The Effect Of Zoledronate On The Osteoblasts Of Osteoporosis Rats

The decreasing estrogen level in postmenopausal women would lead to postmenopausal osteoporosis（PMO）,and bisphosphonate（BPs）is widely used in the treatment of PMO.Bisphosphonate-related osteonecrosis of the jaws（BRONJ）is the severe complication of this medicine,but the pathophysiology mechanism of this complication has not been illustrated clearly.Periodontitis can lead to the resorption of alveolar ridge and cause tooth loosening and loss.Recent researches have indicated that periodontitis is the hazard of BRONJ,but the detailed effect of periodontitis on the development of BRONJ remains to be elucidated.ObjectiveOsteoblasts of the mandible（MOBs）and femur（FOBs）were isolated from rats in vitro to compare the variations of biological behaviors between osteoblasts from different embryonic layers.Then postmenopausal osteoporosis（PMO）rat model was established to isolate and culture MOBs and FOBs from PMO rats,and zoledronate（ZOL）was used to stimulate with cells to compare the effect of ZOL on biological behaviors of different embryonic layers osteoblasts under PMO condition.MOBs from PMO rats culturing with ZOL and Porphyromonas gingivalis lipopolysaccharide（P.gingivalis LPS）,expression levels of osteogenic-,osteoclastic-and angiogenic-related genes were detected to investigate the effect of ZOL and P.gingivalis LPS on MOBs of PMO rats.Methods1.Isolating and culturing MOBs and FOBs of normal rats,Flow Cytometric was performed to identify the cell surface markers and cell counting kit-8（CCK-8）assay was used for detecting proliferation ability.2.Culturing with osteogenic-inducing medium,alkaline phosphatase（ALP）staining and activity,alizarin red S staining and mineralization quantification,real-time fluorescent quantitative polymerase chain reaction（Real-time PCR）for osteogenic gene expressions were conducted to compare the osteogenesis capability between MOBs and FOBs.3.Real-time PCR and enzyme-linked immunosorbent assay（ELISA）were performed to detect the expression level of angiogenic factors in MOBs and FOB.4.The expression of angiogenic genes and proteins of human umbilical vein endothelial cells（HUVECs）co-cultured with conditioned media from MOBs and FOBs were evaluated by Real-time PCR and Western Blot,then CCK-8,wound-healing,Transwell and in vitro Matrigel tube formation assays were conducted to compare the relative functions of HUVECs cultured with the conditioned media.5.PMO rat model was established by bilateral ovariectomy,MOBs and FOBs were isolated and ZOL was used to stimulated with the cells to compare their proliferative ability and expression of osteogenesis,osteoclastogenesis and angiogenesis genes.6.MOBs of PMO rats were cultured with ZOL and P.gingivalis LPS,and Real-time PCR was conducted to detect the expression level of osteogenic-,osteoclastic-and angiogenic-related genes.Results1.There were no significant differences in morphology of rats MOBs and FOBs,and they were all negative for CD31 and CD45,but MOBs showed stronger proliferation ability than FOBs.2.Culturing with osteogenic-inducing media,FOBs displayed stronger ALP activity and mineralization nodules formation ability and expressed more osteogenic genes of Alpl,Runt-related transcription factor 2（Runx2）,osteocalcin（Oc）and bone sialoprotein（Bsp）.3.The results of Real-time PCR and ELISA indicated that MOBs produced more angiogenic factors of angiopontin-1（ANGPT1）and fibroblast growth factor 2（FGF2）,and fewer vascular endothelial growth factor（VEGF）,but FOBs expressed more chemokine（C-X-C motif）ligand 9（CXCL9）.4.Compared to culturing with FOBs conditioned media,the group of HUVECs culturing with MOBs conditioned media could produce more angiogenesis factors of hypoxia-inducible factor 1 alpha（HIF1A）,endothelial nitric oxide synthase（eNOS）,kinase domain region（KDR）,endothelial-specific receptor tyrosine kinase 2（TIE2）,and showed stronger proliferation,migration and tube formation capability.5.ZOL could inhibit the proliferation of MOBs and FOBs from PMO rats in a dose-dependent manner,and MOBs presented more sensitive than FOBs to the stimulation of ZOL with weaker osteogenic genes expression and increase in expression of osteoclastic and angiogenic genes.6.PMO rats MOBs culturing with P.gingivalis LPS and 10^-5 M showed more significantly decrease in osteogenesis-related genes,while obviously stronger osteoclastic and angiogenic genes expressions.Conclusions1.There were skeletal-site specific differences in osteoblasts from different embryonic layers,MOBs possessed stronger proliferation and angiogenesis ability,while FOBs showed excellence in osteogenesis.2.MOBs from PMO rats were more sensitive than FOBs when stimulated with ZOL.3.P.gingivalis LPS could aggravate the effect of ZOL on MOBs of PMO rats with the result that the osteogenesis of osteoblasts was further inhibited,while the regulation on osteoclastogenesis and angiogenesis were promoted more significantly.