Relaxin-2 overexpression in hUC-MSCs accelerates tendon-bone integration by activating osteogenic Wnt/β-catenin and angiogenic JAK2/STAT3 cascades.

Rotator cuff injuries frequently result in poor tendon-bone healing and high retear rates post-surgery, necessitating novel therapeutic strategies. This study investigates whether human umbilical cord mesenchymal stem cells overexpressing Relaxin-2 (hUC-MSCs-Relaxin-2) enhance tendon-bone healing and elucidates the underlying mechanisms. hUC-MSCs were transduced with Relaxin-2 lentivirus. Osteogenesis was evaluated by Alizarin Red staining and qRT-PCR/Western blot for BMP-2, Runx2, and Osterix. Angiogenesis was assessed via co-culture with EA.hy926 endothelial cells using Transwell migration, VEGF ELISA, and a tube formation assay. Pathway involvement was confirmed with 9-ING-41 (GSK-3β inhibitor) and CEP-33779 (JAK2 inhibitor). In a rat rotator cuff reconstruction model, animals received local injections of vehicle, hUC-MSCs, or hUC-MSCs-Relaxin-2 (n = 10/group). At 8 weeks, healing was evaluated by biomechanics, histology, quantitative immunohistochemistry, and molecular analyses. In vitro, Relaxin-2 overexpression enhanced osteogenesis via Wnt/β-catenin activation and promoted endothelial migration and VEGF production via JAK2/STAT3 signaling, effects blocked by respective inhibitors. In vivo, hUC-MSCs-Relaxin-2 significantly improved maximal failure load (16.5 ± 1.8 vs. 10.3 ± 1.2 N) and stiffness (9.7 ± 1.0 vs. 6.5 ± 0.8 N/mm), reduced inflammation, and increased osteogenic and angiogenic markers at the repair site. Collectively, hUC-MSCs-Relaxin-2 promote tendon-bone healing through coupled osteogenesis and angiogenesis via Wnt/β-catenin and JAK2/STAT3 pathways, offering a promising cell-based therapy to reduce retear rates after rotator cuff repair.