Preliminary Research For Constructing Tissue-engineered Oral Mucosa Using Silk Fibroin Scaffold

Objective: To evaluate the biocompatibility and histocompatibility of different aperture (200~300μm and 10~20μm) silk fibroin scaffold (SF), to investigate the feasibilities of silk fibroin as scaffold for tissue-engineered oral mucosa and to provide experimental basis for the futher development of tissue-engineered oral mucosa.Methods: In vitro experiments– the SD rat's oral fibroblast cells (OFC) were cultured by tissue adherent method. Third generations of cells were identified by waveform protein and were used as seed cells. The cultivated OFC was inoculated on different aperture silk fibroin scaffolds and culture plates, then cells were observed daily using inverted microscope. The silk fibroin-oral fibroblasts (SF-OFC) fixed by 4% paraformaldehyde and 2.5% glutaral were stained by HE histological staining. We observe the cells'growth and proliferation using inverted microscope and scanning electron microscopy (SEM) and count the cells of SF-OFC by MTT test, to evaluate the silk fibroin-cell compatibility. In vivo experiments - different aperture silk fibroin scaffolds were implanted submucosa of male SD rats in oral and also set up a blank control group simultaneously. We observe the operative areas at 1, 2, 3 and 4 week in general observation. The silk fibroin-mucosa complexes were fixed for HE, masson and CD34 histological staining, to evaluate the silk fibroin-tissue compatibility. All the data were analyzed by SPSS 11.5 software package.Results: In vitro experiments - we successfully obtained the SD rat's oral fibroblast cells (OFC) by tissue adherent method. The cells were identified as positive after vimentin antibody staining. We observed the cells could attach, extend and proliferate normally on the silk fibroin using inverted microscope and scanning electron microscopy. HE-stained sections of SF-OFC showed the individual cells or cell colonies along the silk fibroin scaffold. MTT test showed that the silk fibroin is non-toxic and suitable for cell growth. All the results showed the silk fibroin has good biocompatibility, and the large aperture silk fibroin is better than the small one. In vivo experiments– the SD rats were in good condition after surgery. The mucosa implanted silk fibroin scaffold was higher than the surrounding area. The operative region is no significant degeneration or necrosis. The operative incision is primary healing in each group. On HE-stained sections, the number of the oral fibroblasts and inflammatory cells within the scaffolds were determined; secretion of collagen was observed by masson staining; the neovascularization were detected by CD34 immunohistochemistry. The three results indicated that silk fibroin has good histocompatibility, and the large aperture group is better than the small one.Conclusion: The experiment has proved that porous silk fibroin has good biocompatibility. The large aperture group-3D silk fibroin scaffolds are better for future applications in oral surgery. The results provide promising experimental basis for further constructing the tissue-engineered oral mucosa.