In Vitro Biocompatibility Of Chitosan-based Materials To Primary Culture Of Hippocampal Neurons

Objective This study aimed to investigate in vitro biocompatibility of biomaterial chitosan to primarily cultured hippocampal neurons, in order to provide scientific evidence of chitosan-based scaffold used as a candidate biomaterial for cell replacement therapy of central nervous system (CNS) injury.Methods1,The primarily hippocampal neurons were cultured and neuron purity were identified by NF-200 immunocytochemistry. The adhesion, growth and distribution of protein expression of hippocampal neurons cultured on the substrate made up of chitosan fibers was observed under an inverted microscope, scanning electron microscopy, transmission electron microscopy and immunocytochemistry (NF-200, GAP-43, MAP-2,β-tubulin III).2,MTT tests was performed to determine the cell viability of hippocampal neurons cultured in chitosan fiber extract for different periods (12 h,24 h,48 h,72 h).3,Western blot analysis was conducted to determine the expression levels of GAP-43 andβ-tubulin III proteins in hippocampal neurons that were cultured in chitosan fiber extract for different periods (12 h,24 h,48 h,72 h).4,The adhesion, growth and distribution of protein expression of hippocampal neurons cultured on the substrate made up of chitosan membranes was observed under an inverted microscope, scanning electron microscopy, transmission electron microscopy and immunocytochemistry (NF-200,β-tubulin III, synaptophysin). The Leica Qween soft were performed to measure average and total length of neuritis for hippocampal neurons cultured on the poly-lysine-coated coverslips and the substrate made up of chitosan membranes.5,MTT tests was performed to determine the cell viability of hippocampal neurons cultured in chitosan membranes extract for different periods (12 h,24 h,48 h,72 h).6,Western blot analysis was conducted to determine the expression levels of GAP-43 andβ-tubulin III proteins in hippocampal neurons that were cultured in chitosan membranes extract for different periods (12 h,24 h,48 h,72 h).Results1,The hippocampal neurons were cultured and neuron purity identified by NF-200 immunocytochemistry was 96.3%. The results of inverted microscope, scanning electron microscopy and immunocytochemistry (NF-200, GAP-43, MAP-2,β-tubulin III) indicated that hippocampal neurons were able to adhere to and grow on the chitosan fibers, the cell morphology and the expression of neuron-specific marker proteins were normal. Observation under transmission electron microscopy showed that hippocampal neurons were adhered and grew on the chitosan fibers and the cell ultrastructures were completely normal.2,MTT assay indicated that the cell viability of hippocampal neurons in chitosan fiber extract were not significantly different from that in plain neuronal medium or hydroxyapatite extract, but significantly higher than that in organotin extract after culture for different times.3,Western analysis revealed that no significant difference in the protein level of growth-associated protein-43 andβ-tubulin III was detected between hippocampal neurons cultured in chitosan fibers extract and in plain neuronal culture medium.4,The results of inverted microscope, scanning electron microscopy and immunocytochemistry (NF-200,β-tubulin III, synaptophysin) indicated that hippocampal neurons were able to adhere to and grow on the chitosan membranes well, the cell morphology and the expression of neuron-specific marker proteins were normal. Observation under transmission electron microscopy showed that hippocampal neurons adhered and grew on the chitosan membranes and the cell ultrastructures were completely normal. The Leica Qween soft measurement revealed that no significant difference in average and total length of neuritis for hippocampal neurons cultured on the poly-lysine-coated coverslips and the substrate made up of chitosan membranes. 5,MTT assay indicated that the cell viability of hippocampal neurons in chitosan membranes extract were not significantly different from that in plain neuronal medium or hydroxyapatite extract, but significantly higher than that in organotin extract after culture for different times.6,Western analysis revealed that no significant difference in the protein level of GAP-43 andβ-tubulin III was detected between hippocampal neurons cultured in chitosan fibers extract and in plain neuronal culture medium.Conclusions1,The results collectively demonstrate that biomaterial chitosan is biocompatible to primary culture of hippocampal neurons and without any change on cell phenotype and functions.2,The hippocampal neurons cultured in chitosan material extract show no change of morphology, viability and the expression of specific proteins which are quite important in the process of neuronal growth, namely, GAP-43 andβ-tubulin III proteins. The results demonstrate that chitosan material without cytotoxic effects to primary culture of hippocampal neurons, raising a potential possibility of using chitosan for CNS therapy.