Preparation Of Metformin Sustained Release Coating And Its Regulatory Effect On Osteogenic Differentiation Of PDLSCs Under High Glucose Condition

Objective: Periodontitis is a chronic inflammatory disease that occurs in the supporting structure of teeth(about 10% of adults have advanced periodontitis),and can lead to tooth loosening and loss.Diabetes is the main risk factor of periodontitis,which leads to the increase of the prevalence and severity of periodontitis.Due to the oxidative stress induced by hyperglycemia in diabetic state impairs the regeneration function of human periodontal ligament stem cells(h PDLSCs),the clinical application of traditional transplantation materials and barrier membranes in periodontal regeneration of diabetic patients is difficult to achieve satisfactory results.In addition,due to the increase of Enterobacteriaceae,Enterococcus,Staphylococcus and other pathogens in the oral cavity of diabetic patients,the postoperative infection is serious,which eventually leads to the failure of regenerative therapy.Therefore,in order to overcome these problems,it is urgent to design antioxidant stress and antibacterial surfaces on biomaterials for diabetes.Methods: 1.Construction and characterization of metformin loaded self-assembled sustained-release coating: Firstly,the surface of polystyrene(PS)was pretreated with polydopamine,and then the antibacterial polymer carboxymethyl chitosan(CMC,negative polyelectrolyte)and polylysine(PLL,positive polyelectrolyte)were used as building units to establish layer by layer self-assembled functional coating,and genipin was used as crosslinking agent to further crosslink the monolayers.The surface morphology of the coating was observed by field emission scanning electron microscope(FE-SEM),and the chemical composition was detected by X-ray photoelectron spectroscopy(XPS).The above physical and chemical properties characterization were used to evaluate whether PLL/CMC coating is successfully modified on PS surface.2.In vitro cytocompatibility evaluation of metformin loaded self-assembled sustained-release coating: The early adhesion morphology of h PDLSCs on the coating surfaces was evaluated by living cell staining,cytoskeleton staining and focal adhesion protein staining,and the cell proliferation was detected by CCK-8,and the cytocompatibility of functional modified coating was further evaluated by LDH detection and TUNEL apoptosis experiment.3.Evaluation of the protective effect of metformin loaded self-assembled sustained-release coating on h PDLSCs injured by high glucose: After h PDLSCs were stimulated with high glucose,ROS quantitative,qualitative detection of ALP and calcium content were detected,RT-q PCR was used to detect the expression of Runx2,ALP and OCN,and immunofluorescence staining was used to detect the expression of OPN and COL1A1,so as to evaluate whether metformin released from the functionalized coating can rescue oxidative stress and osteogenic damage of h PDLSCs under high glucose condition.4.In vitro antibacterial performance evaluation of self-assembled sustained-release coating loaded with metformin: the number of bacteria(E.coli,S.aureus,S.mutans)adhered to the coating surface after 4 h of culture was quantitatively detected by microbial activity.And the living bacteria after 4 h of culture were observed by fluorescence staining to evaluate the antibacterial performance of functionalized modified surface.Results: 1.SEM results showed that the surface roughness of the coating was improved after loading,which proved the loading effect of the coating.XPS results showed that the content of N1 s and O1 s increased and the content of C1 s decreased after loading PLL/CMC,indicating the successful coating;meanwhile,compared with PS-PLL/CMC group,the nitrogen carbon ratio of PS-PLL/CMC/Met group further increased,and the analysis results of nitrogen spectrum and carbon spectrum all proved the successful loading of metformin.In vitro sustained release experiments showed that the coating could achieve slow and sustained drug release.2.The results of living cell staining showed that the number of cells detected in PS-PLL/CMC/Met group was close to that in PS group,indicating that metformin sustained-release coating had good cell affinity.Cytoskeleton staining and focal adhesion protein staining showed that,compared with PS-PLL/CMC group,PS-PLL/CMC/Met group had similar healthy spreading morphology as PS group,suggesting that metformin may improve the inflammatory effect of the coating.CCK-8 test showed that the OD value of each group increased with time,indicating that the coating could promote the normal growth of h PDLSCs.The results of TUNEL staining and LDH test further confirmed that the coating had good biocompatibility.3.ROS detection showed that the production of ROS in h PDLSCs was significantly increased after high glucose stimulation,while the production of ROS was decreased after loading metformin.ALP activity assay showed that high glucose stimulation decreased ALP activity of h PDLSCs,but ALP activity increased after drug loading.The trend of alizarin red staining was consistent with ALP evaluation.RT-q PCR showed that the expression of osteogenic related genes(Runx2,ALP and OCN)was down regulated by high glucose stimulation,and the immunofluorescence staining of OPN and Col1a1 showed the same results.The osteogenic related genes in PS-PLL/CMC/Met group were significantly increased compared with the other two groups.4.The results of microbial activity quantitative test showed that in the initial adhesion stage(4 h),the number of three kinds of bacteria detected in PS-PLL/CMC group and PS-PLL/CMC/Met group decreased significantly compared with PS group.The results of live bacteria staining showed that a large number of bacteria survived in PS group,while only a small number of live bacteria were detected in PS-PLL/CMC and PS-PLL/CMC/Met groups,which was consistent with the quantitative results,suggesting that PLL/CMC can significantly inhibit the adhesion and proliferation of periodontal pathogens.Conclusions: 1.In this study,layer by layer self-assembly technology,polydopamine covalent grafting and genipin biological crosslinking were used to construct the self-assembled sustained-release coating of PLL/CMC loaded with metformin.Drug release experiments showed that the PLL/CMC coating could release metformin continuously in vitro.2.Metformin loaded self-assembled sustained-release coating has good cell compatibility,can promote the normal proliferation of h PDLSCs,and metformin has a positive effect on cell spreading.3.Metformin loaded self-assembled sustained-release coating can enhance the antioxidant capacity of h PDLSCs under high glucose condition,and can restore the high glucose induced osteogenic damage.4.Metformin loaded self-assembled sustained-release coating has excellent antibacterial activity,and the colonization and growth of periodontal disease-related pathogens on the coating surface are inhibited.