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Injuries to the anterior cruciate ligament (ACL) often require surgical reconstruction utilizing tendon grafts to restore knee function and stability. Some current graft options for ACL repair are associated with poor long-term outcomes. Our laboratory has fabricated tissue-engineered bone-ligament-bone (BLB) constructs that demonstrate native ligament regeneration and advancement toward native ACL mechanical properties in a sheep ACL reconstruction model. Prior work has shown that freezing BLBs as a method of preservation resulted in similar outcomes compared with fresh BLBs after 6-month implantation. The purpose of this study was to evaluate the long-term efficacy of fresh and frozen BLBs. We hypothesized that both fresh and frozen BLBs would show continued regeneration of structural and functional properties toward those of native ACL after a 2-year implantation. Following removal of the native ACL, fresh ( n  = 2) and frozen ( n  = 2) BLBs were implanted arthroscopically. After 2 years of recovery, sheep were euthanized and both the experimental and contralateral hindlimbs were removed and radiographs were performed. Explanted knees were initially evaluated for joint laxity and were then further dissected for uniaxial tensile testing of the isolated ACL or BLB. Following mechanical testing, explanted contralateral ACL (C-ACL) and BLBs were harvested for histology. Two years post-ACL reconstruction, fresh and frozen BLBs exhibited similar morphological and biomechanical properties as well as more advanced regeneration compared with our 6-month recovery study. These data indicate that an additional 1.5-year regeneration period allows the BLB to continue ligament regeneration in vivo . In addition, freezing the BLBs is a viable option for the preservation of the graft after fabrication.

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Fresh and Frozen Tissue-Engineered Three-Dimensional Bone–Ligament–Bone Constructs for Sheep Anterior Cruciate Ligament Repair Following a 2-Year Implantation