Background: Tendon–bone healing after anterior cruciate ligament reconstruction (ACLR) remains limited by insufficient bone formation and an unfavorable inflammatory microenvironment. Although bone marrow–derived mesenchymal stem cells (BMSCs) have therapeutic potential, their osteogenic and immunomodulatory effects are suboptimal. Additionally, the role of R-spondin 2 (RSPO2) in enhancing BMSC-mediated tendon–bone healing remains unclear. This study aims to investigate the role of RSPO2 in promoting the osteogenic differentiation of BMSCs and its effect on M2 macrophage polarization, as well as the potential of RSPO2 combined with BMSCs to accelerate tendon-bone healing after ACLR.

Methods: During the osteogenic induction of BMSCs, low, medium, and high concentrations of RSPO2 recombinant protein (10, 20, 40 nmol/L) were added, respectively, followed by alkaline phosphatase (ALP) staining and the measurement of osteogenesis-related protein expression. RAW264.7 cells were differentiated into macrophages using PMA, and the effects of BMSCs and/or RSPO2 on M1/M2 macrophage polarization were analyzed using qRT-PCR. A rat ACLR model was established, and the rats were divided into four groups: Control, BMSCs, BMSCs+Low RSPO2 (co-injection), and BMSCs+Medium RSPO2 (co-injection). Micro-CT imaging was performed to analyze bone tunnel area and mineralized tissue formation at 4 and 8 weeks post-ACLR. Histological analysis, including HE staining, Safranin O-Fast Green staining, and COL2α1 immunohistochemistry, was used to evaluate tendon-bone healing. Immunofluorescence staining was conducted to detect the expression of M1 (iNOS) and M2 (CD163) macrophage markers at the tendon-bone interface.

Results: RSPO2 significantly promoted the osteogenic differentiation of BMSCs, with the medium concentration of RSPO2 showing the most pronounced effect. RSPO2 and BMSCs also promoted M2 macrophage polarization and inhibited M1 polarization, with the medium concentration of RSPO2 demonstrating the most significant impact. Micro-CT results indicated that RSPO2 and BMSCs treatment significantly reduced the bone tunnel area and increased the formation of mineralized tissue compared to the control group. Histological analysis revealed that RSPO2 and BMSCs treatment improved tendon-bone interface healing, characterized by more orderly fiber tissue arrangement and an increase in chondrocytes and fibrocartilaginous tissue. Immunofluorescence staining results showed that RSPO2 and BMSCs promoted M2 macrophage polarization at the tendon-bone interface.

Conclusions: RSPO2 enhances BMSC-induced osteogenic differentiation and promotes M2 macrophage polarization, thereby improving tendon–bone healing after ACLR surgery in a small-animal model. The combined treatment of RSPO2 and BMSCs demonstrates promising preclinical potential in accelerating bone formation and tendon–bone interface repair, with the medium concentration of RSPO2 showing the most notable effect. However, these findings are based solely on a small-animal model, and further validation in large-animal models and clinical studies is required before potential clinical translation.