Enhancement of Tendon-Bone Integration of Anterior Cruciate Ligament Grafts with Bone Morphogenetic Protein-2 Gene Transfer

Abstract

The integration of tendon grafts used for replacement of the anterior cruciate ligament is still sometimes unsatisfactory and may be associated with postoperative anterior-posterior laxity. The goal of this study was to examine the capacity of bone morphogenetic protein-2 (BMP-2) gene transfer to improve the integration of semitendinosus tendon grafts at the tendon-bone interface after reconstruction of the anterior cruciate ligament in rabbits. The anterior cruciate ligaments of adult New Zealand White rabbits were replaced with autologous double-bundle semitendinosus tendon grafts. The semitendinosus tendon grafts had been infected in vitro with adenovirus-luciferase, adenovirus-LacZ (AdLacZ), or adenovirus-BMP-2 (AdBMP-2); untreated grafts served as controls. The grafts were examined histologically at two, four, six, and eight weeks after surgery. In additional experiments, the structural properties of the femur-anterior cruciate ligament graft-tibia complexes, from animals killed eight weeks postoperatively, were determined from uniaxial tests. The stiffness (N/mm) and ultimate load to failure (N) were determined from the resulting load-elongation curves. Genetically engineered semitendinosus tendon grafts expressed reporter genes as well as BMP-2 in vitro. The AdLacZ-infected grafts showed two different histological patterns of transduction. Intra-articularly, infected cells were mostly aligned along the surface, and they decreased in number between two and eight weeks after surgery. In the intra-tunnel portions of the grafts, the number of infected cells did not decrease during the observation period. Moreover, a high number of transduced cells was found in the deeper layers of the tendons. In the control group, granulation-type tissue at the tendon-bone interface showed progressive reorganization into a dense connective tissue, and a later establishment of fibers resembling Sharpey fibers. In the specimens with an AdBMP-2-infected anterior cruciate ligament graft, a broad zone of newly formed matrix resembling chondro-osteoid had formed at the tendon-bone interface at four weeks after surgery. This area was increased at six weeks, showing a transition from bone to mineralized cartilage and nonmineralized fibrocartilage. In addition, in the AdBMP-2-treated specimens, the tendon-bone interface in the osseous tunnel was similar to that of a normal anterior cruciate ligament insertion. The stiffness (29.0 +/- 7.1 N/mm compared with 16.7 +/- 8.3 N/mm) and the ultimate load to failure (108.8 +/- 50.8 N compared with 45.0 +/- 18.0 N) were significantly enhanced in the specimens with an AdBMP-2-transduced graft when compared with the control values (p < 0.05). This study demonstrates that BMP-2 gene transfer significantly improves the integration of semitendinosus tendon grafts in bone tunnels after reconstruction of the anterior cruciate ligament in rabbits. Novel technologies including gene therapy and tissue engineering, such as those described in this study, may provide useful therapeutic procedures to enhance biological healing after reconstruction of the anterior cruciate ligament.