| Abstract:Trauma,cancer,inflammation of oral and maxillofacial region may cause facial nerve damages and have a poor self-healing ability after nerve injury. And so, how to repair damaged facial nerve simply and effectively is an urgent problem in oral and maxillofacial surgery and even micro plastic surgery now. Emergence of tissue engineering technology has brought new ideas for the facial nerve and other peripheral nerve repair. Tissue engineering is combined with biomaterials and cell therapy to develop new therapeutic strategies for neurodegenerative diseases. Adipose-derived mesenchymal stem cells (ADSCs) have the corresponding property that they can differentiate into neuron-like cells. Chitosan materials have good physical and chemical properties, and have special biomedical characteristics like good biocompatibility, biodegradability, non-immunogenicity and low toxicity, and wide array of sources, so they are the nerve tissue engineering material with good prospects. This experiment is to explore the influence of chitosan material on BDNF expression in neural induction process of rat adipose-derived mesenchymal stem cells, and then clarify the effect of chitosan material to the proceed adipose-derived mesenchymal stem cells into nerve cell. Methods:The proliferation of the adipose-derived mesenchymal stem cell that obtained was measured by CCK-8 and was identified by immunohistochemical method. ADSCs were induced into neural cells by neural induction medium that is added 20ng/mL bFGF and 20ng/mL EGF and 2% B27 to neurobasal medium, and these induced cells were identified by morphology and immunofluorescence. The influence of chitosan material on the viability of ADSCs was measured by CCK-8. Compare differentiation of BDNF expression in the induction processes between the non-chitosan film group and the group of chitosan membranes by Quantitative Real-time PCR.Resμlts:The shape of the primary cμltured cells is elongate and fusiform, which is difficμlt to distinguish with BMSCs on the shape. Nucleus is in the center, and it grows adherently. Associated antigens as CD90, CD 105 which are detected by immunohistochemistry are positive and CD45, associated antigen of hematopoietic stem cell, is negative. It has mμltilineage differentiation capacity, such as:induction into adipocytes and into chondrocytes, and we can find that ADSCs have actively growing, strong proliferative capacity through the growth curve. Because these cells derived from fat, above that can be defined as ADSCs.ADSCs were induced into neurospheres in nine days by neurobasal medium added with the 20ng/mL bFGF and 20ng/ mL EGF and 2% B27. The obtained neurospheres is positive for nestin-antibody by immunofluorescence staining. The obtained cells through further induction of differentiation from neurospheres are also positive for MAP-2-antibody by immunofluorescence staining. Combining with Morphology, we can basically determine that ADSCs have successfμlly induced into neural cells. The resμlts from CCK-8 indicated that application of chitosan material or not have some influence on the proliferation of ADSCs, the between-group difference is statistically significant (P<0.05). Quantitative Real-time PCR has analyzed the affect on the expression of BDNF,which applied of chitosan membrane or not in the neural differentiation process. Neural differentiation potential of ADSCs was evaluated by the change of quantity of BDNF. Quantitative Real-time PCR measured the quantity of BDNF mRNA after successful induction in control group (no-chitosan film applications) was 1.16556; in the experimental group (application of chitosan film) was 2.66166. And there are obvious differences between the control group and the experimental group, the numerical of BDNF mRNA in the experimental group was 2.28 times higher than the control value (P<0.05). Conclusion: ADSCs has a relatively strong proliferative capacity, and can be induced to differentiate into nerve cells. The experimental analysis cells after differentiation in the level of morphology and phenotype, which showed a significant change; Application of chitosan biological material has affected the expression of BDNF factor in the process of ADSCs induced into neural cells, it further illustrates that chitosan biological material is favorable for ADSCs induced into neural cells, and last because it has a high compatibility. So it can become an ideal material for the treatment of neurological damage in vivo. |
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