Investigation Of Silk Fibroin-chitosan Blend And Submandibular Glands Cell Co-culture In Vitro

ObjectiveIn order to provide the feasibility of silk fibroin-chitosan blend (SFCS) and submandibular gland cells co-culture in vitro, to building the reconstruction of submandibular gland.MethodsPrimary in vitro explant culture 3 days old SD rat submandibular gland cells (RSMG-cell), immunohistochemistry identification of cell origin, passage.The first two generation of submandibular gland cells with the concentration of 2.0×104/ml were seeded onto the 5mm×5mm×2mm SFCS in vitro, as the experimental group. While 2.0×104/ml in the first two generation of submandibular gland cells were seeded onto the culture plate in vitro, as a control group.After inoculation, respectively, 1h,2h,4h,8h,24h, determination of the experimental group and control group SD rat submandibular gland cell adhesion rate. Inverted phase contrast microscope observation of two groups periodically SD rat submandibular gland cell growth and morphological changes, and in the first 3,7 days of composite culture drawn, scanning electron microscopy the ultra structure of submandibular gland cells.Group 2 were detected by MTT SD rat submandibular gland cell proliferation. Composite culture were taken 1 day to 10 days of culture supernatant to detect amylase content, testing group 2 SD rat submandibular gland cell function. Hoechst-PI staining to detect cell viability. Using SPSS for windows 13.0 statistical analysis software on the results of two independent samples t test, test level a=0.05, P <0.05 considered significant difference. ResultThe success of the SD rat submandibular gland cells (RSMG-cell) and preparation out of silk fibroin-chitosan blend membranes culture in vitro. Experimental group was inoculated 1 h, when RSMG-cell began to attach to silk fibroin-chitosan blend membrane,4 h, when more than 60%,8 h, when more than 80% RSMG-cell adhesion, 24 h, when 90% of above RSMG-cell adhesion. In the control group RSMG-cell 2h begins to attach to culture plate,4h reached 45%,8 h, when more than 80% RSMG-cell adhesion,24 h, when 95% of above RSMG-cell adhesion.. Inverted phase contrast microscope two groups RSMG-cell are growing well, the cell morphology was no significant difference in the experimental group the first three days will see the RSMG-cell extension, cell numbers increased slightly protruding began to clear. Chapter 7 days a marked increase in cell numbers, stretching full, protruding clear that the cytoplasm of the secretory granules can be seen clearly. Submandibular gland of the three kinds of cells, namely, ductal cells, myoepithelial cells and acinar epithelial cells throughout the growth period of diverse. Scanning electron microscope composite cultured for 7 days RSMG-cell stretching in the silk fibroin-chitosan blend membraness mesh structure, showing polygonal, spindle-shaped, with multiple processes, the cell surface, there are many secretory granules, filamentous fibers and extracellular matrix. With the extension of composite culture time, cell supernatant amylase levels were increased in varying degrees. Hoechst-PI staining, see RSGCs and SF-blend films cocultured 3 days RSMG-cell nuclei showed diffuse homogeneous blue fluorescence. On day 5 cells significantly increased the amount of fluorescence, the fluorescence at day 7 cells significantly increased cell density into afilm fluorescence.9 days began to apoptosis, red fluorescence, but still visible dividing cells.ConclusionsSubmandibular gland cells and SF-blend films have good biocompatibility, submandibular gland cells in SF-chitosan film grow and proliferate well.