Exosomes secreted by hypoxia-stimulated bone-marrow mesenchymal stem cells promote grafted tendon-bone tunnel healing in rat anterior cruciate ligament reconstruction model

BACKGROUND: After anterior cruciate ligament (ACL) reconstruction in clinic, firm and rapid integration of the grafted tendon into the bone tunnel remains a challenge. Exosomes from hypoxia-treated stem cells are beneficial for promoting angiogenesis and then coupling with osteogenesis. Therefore, exosomes from hypoxia-cultured bone-marrow mesenchymal stem cells (Hypo-Exos) may be a cell-free therapy for enhancing graft-bone incorporation after ACL reconstruction. METHODS: Exosomes from normoxia-cultured bone-marrow mesenchymal stem cells (Norm-Exos) or Hypo-Exos were respectively cultured with human umbilical vein endothelial cells (HUVECs) for in-vitro evaluating their functions in HUVECs proliferation, migration, and tube formation. A total of 87 rats with single-bundle ACL reconstructions in the right knee were randomly allocated into 3 different treatments: phosphate-buffered saline (PBS) with the adhesive hydrogel injection as control (Ctrl), Norm-Exos with the adhesive hydrogel injection (Norm-Exos), and Hypo-Exos with the adhesive hydrogel injection (Hypo-Exos). At postoperative weeks 2, 4, or 8, the ACL graft-bone integrations were evaluated. RESULTS: Hypo-Exos was a better stimulator for in-vitro HUVECs proliferation, migration, and tube formation compared to PBS or Norm-Exos. Hypo-Exos within the adhesive hydrogel could be sustained-released at least 14 days around the peri-graft site. Radiologically, at week 4 or 8, femoral or tibial bone tunnel areas (BTA), as well as bone volume/total volume ratio (BV/TV) of the femoral or tibial peri-graft bone in the Hypo-Exos group, improved significantly better than these parameters of the Ctrl and Norm-Exos groups (P<0.05 for all). Histologically, the grafted tendon-bone interface in the Hypo-Exos group showed significantly higher histologic scores at week 4 or 8 as compared with the other groups (P<0.05 for all). Immunofluorescent staining verified that type H vessels were more abundant in the Hypo-Exos group when compared to the Ctrl or Norm-Exos group at week 2. Biomechanically, the Hypo-Exos group exhibited a significantly heightened failure load compared with the Ctrl and Norm-Exos groups (P<0.05 for all) at 8 weeks. Meanwhile, the stiffness in the Hypo-Exos group was the greatest among the three groups. CONCLUSION: Peri-graft Hypo-Exos injection accelerates grafted tendon-bone tunnel integration after ACL reconstruction by improving peri-graft bone microarchitecture.