Preliminary Study On The Application Of Silk Fibroin Porous Scaffolds In Oral Soft Tissue Thickening

Objective:to investigate the physical properties,degradation characteristics,cellular compatibility and histocompatibility of three kinds of silk fibroin porous scaffolds with different concentrations in vitro and in vivo,and to evaluate the feasibility of the scaffolds for oral soft tissue thickening.Methods:three kinds of fibroin scaffolds with different concentrations of SF1（1wt%）,SF3（3wt%）and SF5（5wt%）were prepared by freeze-drying.1.By Scanning electron microscopy（SEM）,Fourier transform infrared spectroscopy,（FTIR）,X-ray diffraction（XRD）,Differential scanning calorimetry（DSC） characterization of materials and secondary structure was analyzed,then pore size,porosity and in vitro degradation rate of each scaffold was measured.2.Human gingival fibroblasts（HGFs）were inoculated on three kinds of scaffolds,on day 3 and day 5 of co-culture,Cell counting kit（cck-8）was used to evaluate the influence of scaffolds on the proliferation activity of HGFs during the culture cycle,and the relative Cell activity of HGFs on scaffolds in each group was ompared.3.The three groups of scaffolds were implanted underneath the oral mucosa of New Zealand white rabbits,and the general morphology of the perative area was observed after 3 months.The changes of the mucosal thickness of each group before and after the operation were measured and compared,and the thickening effect of each group of materials on the oral mucosa of rabbits was evaluated.Specimens were taken from the operative area and pathological sections were made.HE and Masson staining were used to observe the degradation of caffolds,inflammatory reaction,neovascularization and newly formed ollagen fibers,and to compare and analyze the histocompatibility of silk fibroin scaffolds in each group.Results:1.The SEM images show that the three kinds of material all have nterconnected porous structure,the pore size were 80.61±30.63μm（SF1）,133.40±22.85μm（SF3）,34.88±15.96μm（SF5）,respectively,FTIR,XRD,DSC test results showed that the secondary structures of SF1,SF3 and SF5 were similar and the main conformation was SilkⅡtype,The porosity was95.13±4.12%（SF1）,90.05±6.68%（SF3）and 86.85±5.43%（SF5）, espectively.After being immersed in PBS containing 1 UmL^-1 for 24 hours,the residual mess of the three scaffold materials was 83.55%（SF1）88.63%（SF3）and 91.84%（SF5）,respectively.2.CCK-8 results showed that from day 3 to day 5 of co-culture,HGFs on scaffolds in each group presented a proliferation rend.The relative cell activity of HGFs on scaffolds was compared（SF1>SF3>SF5）（P<0.05）.3.Compared with preoperative（T₀）,3 months after mplantation（T₂）,the implantation sites showed increased mucosal thickness（P<0.05）,and the thickening effect was as follows:SF1<SF3<SF5（P<0.05）.Compared with immediately after the surgery（T₁）,3 months after implantation（T₂）,the mucosal thickness of the SF1 group was significantly reduced（P<0.05）,while the mucosal thickness of the SF5 group was almost unchanged.HE and Masson staining results showed that the SF1 scaffold material had been ompletely degraded 3 months after the surgery,with no inflammatory reaction observed.In the SF3 group,only a small amount of scaffold fragments was bserved,with a small number of inflammatory cells around the fragments,a arge amount of new blood vessels and collagen fibers were observed.Most of the scaffolds in SF5 group were still not degraded,and the scaffold was surrounded by newly formed collagen fibers and large number of inflammatory cells could be seen in these fibers.Conclusion:the results of physical properties test and cell proliferation activity test showed that all the three materials had the potential to be used as tissue engineering scaffolds,however,the results of animal experiments showed that SF1 had poor olume stability after implantation thus failed to play a good role as scaffolds. lthough SF5 has the most obvious thickening effect on mucosa,the foreign body reaction is evident and the histocompatibility is poor.While SF3 showed atisfactory thickening effect as well as good histocompatibility within 3 months.Therefore,SF3 silk fibroin porous scaffold may be a more ideal material for oral soft tissue thickening.