Background

Rotator cuff injury (RCI) is a common shoulder injury, which is difficult to be completely repaired by surgery. Hence, new strategies are needed to promote the healing of tendon-bone.

Objective

We aimed to investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) overexpressing RUNX1 on the tendon-bone healing after RCI, and to further explore its mechanism.

Methods

Lentiviral vector was used to mediate the overexpression of RUNX1. RUNX1-overexpressed UCB-MSCs (referred to as MSC-RUNX1) were co-cultured with osteoclasts, and TRAP staining was performed to observe the formation of osteoclasts. Then MSC-RUNX1 was cultured in osteogenic differentiation medium, Alizarin red staining was conducted to detect osteogenic differentiation. The expression of markers of osteogenesis and osteoclast was detected by RT-qPCR. EA. hy926 cells were co-cultured with MSC-RUNX1. Transwell assay was used to detect the migration, and the expression of angiogenesis related-genes VEGF and TGF-β was detected by RT-qPCR. The rat rotator cuff reconstruction model was established and MSCs were injected at the tendon-bone junction. Biomechanical test and micro-CT scanning were performed, and HE, Masson and Alcian Blue staining were used for histological evaluation of tendon-bone healing. TUNEL and PCNA immunofluorescence (IF) staining were performed to evaluate apoptosis and proliferation at the tendon-bone healing site. The levels of TNF-α, IL-6 and IL-8 in serum were detected by ELISA. The expression of CD31 and Endomucin that related to angiogenesis was detected by IF. Safranin O-fast and TRAP/CD40L immunohistochemical staining were used to assess the levels of osteoclasts and osteoblasts at the tendon-bone healing site.

Results

hUC-MSCs overexpressing RUNX1 inhibited osteoclast formation and promoted osteogenic differentiation. MSC-RUNX1 could promote the migration and tube formation of EA. hy926 cells, and up-regulate the levels of VEGF and TGF-β. Model mice treated with MSC-RUNX1 partially restored the biomechanical indexes. Treatment of MSC-RUNX1 obviously increased the bone density, accompanied by the formation of new bone. In vivo experiments showed that MSC-RUNX1 treatment could promote tendon-bone healing and inhibit inflammatory response in rats. MSC-RUNX1 treatment also promoted angiogenesis at the tendon-bone healing site, while inhibiting osteoclast formation and promoting osteogenic differentiation.

Conclusion

hUC-MSCs overexpressing RUNX1 can inhibit the formation of osteoclasts and differentiation of osteoblasts, promote angiogenesis and inhibit inflammation, thereby promoting tendon-bone healing after RCI.

中文翻译：

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过表达RUNX1的人脐带间充质干细胞通过抑制骨溶解、增强成骨和促进血管生成促进腱骨愈合

背景

肩袖损伤（RCI）是一种常见的肩部损伤，很难通过手术完全修复。因此，需要新的策略来促进腱骨的愈合。

客观的

我们的目的是探讨过表达RUNX1的人脐带间充质干细胞（hUC-MSCs）对RCI后腱骨愈合的影响，并进一步探讨其机制。

方法

使用慢病毒载体介导RUNX1的过表达。将RUNX1过表达的UCB-MSCs（简称MSC-RUNX1）与破骨细胞共培养，进行TRAP染色观察破骨细胞的形成情况。然后将MSC-RUNX1在成骨分化培养基中培养，茜素红染色检测成骨分化。 RT-qPCR检测成骨和破骨细胞标志物的表达。 EA。 hy926细胞与MSC-RUNX1共培养。采用Transwell实验检测迁移情况，RT-qPCR检测血管生成相关基因VEGF、TGF-β的表达。建立大鼠肩袖重建模型，并在肌腱-骨连接处注射间充质干细胞。进行生物力学测试和显微CT扫描，并使用HE、Masson和Alcian Blue染色对腱骨愈合进行组织学评价。采用 TUNEL 和 PCNA 免疫荧光 (IF) 染色来评估腱骨愈合部位的细胞凋亡和增殖。采用ELISA法检测血清中TNF-α、IL-6、IL-8水平。 IF检测与血管生成相关的CD31和Endomacin的表达。采用 Safranin O-fast 和 TRAP/CD40L 免疫组织化学染色来评估腱骨愈合部位破骨细胞和成骨细胞的水平。

结果

hUC-MSC 过表达 RUNX1 抑制破骨细胞形成并促进成骨分化。 MSC-RUNX1可以促进EA的迁移和管形成。 hy926 细胞，并上调 VEGF 和 TGF-β 的水平。 MSC-RUNX1治疗的模型小鼠生物力学指标部分恢复。 MSC-RUNX1的治疗明显增加了骨密度，并伴随着新骨的形成。体内实验表明，MSC-RUNX1治疗可以促进大鼠腱骨愈合并抑制炎症反应。 MSC-RUNX1治疗还促进腱骨愈合部位的血管生成，同时抑制破骨细胞形成并促进成骨分化。

结论

过表达RUNX1的hUC-MSCs可以抑制破骨细胞的形成和成骨细胞的分化，促进血管生成并抑制炎症，从而促进RCI后腱骨的愈合。