Distraction osteogenesis (DO) is a highly efficacious form of reconstructive bone regeneration, but its clinical utility is limited by the prolonged period required for bone consolidation to occur. Understanding the mechanistic basis for DO and shortening this consolidation phase thus represent promising approaches to improving the clinical utility of this procedure. A mandibular DO (MDO) canine model was established, after which small RNA sequencing was performed to identify relevant molecular targets genes. Putative miRNA target genes were identified through bioinformatics and confirmed through qPCR, Western blotting, and dual-luciferase reporter assays. Peripheral blood samples were collected to isolate serum and endothelial colony-forming cells (ECFCs) in order to measure miR-205, NOTCH2, and angiogenic cytokines expression levels. Lentiviral constructs were then used to inhibit or overexpress miR-205 and NOTCH2 in isolated ECFCs, after which the angiogenic activity of these cells was evaluated in migration, wound healing, proliferation, tube formation, and chick chorioallantoic membrane (CAM) assay. Autologous ECFCs transfected to knockdown miR-205 and were injected directly into the distraction callus. On days 14, 28, 35 and 42 after surgery, bone density was evaluated via CBCT, and callus samples were collected and evaluated via histological staining to analyze bone regeneration and remodeling. MiR-205 was identified as being one of the miRNAs that was most significantly downregulated in MDO callus samples. Downregulation of miR-205 was also observed in DO-ECFCs and serum of animals undergoing MDO. Inhibiting miR-205 markedly enhanced angiogenesis, whereas overexpressing miR-205 had the opposite effect in vitro. Importantly, NOTCH2, which is a unique regulator in bone angiogenesis, was identified as a miR-205 target gene. Consistent with this regulatory relationship, knocking down NOTCH2 suppressed angiogenesis, and transduction with a miR-205 inhibitor lentivirus was sufficient to rescue angiogenic activity. When ECFCs in which miR-205 had been inhibited were transplanted into the MDO callus, this significantly bolstered osteogenesis, and remodeling in vivo. MiR-205 is a significant regulator of the MDO process, and inhibiting this miRNA can accelerate MDO-related mineralization. Overall, these results offer new insights into the mechanistic basis for this procedure, highlighting potential targets for therapeutic clinical intervention.

中文翻译：

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MicroRNA-205通过靶向转录调节物NOTCH2介导内皮祖细胞在分散成骨作用中发挥作用

分心成骨术（DO）是一种高效的重建性骨再生形式，但其临床实用性受到骨巩固发生所需的延长时间的限制。因此，了解溶解氧的机理基础并缩短该巩固阶段代表了改善该过程临床实用性的有前途的方法。建立下颌DO（MDO）犬模型，然后进行小RNA测序以鉴定相关的分子靶基因。通过生物信息学鉴定了推定的miRNA靶基因，并通过qPCR，Western印迹和双荧光素酶报告基因检测法进行了确认。收集外周血样品以分离血清和内皮集落形成细胞（ECFC），以测量miR-205，NOTCH2和血管生成细胞因子的表达水平。然后将慢病毒构建体用于抑制或过度表达分离的ECFC中的miR-205和NOTCH2，然后在迁移，伤口愈合，增殖，管形成和鸡尿囊尿膜（CAM）分析中评估这些细胞的血管生成活性。自体ECFCs转染至miR-205，并直接注入干扰物愈伤组织中。在手术后的第14、28、35和42天，通过CBCT评估骨密度，并通过组织学染色收集和评估愈伤组织样本，以分析骨再生和重塑。MiR-205被确定为MDO愈伤组织样品中最显着下调的miRNA之一。在DO-ECFC和经历MDO的动物血清中也观察到miR-205的下调。抑制miR-205可以显着增强血管生成，而过表达miR-205在体外则相反。重要的是，NOTCH2是骨血管生成中的独特调节剂，被鉴定为miR-205靶基因。与此调节关系一致，敲除NOTCH2可抑制血管生成，而用miR-205抑制剂慢病毒进行转导足以挽救血管生成活性。将其中抑制了miR-205的ECFC移植到MDO愈伤组织中时，这显着增强了成骨作用，并在体内重塑。MiR-205是MDO过程的重要调节剂，抑制该miRNA可以加速MDO相关的矿化作用。总体而言，这些结果为该手术的机理基础提供了新见解，突出了治疗性临床干预的潜在目标。NOTCH2是骨血管生成中的独特调节剂，被鉴定为miR-205靶基因。与此调节关系一致，敲除NOTCH2可抑制血管生成，而用miR-205抑制剂慢病毒进行转导足以挽救血管生成活性。将其中抑制了miR-205的ECFC移植到MDO愈伤组织中时，这会大大促进成骨作用并在体内重塑。MiR-205是MDO过程的重要调节剂，抑制该miRNA可以加速MDO相关的矿化作用。总体而言，这些结果为该手术的机理基础提供了新见解，突出了治疗性临床干预的潜在目标。NOTCH2是骨血管生成中的独特调节剂，被鉴定为miR-205靶基因。与此调节关系一致，敲除NOTCH2可抑制血管生成，而用miR-205抑制剂慢病毒进行转导足以挽救血管生成活性。将其中抑制了miR-205的ECFC移植到MDO愈伤组织中时，这会大大促进成骨作用并在体内重塑。MiR-205是MDO过程的重要调节剂，抑制该miRNA可以加速MDO相关的矿化作用。总体而言，这些结果为该手术的机理基础提供了新见解，突出了治疗性临床干预的潜在目标。与此调节关系一致，敲除NOTCH2可抑制血管生成，而用miR-205抑制剂慢病毒进行转导足以挽救血管生成活性。将其中抑制了miR-205的ECFC移植到MDO愈伤组织中时，这会大大促进成骨作用并在体内重塑。MiR-205是MDO过程的重要调节剂，抑制该miRNA可以加速MDO相关的矿化作用。总体而言，这些结果为该手术的机理基础提供了新见解，突出了治疗性临床干预的潜在目标。与此调节关系一致，敲除NOTCH2可抑制血管生成，而用miR-205抑制剂慢病毒进行转导足以挽救血管生成活性。将其中抑制了miR-205的ECFC移植到MDO愈伤组织中时，这会大大促进成骨作用并在体内重塑。MiR-205是MDO过程的重要调节剂，抑制该miRNA可以加速MDO相关的矿化作用。总体而言，这些结果为该手术的机理基础提供了新见解，突出了治疗性临床干预的潜在目标。将其中抑制了miR-205的ECFC移植到MDO愈伤组织中时，这会大大促进成骨作用并在体内重塑。MiR-205是MDO过程的重要调节剂，抑制该miRNA可以加速MDO相关的矿化作用。总体而言，这些结果提供了对该程序的机械基础的新见解，突出了治疗性临床干预的潜在目标。将其中抑制了miR-205的ECFC移植到MDO愈伤组织中时，这会大大促进成骨作用并在体内重塑。MiR-205是MDO过程的重要调节剂，抑制该miRNA可以加速MDO相关的矿化作用。总体而言，这些结果为该手术的机理基础提供了新见解，突出了治疗性临床干预的潜在目标。