Background:The insufficient regeneration of fibrocartilage at the tendon enthesis is the primary cause of retearing after surgical reattachment of the rotator cuff. Exosomes derived from bone marrow–derived mesenchymal stem cells (BMSC-Exos) and kartogenin (KGN) have been demonstrated to induce fibrocartilage formation. Loading drugs into exosomes may lead to a synergistic effect, significantly enhancing the inherent activity of both components. However, further investigation is necessary to determine whether loading KGN into BMSC-Exos could yield superior efficacy in promoting tendon enthesis healing.Purpose:To study the effect and mechanism of KGN-loaded BMSC-Exos (Kl-BMSC-Exos) on tendon enthesis repair and biomechanical properties in a rat rotator cuff injury (RCI) model.Study Design:Controlled laboratory study.Methods:The characteristics and in vivo retention of exosomes were demonstrated using nanoflow cytometry, transmission electron microscopy, and in vivo imaging of a small animal. The differentiation markers of BMSCs were assessed through quantitative polymerase chain reaction and immunofluorescence assays. Unilateral supraspinatus tenotomy and repair were performed in rats to establish the RCI model. Gelatin sponges were utilized to contain and deliver exosomes. In total, 44 rats were randomly assigned to 4 groups: sham, RCI, BMSC-Exos, and Kl-BMSC-Exos. Tendon enthesis regeneration and biomechanical properties were evaluated 8 weeks after surgery. RNA sequencing of BMSCs was performed to elucidate the underlying mechanism through which Kl-BMSC-Exos enhance tendon enthesis healing.Results:No discernible disparities in fundamental characteristics were evident between BMSC-Exos and Kl-BMSC-Exos. Incorporating exosomes into a gelatin sponge extended the in vivo retention time from 7 to 14 days. Kl-BMSC-Exos were more effective in inducing differentiation markers of BMSCs, improving fibrocartilage regeneration, organizing collagen fiber arrangement, and enhancing the biomechanical properties of tendon enthesis. Furthermore, transcriptomics suggested that Mospd1 was involved in Kl-BMSC-Exos–mediated tendon enthesis healing by enhancing fibrocartilage regeneration.Conclusion:The incorporation of exosomes into a gelatin sponge significantly enhances their in vivo retention time. Kl-BMSC-Exos can expedite the healing of RCI by enhancing chondrogenesis and fibrocartilage regeneration, providing more organized collagen fiber arrangement and superior biomechanical properties of the rotator cuff enthesis. The promotion of rotator cuff enthesis regeneration may contribute to enhancing the chondrogenic potential in BMSCs through Kl-BMSC-Exos–mediated upregulation of Mospd1.Clinical Relevance:As a cell-free therapeutic approach, Kl-BMSC-Exos displayed a better therapeutic effect on tendon enthesis healing than BMSC-Exos did, and these can be used as a biologic augmentation to enhance the healing of rotator cuff enthesis.

中文翻译：

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源自骨髓间充质干细胞的 Kartogenin 负载外泌体在肩袖损伤大鼠模型中增强软骨形成并加速肌腱附着点愈合


背景： 肌腱附着点纤维软骨再生不足是肩袖手术复位后再撕裂的主要原因。来自骨髓来源的间充质干细胞 （BMSC-Exos） 和核晶素 （KGN） 的外泌体已被证明可诱导纤维软骨形成。将药物加载到外泌体中可能会导致协同效应，显着增强两种成分的内在活性。然而，需要进一步研究以确定将 KGN 加载到 BMSC-Exos 中是否可以在促进肌腱附着点愈合方面产生卓越的疗效。目的：研究KGN负载BMSC-Exos （Kl-BMSC-Exos） 对大鼠肩袖损伤 （RCI） 模型中肌腱附着点修复及生物力学特性的影响及机制。研究设计： 对照实验室研究。方法： 使用纳流式细胞术、透射电子显微镜和小动物体内成像证明外泌体的特性和体内保留。通过定量聚合酶链反应和免疫荧光测定评估 BMSCs 的分化标志物。对大鼠进行单侧冈上肌肌腱切开术和修复，建立 RCI 模型。明胶海绵用于容纳和递送外泌体。总共将 44 只大鼠随机分为 4 组： sham、 RCI 、 BMSC-Exos 和 Kl-BMSC-Exos。术后 8 周评估肌腱附着点再生和生物力学特性。对 BMSCs 进行 RNA 测序以阐明 Kl-BMSC-Exos 增强肌腱附着点愈合的潜在机制。结果： BMSC-Exos 和 Kl-BMSC-Exos 之间基本特征无明显差异。 将外泌体掺入明胶海绵中可将体内保留时间从 7 天延长至 14 天。Kl-BMSC-Exos 在诱导 BMSCs 的分化标志物、改善纤维软骨再生、组织胶原纤维排列和增强肌腱附着点的生物力学特性方面更有效。此外，转录组学表明 Mospd1 通过增强纤维软骨再生参与 Kl-BMSC-Exos 介导的肌腱附着点愈合。结论： 外泌体掺入明胶海绵中显著提高了其体内保留时间。Kl-BMSC-Exos 可以通过增强软骨生成和纤维软骨再生来加速 RCI 的愈合，提供更有组织的胶原纤维排列和肩袖附着点的卓越生物力学特性。促进肩袖附着点再生可能有助于通过 Kl-BMSC-Exos 介导的 Mospd1 上调增强 BMSCs 的软骨形成潜力.临床相关性： 作为一种无细胞治疗方法，Kl-BMSC-Exos 对肌腱附着点愈合的治疗效果优于 BMSC-Exos，这些可用作生物增强以增强肩袖附着点的愈合。