The Preliminary Research Of Application Of Chinese Medicine Quercetin On Bone Defects Regeneration

Objectives: Present study focuses on the effects of Chinese medicine quercetin on bone defects regeneration.We explored the biological effects and underlying mechanism of quercetin on normal and ovariectomized(OVX)rat bone marrow-derived mesenchymal stem cells(r BMSCs).Moreover,the hydroxyapatite bioceramic microspheres with a micro-nano-hybrid surface(n HA bioceramic microspheres)was fabricated to build the quercetin delivery system,and the bone regeneration effect in vivo was evalusted in an OVX rat critical-sized femur defect model.Materials and methods: 1.The effects of quercetin on the proliferation,viability,cellular morphology,osteogenic differentiation and angiogenic factor secretion of r BMSCs were examined by MTT assay,fluorescence activated cell sorter(FACS)analysis,acting staining,Real-time PCR analysis,alkaline phosphatase(ALP)activity and calcium deposition assays,and Enzyme-linked immunosorbent assay(ELISA),respectively,and the optimum drug concentration was chose.2.Whether mitogen-activated protein kinase(MAPK)signaling pathways were involved in the effects of quercetin on r BMSCs was also explored by Western-blot,ALP and Real-time PCR.3.The effects of quercetin on proliferation,osteogenic differentiation and angiogenic factor secretion as well as on the osteoclastogenic factor secretion of OVX r BMSCs were evaluated by MTT assay,Real-time PCR analysis,ALP activity and Western blot,respectively,and the optimum drug concentration was chose.4.Whether MAPK signaling pathways and the protein kinase B(AKT)signaling pathway were involved in the effects of quercetin on OVX r BMSCs was also explored by Western-blot,ALP and Real-time PCR.5.The n HA bioceramic microspheres were fabricated via the hydrothermal transformation method,then the surface and porosity of n HA bioceramic microspheres were evaluated.Vacuum freeze-drying technology was used to fabricate the drug delivery system.And the release property was evaluated.6.The ability to of n HA/quercetin induce osteogenesis,mineralization and angiogenesis in vivo was evaluated in an OVX rat critical-sized femur defect model by Micro-CT,sequential fluorescent labeling and histological assay,respectively.Results: 1.Quercetin significantly enhanced the cell proliferation,osteogenic differentiation and angiogenic factor secretion without effects on cell viability and cellular morphology of r BMSCs in a dose-dependent manner,with a concentration of 2 ?M achieving the greatest stimulatory effect.2.The activation of the ERK and p38 pathways was observed in quercetin-treated r BMSCs,and there was crosstalk between the two signaling pathways.3.Quercetin significantly enhanced the cell proliferation,osteogenic differentiation and angiogenic factor secretion of OVX r BMSCs as well as inhibited the expression of osteoclastogenic factor in a dose-dependent manner,with a concentration of 1 ?M achieving the greatest stimulatory effect.4.The activation of the ERK,p38 and AKT pathways was observed in quercetin-treated OVX r BMSCs,and there was crosstalk among these signaling pathways.5.The n HA bioceramic microspheres had good porosity and release property.6.In the OVX rat critical-sized femur defect model,bone mineral density(BMD),trabecular thickness(Tb.Th),sequence fluorescent labeling area and new bone area for the n HA/quercetin was higher than those for n HA.Conclusions: 1.Quercetin could promote the proliferation,osteogenic differentiation and angiogenic factor secretion of r BMSCs in vitro,and a concentration of 2 ?M achieving the greatest stimulatory effect,partially through the ERK and p38 signaling pathways.2.Quercetin could promote the proliferation,osteogenic differentiation and angiogenic factor secretion of OVX r BMSCs as well as inhibit the osteoclastogenic factor secretion in vitro,and a concentration of 1 ?M achieving the greatest stimulatory effect,partially through the ERK,p38 and AKT signaling pathways.3.The n HA bioceramic microspheres could be used to fabricate the quercetin delivery system,which could promote new bone formation and blood vessel formation in vivo.