Experimental Study Of BSA-coated Cerium Oxide Nanoparticles For Periodontal Bone Regeneration

Objective BSA incubation was used to synthesize cerium oxide nanoparticles（CeO₂@BSA）.We investigated the role of CeO₂@BSA in promoting osteogenic differentiation of human periodontal ligament cells（hPDLCs）,and analyzed the possible molecular mechanism by transcriptome sequencing.We also observed the application of CeO₂@BSA in the repair of periodontal bone defects in SD rats.Methods 1.Primary hPDLCs were isolated and cultured using the tissue block method.CeO₂@BSA was prepared by the BSA incubation method,and the products were assessed by TEM,XRD and DLS.hPDLCs were treated with CeO₂@BSA at different concentrations（0μg/m L,10μg/m L,50μg/m L,100μg/m L,200μg/m L）.After 7 days,cell morphology was observed and live-dead staining was performed.Cell viability was detected by the CCK-8 method at 1,3 and 7 days.2.hPDLCs were treated with different concentrations of CeO₂@BSA.Alkaline phosphatase（ALP）staining and activity assay were performed to determine the changes of ALP activity in different concentrations of CeO₂@BSA.Alizarin red（ARS）staining and quantitative analysis were assessed mineralized nodules in different concentration groups of cells.By performing qPCR and western blot,the mRNA and protein levels（OCN,Col-1,ALP）were detected.hPDLCs were co-cultured with CeO₂@BSA for 7 days and transcriptional sequencing was performed to detect the mechanisms involved at the molecular level.3.The possible mechanism of CeO₂@BSA in the induction of osteogenic process in hPDLCs was explored using the autophagy inhibitor 3-MA.The cells were divided into:1)10μg/m L CeO₂@BSA-treated group;2)10μg/m L CeO₂@BSA+3-MA-treated group.ALP,ARS,qPCR and western blot results of each group were observed separately.4.Tissue-engineered membranes were first constructed and cell adhesion was observed by SEM.A 5 mm x 2 mm x 1 mm defect was made in the mandibular alveolar bone of SD rat using a dental handpiece slow drill.Sixteen SD male rats were randomly divided into two groups:group A with cell-loaded engineered membranes and group B with engineered membranes pre-treated with CeO₂@BSA.The animals were executed after 4 weeks,and the samples were fixed for micro-CT to detect the reconstruction of the defect.Subsequent decalcification was completed and sections were stained for HE,Masson and immunohistochemistry.Results 1.The synthesis of CeO₂@BSA nanoparticles by BSA incubation method was characterized by XRD,TEM and DLS,which showed that the synthesized products had good phenotypes.When the cells were cultured to P3-P5,subsequent experiments were performed.After 7 days of culture at different concentrations,cell morphology and live-dead staining showed no significant cytotoxicity of CeO₂@BSA,which was further confirmed by the CCK-8 results as having good biocompatibility.2.ALP activity showed the best osteogenic effect at low concentration state（10μg/m L,50μg/m L）.ARS staining and quantification results showed the same trend as ALP activity.Compared with OS group,m RNAs and proteins（OCN,ALP,Col-1）expression were significantly higher in 10μg/m L and 50μg/m L CeO₂@BSA groups.In contrast,the osteogenic ability of hPDLCs was inhibited at high CeO₂@BSA concentrations（100μg/m L,200μg/m L）.Transcriptome sequencing and validation of the results showed that the possible mechanism of promoting osteogenesis of hPDLCs by low concentration of CeO₂@BSA was mediated through TGF-βsignaling pathway.Meanwhile,autophagy-related genes were significantly upregulated.3.qPCR and western blot results showed that levels of LC3 and Beclin-1（autophagy key genes）and proteins（LC3-II and p62）were significantly changed.Also,after autophagy was blocked,ALP,ARS staining,qPCR and western blot results showed that the osteogenic ability of CeO₂@BSA was significantly inhibited.4.SEM showed that the cells were completely covered on the surface of the tissue-engineered membrane after 7 days.After 4 weeks of membrane implantation,micro-CT showed that bone tissue repair in the CeO₂@BSA group was superior to that in the control group.The analysis of quantitative results of various bone parameters confirmed the good bone repair ability of CeO₂@BSA.HE and Masson staining results further confirmed the repair ability of the CeO₂@BSA group.IHC results also showed that expressions of osteogenic and autophagy-related proteins were higher in the CeO₂@BSA group than in the control group.Conclusion CeO₂@BSA has good biocompatibility and promotes the osteogenic differentiation of hPDLCs at low concentrations,and the related mechanism may be regulated by the TGF-βsignaling pathway.Meanwhile,it was able to activate the autophagic process of hPDLCs,and the high concentration of CeO₂@BSA led to the over-activation of autophagy,which inhibited their osteogenic ability.It can be concluded that CeO₂@BSA may promote the osteo-differentiation of hPDLCs through the autophagy and TGF-βsignaling pathway.The in vivo experiments further confirmed that CeO₂@BSA nanoparticles could promote the repair of periodontal bone defects in SD rats.