The New Model Of Tissue Engineering In Vitro Skin Stimulate Detection

Objectives To construct seed cells of tissue engineering skin model which immortalized human keratinocyte HaCaT cells, silk fibroin modified polyester/polyamide. And compared with the experimental animal, observed the sensitivity, specificity and accuracy. The purpose of provide experimental evidence is the tissue engineering skin model to replace animal model.Methods 1 High serum glucose medium on proliferation of HaCaT cells were cultured, until the cell growth reached 80%, seeded in silk fibroin modified polyester/polyamide nonwoven scaffold, results are scanning by electron microscopy with the first day after inoculation, growth of HaCaT cells third day on the stand with the blank control stents, HaCaT cells detected on the holder material adhesion and proliferation. 2 After 4 days in which silk fibroin modified polyester/polyamide nonwoven scaffolds, thermoplastic forming tray-shaped, sized to fit in a 24 well plate, fibroblasts were cultured for and then vaccination immortalized human keratinocytes cultured until 3 days when merging cells overgrow bracket model gas- liquid cultured for 2 weeks to construct the tissueengineered skin models used for detection. The cultured skin model in different time of paraffin sections and stained by HE, observe the state of skin cell layer model. 3 ECVAM skin irritation from the provisions for substance selected 20 kinds of chemicals, irritants, local model after contact with the skin, MTT was used to determine the relative vitality of cells, conditioned medium was collected and assayed by ELISA for IL-1α factor evaluation construction of skin sensitivity and specificity of the model.Results 1 Fibroin modification formed tray-like thermoplastic polyester/polyamide composite nonwoven scaffold, forming a plurality of porous structure has a certain permeability, HaCaT cells are capable of good growth and can be adhered to the stent without woven fabric fibers. 2 The HaCaT cells and fibroblasts successfully inoculated into fibroin modified polyester/polyamide composite nonwoven scaffold gas- liquid after incubation for 2 weeks, HE staining and scanning electron microscopy of the initial formation of multi-layer cell layer 15 layers and layers of natural cells is very similar, but without the formation of the stratum corneum. 3 20 kinds of chemicals in the tissueengineered skin models were successfully, and the results show the sensitivity of the skin model was 72.7%, specificity 77.8% and accuracy was 75%. 4 There are four chemicals judgment error during skin model and animal model for comparison.Conclusions 1 Successfully prepared silk fibroin modified thermoplastic forming a traylike polyester/polyamide composite nonwovens bracket oriented research, and better scaffold is provided. 2 Growth and differentiation of cells stratified seed can been seen at HaCaT cells in the non-woven silk fibroin modified stent after a period of time, it has begun construction of tissue-engineered skin model. 3 Tissue engineering in vitro skin model constructed for the test substance ECVAM predetermined selected 20 kinds of chemicals, and the results show that the detection accuracy of the model to build their own skin is about 75%.