Study On The Changes And Effects Of Oral Microflora In The Repair Of Mandibular Defects With SPION Scaffold

Objective: To study the changes of oral microflora during the repair of palate-bone defect in SD rats with Superparamagnetic Iron Oxide Nanoparticle(SPION)modified3D-printed PLGA scaffold and its possible effects on the repair of the defect.Method: PLGA scaffolds were prepared by 3D-printing of poly(lactic co-glycolic acid)copolymer(PLGA)by Direct Ink Writing(DIW)technique,and SPION scaffolds were modified by layer-by-layer self-assembly(LBL).The above two kinds of scaffold were observed by Scanning Electron Microscope(SEM),Energy Dispersive Spectrometer(EDS)and Transmission Electron Microscope(TEM).In vivo experiments,bone defects with a diameter of 3mm were prepared at the middle suture of the palatal bone of SD rats,and SPION scaffolds were implanted into the defects.PLGA scaffold implantation and no scaffold material were used as controls.Two weeks after surgery,microflora samples near the operation area were collected and sent to BGI for 16 S r RNA gene sequencing to detect the microflora of the palate in each group.The rats were sacrificed 4 weeks after surgery,and the effect of the scaffold on the repair of the palatal defect was studied by imaging and histological examination.To investigate the effects of SPION scaffold on serum iron level markers and bone metabolism level markers in rats.Blood was collected from the rats before death.Serum Iron(SI),Hepcidin and Procollagen type 1 N-terminal Propeptide(P1NP)were determined by Enzyme Linked Immunosorbent Assay(ELISA).In order to explore the possible reasons for the above bacterial flora results,characteristic bacteria significantly reduced in the SPION scaffold group compared with the other two groups were selected and co-cultured with SPION to observe the influence of SPION on bacterial growth.SEM was used to observe the influence of SPION on the micromorphology of the cultured bacteria.Results: SEM results showed that the fibers of PLGA scaffold and SPION scaffold were arranged regularly,and the surface of SPION scaffold was rougher.EDS results showed that there was iron on the surface of SPION scaffold.TEM results showed that there was a continuous black thin layer on the surface of SPION scaffold,which indicated the successful preparation of SPION scaffold.Imaging and histological results showed that the PLGA scaffold group had a better effect on bone regeneration than the blank control group(p<0.05).Compared with PLGA scaffold group,SPION scaffold group significantly enhanced the repair of bone defects(p<0.05).ELISA results showed that SPION scaffold group had higher serum iron content,faster bone metabolism,and played a role in promoting osteogenesis.The results of 16 S r RNA test showed that SPION scaffold group and blank control group had the largest difference in flora species composition.At the gate level,Proteobacteria was most common in the SPION scaffold group,but significantly decreased in the blank control group.Firmicutes was the highest in the blank control group and significantly decreased in the SPION scaffold group.The relative abundances of these two types of bacteria in the PLGA scaffold group were between the two groups.At the genus level,Gammaproteobacteria was the most common in the SPION scaffold group,but significantly decreased in the blank control group.Clostridia was the highest in the blank control group and significantly decreased in the SPION scaffold group.The relative abundance of both types of bacteria in the PLGA scaffold group was between the two groups.Compared with the other two groups,the diversity of bacteria in the SPION scaffold group was not significantly changed,but the richness was decreased.In the bacterial experiment,Clostridia spp.was selected as the experimental strain.The results showed that SPION had an inhibitory effect on the growth of Clostridia spp.The SEM results showed that SPION could destroy the cell wall of the bacteria.Conclusion: The implantation of SPION scaffold can promote the repair and regeneration of palate-bone defect,and the changed oral may be beneficial to the repair of bone defect by SPION scaffold.