Study On The Effects Of Gold Nanocomposites On Osteogenic Differentiation And Osteo-immunomodulation And The Correlated Mechanisms

Part Ⅰ Synthesis,characterization and cytocompatibility of gold nanoparticles composites Objective: In this study,gold nanocomposites were designed and their effects on cell viability and proliferation were evaluated.Methods: The gold nanocomposites were synthesized and characterized by several analytical techniques(XRD,FT-IR,XPS).Cell viability and proliferation were characterized by CCK-8 test.Results: The results confirmed the successful synthesis of gold nanocomposites.The gold nanocomposites showed good cytocompatibility at the studied concentrations.Conclusion: The gold nanocomposites were synthesized and exerted good cytocompatibility.Part Ⅱ Gold nanoparticles-loaded hydroxyapatite composites guide osteogenic differentiation of human mesenchymal stem cells Objective: In this study,gold nanoparticles(Au NPs)-loaded hydroxyapatite(Au-HA) nanocomposites were designed to guide the osteogenic differentiation of human bone marrowderived mesenchymal stem cells(hMSCs)through the synergistic effects of both Au NPs and HA.Methods: The Au-HA nanoparticles were synthesized and characterized by several analytical techniques(XRD,FT-IR,XPS).Cell viability and proliferation of hMSCs were characterized by CCK-8 test.Cellular uptake of nanoparticles was observed by transmission electron microscope.For the evaluation of osteogenic differentiation,alkaline phosphatase(ALP)staining and activity,Alizarin red staining,and a quantitative real-time polymerase chain reaction(RT-PCR)analysis were performed.In order to examine specific signaling pathways,RT-PCR and Western blotting assay were performed.Results: The results confirmed the successful synthesis of Au-HA nanocomposites.The Au-HA nanoparticles showed good cytocompatibility and internalized into hMSCs at the studied concentrations.The increased level of ALP production,deposition of calcium mineralization,as well as the expression of typical osteogenic genes,indicated the enhancement of osteogenic differentiation of hMSCs.Moreover,the incorporation of Au could activate the Wnt/β-catenin signaling pathway,which seemed to be the molecular mechanism underlying the osteoinductive capability of Au-HA nanoparticles.Conclusion: The Au-HA nanoparticles exerted a synergistic effect on accelerating osteogenic differentiation of hMSCs,suggesting they may be potential candidates for bone repair and regeneration.Part Ⅲ Accelerated Bone Regeneration by Gold-Nanoparticle-Loaded Mesoporous Silica through Stimulating Immunomodulation Objective: In this study,we investigated the modulatory effects of gold nanoparticle(Au NP)-loaded mesoporous silica nanoparticles(Au-MSNs)on macrophages and the subsequent effects on the behavior of osteoblastic lineage cells.Methods: The Au-MSNs nanoparticles were synthesized and characterized by several analytical techniques(XRD,FT-IR,XPS).Cell viability and proliferation of RAW 264.7 cells were characterized by CCK-8 test and flow cytometry.Macrophage polarization and inflammatory response were evaluated by RT-PCT and ELISA.Morphology and proliferation of preosteoblastic cells were evaluated by immunofluorescence and CCK-8 assay.For the evaluation of osteogenic differentiation,ALP activity and staining,ARS,RT-PCR analysis and Western blot were performed.In order to examine bone regeneration in vivo,RT-PCR and Western blotting assay were performed.Results: The results demonstrate that Au-MSNs nanoparticles showed good cytocompatibility and could generate a favorable immune microenvironment by stimulating an anti-inflammatory response and promoting the secretion of osteogenic cytokines by macrophages.As a result,there is an enhancement of osteogenic differentiation in preosteoblastic MC3T3 cells as assessed by the increased expression of osteogenic markers,alkaline phosphatase(ALP)production,and calcium deposition.The immunomodulatory effects and direct osteogenic stimulation by Au-MSNs synergistically increased the osteogenic differentiation capability of MC3T3 cells as a result of crosstalk between Au-MSNconditioned macrophages and Au-MSN-treated osteoblasts in a coculture system.An in vivo study further revealed that Au-MSNs could accelerate new bone formation in a critical-sized cranial defect site in rats based on computed tomography analysis and histological examination.Conclusion: This novel Au-MSNs could significantly promote osteogenic activity by modulating the immune microenvironment,showing its therapeutic potential for bone tissue repair and regeneration.