Platelet-rich Fibrin Inhibits Myofibroblast Transition During Rabbit Schneiderian Membrane Repairing

Objective:To explore the effects and mechanisms of PRF on collagen deposition in rabbit Schneiderian membrane repairing in vivo and in vitro.Methods:1.Five Japanese white rabbits were selected to build the Schneiderian membrane perforation model on both sides of nasal bone.The right side was defined as the PRF treated side and the left side as the control side.2.The number of serous glands in perforated Schneiderian membranes on both sides were compared by H＆E staining 14 days postoperation.3.The collagen deposition in the Schneiderian membranes on both sides were analyzed by Masson trichrome staining 14 days postoperation.4.Primary rabbit Schneiderian membrane-derived cells（rSMDCs）were isolated and cultured.5.RSMDCs were identified by cell immunofluorescence staining.6.PRF extract was prepared and the morphological changes of rSMDCs cultured with PRF extract were observed for 0 h,12 h and 24 h under microscope.7.CCK-8 was used to detect the proliferation of rSMDCs cultured with PRF extract for 24 h,48 h and 72 h.8.Cell scratch test was applied to evaluate the effect of PRF extract on the migration property of r SMDCs.9.The mRNA levels of markers of fibroblast-myofibroblast transition（FMT）（E-cadherin,vimentin,N-cadherin,collagen I,α-SMA）were evaluated by qRT-PCR.10.The protein expressions of FMT markers（E-cadherin,vimentin,collagen I α-SMA）,and TGF-β1/ Smad signaling pathway markers（pSmad2/3,Smad2/3,Smad7）were evaluated by western blot.11.The results were analyzed by Graphpad prism 9 software,and the differences between groups were evaluated by one-way ANOVA and Tukey test,with P < 0.05 as statistical difference.Results:1.The operations were completed by one doctor.There was no death and no infection of experimental animals.The thickness of rabbit PRF membranes were basically equal.2.H＆E staining showed that the numbers of serous glands in perforated Schneiderian membranes in PRF-treated group were significantly higher than that in control group（P < 0.001）3.Masson trichrome staining showed that the collagen deposition in the lamina propria of the perforated rabbit Schneiderian membranes in the PRF-treated group was more localized than that in the control group（P <0.05）,which was widely distributed around the lamina propria.4.After one week of primary cell culture,the r SMDCs around tissue samples could be seen,which was either spindle or angular.With the passage of rSMDCs,the proportion of spindle cells gradually increased,and the morphology was almost the same from passage 3 to passage 5.5.Cell immunofluorescence staining showed that almost all cells were vimentin positive and cytokeratin 7 negative,indicating that they were mesenchymal rather than epithelial cells.6.After treatment with PRF extract for 24 hours,no significant changes in the morphology of r SMDCs were observed under microscope.7.The results of CCK-8 showed that both "3d PRF" and "7d PRF" conditional medium could effectively promote the proliferation of rSMDCs（P < 0.001）,of which "3d PRF" conditional medium showed better performance.8.Cell scratch test showed that both "3d PRF" and "7d PRF" conditional medium could effectively promote the migration of rSMDCs（P < 0.01）,and the migration distance of cells in "3d PRF" conditional medium group was larger（No significant difference with "7d PRF" group however）.9.qRT-PCR results showed that rSMDCs treated with "3d PRF" conditional medium had significantly lower transcription levels of mesenchymal markers（vimentin and N-cadherin）and higher transcription levels of epithelial markers（E-cadherin）.In recombinant TGF-β1-induced FMT of r SMDCs,the transcription levels of α-SMA and collagen I were significantly up-regulated（P < 0.05,P < 0.001）,while the "3d PRF" conditioneal medium could significantly reverse the trends of these genes（P < 0.01,P < 0.001）.10.Western blot showed a similar expression trend as qRT-PCR,rSMDCs treated with "3d PRF" conditional medium showed higher expression level of epithelial marker E-cadherin and lower expression levels of α-SMA and collagen I.Conclusion:Platelet-rich fibrin can effectively repair rabbit Schneiderian membrane perforation without excessive fibrosis,which may be due to the inhibition of TGF-β1-induced FMT.