocytes in vitro and accomplished the identification of biological characteristics.Furthermore, we proved the good compatibility of silk fibroin nanofibers withkeratinocytes which provided a theoretical basis in the skin tissue engineeringapplications. Part II Application of silk fibroin nanofibers in three-dimensional air-liquidculture model and its role in skin engineeringObjective: To establish a three-dimensional air-liquid culture model of normalskin, to explore the feasibility of silk fibroin nanofibers applied as scaffolds withkeratinocytes in the model to construct the tissue-engineered skin and to study the roleof silk fibroin nanofibers in the skin tissue injury repair.Methods: In this study, rat collagen type Ι was used to establish athree-dimensional tissue model of normal skin as a positive control. Then the silkfibroin nanofiber membrane was adopted for the model and the differentiation ofkeratinocytes in vitro was observed by hematoxylin-eosin staining. The silk fibroinnanofibers were transplanted into nude mice skin lesion as epidermal substitution andthe wound healing was observed and recorded by immunofluorescence. Results: Tissue-engineered skin was successfully constructed by using silk fibroinnanofibers and keratinocytes, hematoxylin-eosin staining section showed that humanskin keratinocytes has a preliminary differentiation. It seems that while using the thinnermaterial, the cells demonstrated the more differentiated state. Experiments on nude miceshowed that silk fibroin nanofiber membranes had played an efficient role in woundhealing. The expression of cytokeratin CK10and CK14was detected byimmunofluorescence staining which suggested the intact repair of wound skin.Conclusion: Silk fibroin nanofibers can be used as an ideal material in vitroconstruction of tissue-engineered skin and it is feasible to be applied as theepidermal covering in skin wound repair as well.