Preliminary Study For Building The Lamina Propria Of The Oral Mucosa Using Two Kinds Of Silk Fibroin Scaffold In Vitro

ObjectiveTo investigate the feasibilities of using the containing nanofiber scaffolds, saltingprepared silk fibroin scaffolds which are constructed the lamina propria of the oral mucosain vitro, and to provide experimental evidence for tissue-engineered oral mucosa in thefuture.MethodsThe SD rats’ primary oral mucosal fibroblasts (oral fibroblast cell) were cultured bytissue adherent method. The third generation of oral fibroblast cell (OFC) were identifiedwith Vimentin and stained by DiI and DAPI. The cell morphology was observed indifferent ways. The cultivated OFC was inoculated on two kinds of silk fibroin scaffolds.The cell adhesion rate was checked24hours later and the contractibility rate of those twokinds of silk fibroin scaffolds was calculated within1-7days. The DNA content wasdetected at the1st,4th,7th,10th, and14thday while observing the three-dimensionalstructure and cell proliferation using laser confocal microscopy. The different scaffoldscompounds were scanned by the environment scanning electron microscopy at the4thand7thday. The scaffolds compounds which were cultured for14days were fixed by4%formaldehyde, DAPI staining nuclei, and phalloidin detecting F-actin.ResultsThe primary fibroblast cells were observed in inverted microcopy after9days. Thecells form a long spindle, flat, and oval nuclear shape, the cells structure was complete.The dimension-positive rate of the third OFC was up to99%. DiI and DAPI staining cells were used to observe the cell morphology. The membrane showed orange-red and bluenuclei. The cell adhesion rate was (95%±1.2%) in the containing nanofibers of silkfibroin compared to (86±1.2%) in the the silk fibroin prepared by salting. At the sametime, the containing nanofibers silk fibroin’contraction was not obvious, but saltingprepared silk fibroin scaffold became slightly thicker. It was found that the cells in thecontaining nanofiber scaffolds were significantly higher than ones in the salting preparedsilk fibroin scaffold under laser scanning confocal microscopy. Over a period of time，thecells in the containing nanofiber scaffolds gradually increased, and grew internally. Somecells were obviously aggregated as a cluster. The cells in the salting prepared silk fibroincells were also gradually increased and grew internally, but showed the fewer number ofcells, and slower growth rate, and dispersed more. Scanning electron microscopy revealedthat there were a lot of small villi and folds, formed psedudopodia, surrounded by a largenumber of cell secretions in the surface of the containing nanofiber scaffolds. The cellcharacteristics in the salting prepared silk fibroin surface was not obvious, the cellsecretions was less. We could observe a large number of cord-shaped green fluorescenceand blue nuclei when the containing nanofiber scaffold compounds have been cultivatedfor14days, while Less green fluorescence in the salting prepared silk fibroin underfluorescence microscope,The blue nucleis are not obvious in both scaffolds.ConclusionBoth kinds of the silk fibroin have good biocompatibility, but the containingnanofiber scaffold is significantly better than the salting prepared silk fibroin scaffold. Butit is necessary to research more for both scaffold in tissue engineering materials field.