| Skin is the first defensive barrier for human body. Due to its direct contact with external environment, however, skin is very likely to be harmed by various damages and illnesses and result in skin defect. In addition to conventional therapy, tissue engineered skin products can be used for recovering.have provided a new means for treating skin defect. Composite chitosan tissue engineered skin, which is a relatively ideal 3-dimensional model, has been traditionally cultured in static way. Though the culture technique is relatively mature, it is defected in some ways, such as long time for culturing, heavy workload and occasional pollution. On the other hand, perfusion culture which is a dynamic culture method can avoid many problems that are unavoidable in static culture. Currently, common perfusion culture models include five-layer overlapping model, MINUTH perfusion culture and so on. However, those models are featured with strong pertinence to cells to be culturedpertinence,, limited application and similar vitality of attained cells compared with that of cells attained through static culture. To sum up, those models do not have obvious advantages. The perfusion device applied for this experiment, after being further improved and optimized, is used for achieving better culture effects. This paper has made a preliminary research of perfusion culture of tissue engineered skin, compared cell proliferations of tissue engineered skin under different perfusion flow velocities and studied the influence of different nutrient concentrations on morphological structure as well as cell proliferation and apoptosis of tissue engineered skin. Thus, more bases are provided for further improving optimizing perfusion conditions and a foundation for wider ain-depth application of perfusion culture in the area of tissue engineered skin is laid.Objectives:1. To make a brief analysis of homogeneities and shear forces of low velocity flow field and high velocity flow field of the currently applied perfusion incubator.2. To study influence of different perfusion flow velocities on proliferation of dermal cells of tissue engineered skin and preliminary explore a perfusion speed that is appropriate for culturing tissue engineered derma in a dynamic way.3. To compare influence of different nutrient concentrations on morphological structure as well as cell proliferation and apoptosis of tissue engineered skin and explore for nutrient concentration conditions that are appropriate for cell growth.Approach:1. Make a brief analysis of homogeneities and shear forces of flow fields through calculating 3-dimensional flow fields with two-phase model according to computational fluid dynamics(CFD).2. Enzymatic digestion and separation of samples of young men’s foreskin to attain fibroblast and practice culture and passage; use the second-generation fibroblast to construct composite chitosan tissue engineered derma. Culture compos ite chitosan tissue engineered derma in a dynamic way under low velocity(80ml/min) and high velocity(800ml/min); examine survival states and proliferation states of dermal cells under the two velocities with MTT method.and do statistical analysis.3. Use the second-generation keratinocytes to construct full-thickness composite chitosan tissue engineered skin. Place the skin respectively in 40% and 80% DMEM low-sugar nutrient mediums for perfusion culture. Draw materials after 5 and 10 days respectively for practicing HE staining and observing cellular morphology; then examine cell proliferation state with ki67 and examine cell apoptosis through TUNEL staining; make a statistical analysis.Result:1. Under low flow velocity(such as 80ml/min), the current perfusion structure can guarantee good homogeneity with regard to speed of nutrient solution, dynamic and static pressures, flow field vortex degree, velocity gradient, flow field vector and so on. When the velocity is increased, the whole velocity will increase and dynamic and static pressures increase to different degrees and homogeneity of the flow filed is undesirable.2.Result of MTT shows that: the MTT value in tissue engineered derma increases as ti me goes by under low-velocity perfusion condition, which hints quantity of living cells in t he derma increases constantly and the cells are proliferating; moreover, viability of cells cultivated under low-velocity perfusion condition is obviously higher than that of cells cultivat ed in static way; however, viability of cells cultivated under high-velocity perfusion conditi on is poorer than that of cells cultivated in static way.3. Morphology of tissue engineered skin cells cultured with low-velocity perfusion remains acceptable under HE staining and the bilayer structure of derma is clear in structure, which means tissue engineered skin can still get adequate nutrient under perfusion nutrition.4. Result of ki67 test of the immunity group shows that: quantities of ki67 positive cells of epidermides and dermis,when cultured in 80% DMEA culture mediums, are significantly higher than those of ki67 positive cells of epidermides and dermis cultured in 40% DMEM culture mediums(P<0.05). Compared with quantities of ki67 positive cells of epidermides and dermis cultured in 80% concentration for 5 days, those for 10 days are significantly less(P<0.05).5. 5. Test the impact of concentration of a culture medium on cell apoptosis within double-layered tissue engineered skin with TUNEL. According to statistics, quantities of cell apoptosis within epidermides and dermis cultured in 40% DMEM culture mediums are significantly higher than those within epidermides and dermis cultured in 80% DMEM culture mediums(P<0.05). Compared with quantities of cell apoptosis within epidermides and dermis cultured in 80% DMEM culture mediums for 5 days, those for 10 days are significantly higher(P<0.05).Conclusion:When velocity of the perfusion device is low(80ml/min), all flow indicators of the flow field can guarantee good homogeneity. Therefore, 80ml/min is an appropriate perfusion velocity. Meanwhile, perfusion dynamic culture model is superior to static culture and appropriate for culturing tissue engineered skin. In terms of comparison regarding nutritional condition, 80% concentration culture mediums are superior to 40% concentration culture mediums regarding culturing effect. |
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