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

• Published in: Journal of orthopaedic translation Vol. 36; pp. 152 - 163
• Main Authors: Zhang, Tao, Yan, Shaohang, Song, Ya, Chen, Can, Xu, Daqi, Lu, Bangbao, Xu, Yan
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.09.2022; Chinese Speaking Orthopaedic Society; Elsevier
• Subjects: Anterior cruciate ligament reconstruction; Cell-free therapy; Exosomes; Graft-bone integration; Hypoxia; Original; Graft-bone integration; Hypoxia; Cell-free therapy; Anterior cruciate ligament reconstruction; Exosomes
• ISSN: 2214-031X; 2214-0328
• DOI: 10.1016/j.jot.2022.08.001

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. 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. 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. Peri-graft Hypo-Exos injection accelerates grafted tendon-bone tunnel integration after ACL reconstruction by improving peri-graft bone microarchitecture.