Summary

This study demonstrates that a novel decellularized bovine tendon graft successfully undergoes cellularization and remodeling in an ovine ACL reconstruction model, suggesting its potential as an effective scaffold for ACL reconstruction with excellent biocompatibility and structural integrity.

Abstract

Introduction

Anterior cruciate ligament (ACL) reconstruction presents several challenges. The use of allografts is associated with risks such as disease transmission and inconsistent quality control. Artificial ligaments, while offering good initial strength, often suffer from poor biocompatibility. Autografts, currently the gold standard, also have limitations: donor site morbidity, potential graft volume insufficiency in revision surgeries, multiple ligament reconstructions, or in patients with smaller statures, and time-consuming surgical procedures. To address these issues, we have developed a novel decellularized tendon graft. The aim of this study was to evaluate whether the decellularized bovine tendon graft (D-graft) undergoes cellularization and remodeling in an ovine ACL reconstruction model.

Methods

This study was approved by the animal experimentation review committee of Waseda University. Bovine tendons were decellularized using a proprietary method involving deoxycholic acid solution under pulsatile flow and pressure circulation, combined with microwave irradiation, followed by endonuclease treatment. This process reduced the residual DNA in the grafts to less than 50 ng/mg. Twenty-eight ovine aged three years or older underwent ACL reconstruction using the D-graft. The grafts were trimmed to 6.0 mm in diameter and 60 mm in length. During open surgery, femoral and tibial bone tunnels were made at the native ACL insertion sites. The D-graft was fixed with a titanium button on the femoral side and a double spike plate on the tibial side, with a tension of 40N. Cell infiltration into the D-graft was evaluated using hematoxylin-eosin staining at 3 and 12 months post-surgery. Collagen fiber diameters were measured using a transmission electron microscope (n=3 for the 3-month group, n=5 for the 12-month group). The ultimate tensile strength was assessed at a tensile speed of 300 mm/min (n=7 for both 3-month and 12-month groups). Statistical analysis was performed using Student’s t-test, with significance set at p < 0.05.

Results

At both 3 and 12 months, the D-grafts were covered with ovine synovium. Hematoxylin-eosin staining revealed that the D-grafts were repopulated with cells. Collagen fiber diameters and their occupancy in graft’s cross-sectional area decreased in the 3-month group compared to pre-implantation D-grafts but increased significantly in the 12-month group compared to the 3-month group. The ultimate tensile strength of the 12-month group was significantly higher than that of the 3-month group (p = 0.007).

Discussion

The findings demonstrate that the D-graft successfully undergoes cellularization and remodeling in vivo. The increase in collagen fiber occupancy in the graft’s cross-sectional area and mechanical strength at 12 months supports the successful remodeling of the D-graft over time. This study has limitations, including a small sample size and anatomical differences between ovine and human models. However, the findings in the more severe ovine conditions, due to the lack of rehabilitation compared to human patients, highlight the promise of the D-graft. These results suggest that the D-graft functions effectively as a scaffold for cellularization in ACL reconstruction and undergoes significant structural and mechanical remodeling over time.

Conclusion

This study provides proof of concept for a novel graft for ACL reconstruction, demonstrating excellent biocompatibility and remodeling capabilities.