| Objective:To study the feasibility of tissue engineered mucosa lamina propria with silk fibroin scaffold repairing buccal mucosa defect.Methods :1. Rat oral mucosa fibroblast cells separated from homogenized plasma were collected as single cell suspension for adherent culture. The cells were identified by vimentin at the third subculture and inoculated porous silk fibroin scaffold with pore size 265μm after fluorescent staining. Checked the adhesion rate 24 hour later. Cell adhesion and proliferation were inspected by laser scanning confocal microscope at day 1,4,7 repectively. Scaffold compound were scanned by electron microscopy at day 4 and 7.2. 48 Male SD rats of clean grade were divided into 3groups randomly. Group A was experimental group, n=16,with buccal mucosa circular defect (diameter 10 mm) repaired by tissue engineered mucosa lamina propira . Group B was control group, n=16,with buccal mocusa circular defect repaired only the silk fibroin scaffolds. Group C was negative control ,n=16,Vaseline gauze was used to cover the circular defect. We observed the wound healing ,diameter of the defect changing and examinated the wound tissue by histology and immunohistochemistry were studied at day 14,21 and 28 after operation.Results:1.The culture of the oral mucosa fibroblast cells:①The morphology of the cells were under light microscope with a good dispersion and rapid proliferation. The vimention-positive rate of the third subculture was 99%. The cell adherent rate in the composite was (98.77±1.34)% after 24 hours culture.②The first day after inoculation into the silk fibroin scaffold the cells appeared to scattered along the scaffold pore and the edges in single as round or oval under confocal laser microscopy. 4 days after inoculation the cells number increased and fused together along the scaffold.7 days after inoculation the cells covered more than half of the scaffold surface in spindle and oval shape.③scanning electron microscopy showed that 1 to 4 days after inoculation,the cells spread on the silk fibroin scaffold in spindle or polygon shape. 7 days after inoculation the cells appeared to fine villi and fold, formed psedudopodia like duck web surrounded the fibers of the scaffolds, there were matrix deposited in cell plasma, cells increased in number.2.Wound Examination:①Wound observation: postoperative wound Diameter shrinkage rate was group C>group B> group A.7 days after operation the wound of group A and group B were covered with yellow tayet,the wound of group C with covered light yellow pseadoscorpionidea.②Histology examination showed:the inflammatory cells in group C were much more than in group A and group B at every stage.③immunohistochemistry examination:7 days after operation newly form broad- spectrum cytokeration were positive in all three groups.The Epithelial layers in group A were more than in group B and group C, group C was less but with thick spikes.The epithelium cells in group A and group B were like those in normal mucosa. Epithelization degree in group A was higher than in group B;the thickness of epithelium layer in group C were not even and with much inflammatory cells under that. CD31 stain:the newly formed capillary vessels in group A was much more than in group B and group C ,and even distributed.Conclusion:1.Rat oral mucosa fibroblast could adhere,grow and proliferat to the silk fibroin scaffolds with 265μm aperture and could be edgineered as oral mucosa lamina propria, these can serve as prosthesis for oral mucosa defect. 2.The tissue engineered rat oral mucosa lamina propria can significantly promote the growth of oral epithelial,promote wound healing and inhibit contraction of the wound and can reduce scar contracture.3.It is feasible to use tissue engineered mucosa lamina propria with silk fibroin scaffold for buccal mucosa defect repair,it has certin clinical value. |
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