Introduction

Mechanical stimulation has been proven to promote bone-tendon interface (BTI) healing, but the mechanism remains unclear.

Objective

To investigate the effects of mechanical stimulation on the biological behavior of nestin⁺-bone mesenchymal stem cells (BMSCs) during the BTI healing, and to reveal the mechanisms of mechanical stimulation affecting BTI healing by primary cilia on the nestin⁺-BMSCs.

Methods

Transgenic tracing mice (nestin cre^ERT2:: IFT88^fl/fl/ROSA26 YFP) with primary cilia on nestin⁺-BMSCs conditioned knocked out were constructed, and the littermates (nestin cre^ERT2:: ROSA26 YFP) with normal cilia on nestin⁺-BMSCs were the control. After establishing mouse supraspinatus insertion injury models, samples were collected at week-2 (n = 5 per group), 4 and 8 (n = 15 per group, respectively). In vivo, the repair efficiency was evaluated by histology, imaging, biomechanics, and the migration of nestin⁺-BMSCs, detected by immunofluorescence staining. In vitro, nestin⁺ BMSCs were sorted and stimulated by tensile force to study the mechanisms of primary cilium-mediated mechanosensitive basis.

Results

Mechanical stimulation (MS) accelerated the recruitment of nestin⁺-BMSCs and promoted osteogenic and chondrogenic capacity. Histological, imaging and biomechanical results showed that the BTI healing quality of the IFT88^+/+, MS group was better than that of the other groups. After the conditionally knockout IFT88 in nestin⁺-BMSCs, the repair ability of the BTI was obviously deteriorated, even though mechanical stimulation did not increase significantly (IFT88^-/-, MS group). In vitro results showed the tensile loading enhanced the proliferation, migration and osteogenic or chondrogenic gene expression of nestin⁺-BMSCs with normal cilia. On the other hand, osteogenesis and chondrogenic expression were significantly decreased after inhibiting actin- Hippo/YAP pathway components.

Conclusion

The primary cilia mediated mechanical stimulation regulated osteogenic and chondrogenic differentiation potential of nestin⁺-BMSCs through the actin- Hippo/YAP pathway, and then promoted the BTI healing process.

中文翻译：

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骨-肌腱愈合过程中原发性纤毛介导的机械刺激对巢蛋白+-BMSCs的影响

 介绍

机械刺激已被证明可促进骨-肌腱界面 （BTI） 愈合，但机制仍不清楚。

 目的

探讨机械刺激对 BTI 愈合过程中巢蛋白 ⁺ 骨间充质干细胞 （BMSCs） 生物学行为的影响，并揭示机械刺激影响巢蛋白 ⁺ -BMSCs 上初级纤毛 BTI 愈合的机制。

 方法

构建在巢蛋白⁺-BMSCs条件敲除上具有原代纤毛的转基因示踪小鼠 （nestin cre^ERT2：： IFT88^fl/fl/ROSA26 YFP），并在巢蛋白 ⁺ -BMSCs 上具有正常纤毛的同窝小鼠 （nestin cre^ERT2：： ROSA26 YFP） 为对照。建立小鼠冈上肌插入损伤模型后，在第 2 周 （每组 n = 5 ）、第 4 周和第 8 周 （分别为每组 n = 15 个） 收集样本。在体内，通过组织学、影像学、生物力学和巢蛋白⁺-BMSCs的迁移评价修复效率，免疫荧光染色检测。在体外，通过拉力对巢^{蛋白 +} BMSCs 进行分选和刺激，以研究初级纤毛介导的机械敏感基础的机制。

 结果

机械刺激 （MS） 加速了巢蛋白⁺-BMSCs 的募集，促进了成骨和软骨形成能力。组织学、影像学和生物力学结果显示，IFT88^+/+， MS 组的 BTI 愈合质量优于其他组。在巢蛋白⁺-BMSCs中条件敲除 IFT88 后，BTI 的修复能力明显变差，即使机械刺激没有显着增加 （IFT88^-/-，MS 组）。体外结果表明，拉伸载荷增强了具有正常纤毛的巢^蛋白+-BMSCs 的增殖、迁移和成骨或成软骨基因的表达。另一方面，在抑制肌动蛋白 - Hippo/YAP 通路成分后，成骨和软骨生成表达显著降低。

 结论

初级纤毛介导的机械刺激通过肌动蛋白 Hippo/YAP 通路调节巢^蛋白+-BMSCs 的成骨和成软骨分化潜能，进而促进 BTI 愈合过程。