Kartogenin-Loaded Exosomes Derived From Bone Marrow Mesenchymal Stem Cells Enhance Chondrogenesis and Expedite Tendon Enthesis Healing in a Rat Model of Rotator Cuff Injury.

Abstract

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.