| Objective:The aim of present study was to investigate the osteogenesis and angiogenesis effects of strontium ranelate(SrR)by local delivery method,to comprehensively explore the gene targets that it works on,and to preliminary assess the thrombosis risk of this drug,to give data support for avoiding side effect and better understanding for clinical application.Material and methods:MTT assay were used to assess the influence of SrR on cell proliferation.Qualitative and quantitative ALP assay,alizarin red staining,and realtime PCR were used to evaluate the osteogenesis ability of SrR on OVX-BMSCs.The angiogenesis ability of SrR was studied by immunofluorescence staining of CD31 and v WF,transwell,tubeformation,realtime PCR assay and western Blot assay.Signaling pathway of PI3K/AKT/m TOR was also studied.Then RNA-Seq was conducted and the quality of sequencing was firstly evaluated.Aftere we got the different expressed genes,enrichment analysis of Kyoto Encyclopedia of Genes and Genome(KEGG)and functional annotation of Gene Ontology(GO)were analyzed.ROCK1 gene interfered by lentivrus transfection and the effect of SrR on ROCK1 gene followed osteogenesis differentiation was studied.At last,Osteoporosis model SD rat were used for animal experiment.Cylindrical bone defects were made on the outside of femur condylar and nano-scaled HAp particles loaded with or without SrR were filled in the defect.After 4-8 weeks observing time,samples were got and micro CT scanning,Van Gieson staining,fluorescence staining,HE staining,Tartrate resistant acid phosphatase(TRAP)staining and CD31 immumohistochemical staining were used to investigate the bone regeneration and blood vessel regeneration conditions.Result:The results demonstrated that 0.125-0.5m M SrR could promote cell proliferation,and promote the osteogenesis ability in a dose dependent way,in which concentration range from 0.25-1.0m M made the best performance.Immunofluorescence staining,transwell and tube formation assay also showed that this range of SrR could enhance the angiogenesis ability.The gene expression of ET-1,PAI-1 and t-PA those related to the thrombotic protein were unregulated followed with the increased concentration.Moreover,SrR could antagonize the PI3K-Akt-m TOR signaling pathway.And according to the quality confirmed result of RNA-Seq,the significant different expressed genes after treated by SrR were 645 in total.KEGG analysis and GO annotation analysis demonstrated that the significant annotation of signaling pathways or genes were gathered on cell metabolism,cell adhesive,migration,morphological change,cytoskeleton reconstruction and cell differentiation.ROCK1 gene,functions of promoting osteogenesis ability and inhibit cell apoptosis ability was verified for one of the SrR drug effect on OVX-BMSCs.The in-vivo study presented that the higher BMD,BV/TV,trabecular thickness and number,faster new bone deposition rate,and higher blood vessel fraction were observed in groups with SrR-loaded HAp nano-particles than groups without drug loading.VG staining and HE staining showed that bigger new bone area in SrR-load HAp group.TRAP staining showed a better osteoclast activity for SrR-Loaded HAp group during early phase,and became inactive at 8 weeks.CD31 immumohistochemical staining presented a higher density of blood vessel in SrR-Loaded HAp group.Conclusions:1.Low concentration(0.25-0.5mM)of strontium ranelate could promote the osteogenic differentiation and angiogenesis ability of cells,and have no effect on thrombosis related gene expressions.2.Strontium ranelate has a positive influence on cell metabolism,cell adhesive,migration and cell differentiation.ROCK1 gene is one of the targets that SrR promotes the osteogenic differentiation and inhibits cell apoptoisis on OVX-BMSCs.3.Strontium ranelate showed a favourable osteogenesis and angiogenesis effect on OVX rat by sustain-released local delivery method. |
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