We attempted to generate a bone-tendon-bone structure by injecting human-type recombinant human bone morphogenetic protein-2 (rhBMP-2) into the semitendinosus tendon, and an anterior cruciate ligament (ACL) defect was reconstructed by grafting the engineered bone-tendon-bone graft. Two ossicles with a separation distance of 1 cm were generated within the left semitendinosus tendon of a rabbit 6 weeks after the injection of rhBMP-2 (15 µg at each site). The engineered bone-tendon-bone graft was transplanted in order to reconstruct the ACL by passing the graft through the bone tunnels. In the control group, the ACL was reconstructed with the semitendinosus tendon without BMP-2 using the same methods as those used in the experimental group. The animals were harvested at 4 or 8 weeks after surgery and examined by radiographic, histological, and biomechanical methods. In the experimental group, ossicles in the bone-tendon-bone graft were successfully integrated into the host bone of the femur and tibia. Histological analysis revealed that characteristic features identical to the normal direct insertion morphology had been restored. Biomechanical pull-out testing showed that the ultimate failure load and stiffness of the reconstructed ACL in the experimental group were significantly higher than those in the control group at both 4 and 8 weeks (p < 0.05). These results indicate the potential of regenerative reconstruction of the ACL, and the reconstruction resulted in the restoration of morphology and function equivalent to those of the normal ACL.
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