RESUMEN
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.
RESUMEN
Bone defects resulting from trauma or tumor are one of the most challenging problems in clinical settings. Current tissue engineering (TE) strategies for managing bone defects are insufficient, owing to without using optimal osteoconductive material and seeding cells capable of superior osteogenic potential; thus their efficacy is instable. Herein, a novel TE strategy was developed for treating bone defects. First, the decellularized bone matrix (DBM) was manufactured into powders, and these DBM powders preserved the ultrastructural and compositional properties of native trabecular bone, are non-cytotoxic and low-immunogenic, and are capable of inducing the interacted stem cells differentiating into osteogenic lineage. Then, a subtype of osteoprogenitors was isolated from mouse long bones, and its high osteogenic potential was identified in vitro. After that, we constructed a "bone-forming unit" by seeding the special subtype of osteoprogenitors onto the DBM powders. In vivo performance of the "bone-forming units" was determined by injecting into the defect site of a mouse femoral epiphysis bone defect model. The results indicated that the "bone-forming unit" was capable of enhancing bone defect healing by regulating new bone formation and remodeling. Overall, the study establishes a protocol to construct a novel "bone-forming unit," which may be an alternative strategy in future bone TE application.