Enhanced Achilles Tendon Healing With Novel Pre-Vascularized BMSC Sheets in a Rabbit Model

Acute Achilles tendon (AT) ruptures affect approximately 18 individuals per 100,000 annually. The tendon's low cellularity and vascularity significantly hinder the healing process, often associated with undesirable scarring. While bone marrow-derived mesenchymal stem cell sheet (BMSCS) has shown promise in enhancing AT healing, the potential benefits of prevascularization have not been investigated. This study aimed to develop a novel pre-vascularized BMSCS (PBMSCS) by incorporating endothelial cells (ECs), hypothesizing it would outperform BMSCS in promoting AT healing. Cells were isolated, and sheets were prepared following established protocols 1,2 . BMSCs (500,000 cells/well) were seeded in 6-well plates and treated with L-ascorbic acid. After four days, ECs (20,000 cells/cm 2 ) were co-cultured with BMSCS for two days to form PBMSCS. Twenty female New Zealand White rabbits (2.5–3.0 kg) were randomly divided into BMSCS and PBMSCS groups (n=10 per group). Bilateral medial gastrocnemius tendons were tenotomized 2 cm proximal to the calcaneal tubercle and repaired using a modified Kessler suture. PBMSCS was wrapped around the repair site. After six weeks, AT healing was assessed via histology, RT-PCR, and biomechanical testing. Comparisons between groups were using Student's test with SPSS ( P < 0.05). The PBMSCS group demonstrated well-organized and oriented collagen fibers, in contrast to the immature and disorganized fibers observed in the BMSCS group. Biomechanical testing showed that PBMSCS led to significantly higher ultimate failure load (216.5 ± 56.90 N vs. 162.2 ± 43.99 N, P = 0.028), cross-sectional area (6.373 ± 0.69 mm 2 vs. 5.447 ± 0.75 mm 2 , P = 0.010), and stiffness (73.98 ± 17.31 N/mm vs. 54.13 ± 11.73 N/mm, P = 0.0076). Additionally, the expression levels of tenogenic and angiogenic genes were significantly elevated in the PBMSCS group compared to the BMSCS group. Our study demonstrated that PBMSCS improved histological and mechanical outcomes following AT repair and upregulated tenogenic and angiogenic gene expressions. These benefits are likely due to the formation of a functional microvascular network, enhancing the delivery of pro-healing cells and factors to the injury site.