Abstract
Objectives:
Anatomic anterior cruciate ligament (ACL) reconstruction is defined as the restoration of the native insertion sites, tissue dimensions, and collagen orientation. However, the microstructure and composition of standard tendon grafts used for ACL reconstruction differs from ligament. A bone-ACL-bone allograft (B-ACL-B) could represent a potential solution to ACL reconstruction, and thus the purpose of this study is to evaluate incorporation of a B-ACL-B graft used for ACL reconstruction in a rabbit model.
Methods:
Our study was approved by the Institutional Animal Care and Use Committee. A total of 71 New Zealand white rabbits were used, with 30 donor rabbits for harvesting bilateral B-ACL-B allografts, and recipient rabbits undergoing unilateral ACL resection followed by reconstruction with B-ACL-B allograft. Animals were euthanized for biomechanical testing (n = 14) at 4 and 8 weeks after surgery, and for micro–computed tomography (μCT), multi-photon microscopy, transmission electron microscopy (n=12), and histological analysis (n= 15) at 2, 4 and 8 weeks after surgery. Multi-photon microscopy analysis used Fast Fourier Transform in imageJ to calculate the proportion of fibers that deviated from orthogonal as a measure of collagen alignment and organization. One-way analysis of variance (ANOVA) with the post-hoc Tukey test was used to analyze differences among groups.
Results:
Gross inspection and radiographs confirmed intact ACL allograft in the proper anatomic position without evidence of complication. There was no significant difference between 4 and 8 weeks in mean failure force (4 weeks: 18.21 ± 8.74 N, 8 weeks: 21.75 ± 4.21 N, p=0.39) or stiffness (4 weeks: 8.08 ± 3.36 N/mm, 8 weeks: 10.88 ± 2.88 N/mm, p=0.15) for the B-ACL-B allografts. Progressive healing occurred between the bone block and the bone tunnel as demonstrated by a gradual increase in average bone-volume fraction (BVF) and total mineral density (TMD) between the bone block and tunnel wall in both femoral and tibial tunnels at 4 and 8 weeks. There was a significant decrease in TMD of both femoral and tibial bone blocks compared to day 0, indicating remodeling of the bone block (both p<0.001). Histological analysis showed bone block resorption at 2 weeks, followed by gradual new bone formation at the bone block-tunnel interface at 4 and 8 weeks. There was a gradual decrease in cellularity at the interface between bone block and tunnel wall (p=0.014), and increase in cellularity along the superficial aspect of the intra-articular ligament (p =0.008). Multi-photon microscopy demonstrated that the native control ACL demonstrated a mean % deviation from orthogonal value of 9.03± 1.81, 13.5±3.83 and 9.24±3.42 for femoral enthesis, mid-ACL, and tibial enthesis, respectively. The two-week specimens had a mean value of 6.50±3.34 (femoral enthesis), 10.4±1.73 (mid-ACL) and 10.97±3.666 (tibial enthesis) with none being statistically significantly different from control. The four-week samples had a mean of 9.10±1.87 for femoral enthesis, 8.39±3.28 for mid-ACL and 8.36±4.65 for tibial enthesis. Lastly, the 8-week specimens demonstrated that the mean deviation for the femoral enthesis was 7.26±1.67, mid-ACL was 7.89±1.58, and tibial enthesis was 10.8±2.55. The values for the mid-ACL was significantly less than controls at 4 and 8 weeks (p=0.0126 and 0.0063, respectively), suggesting maintenance of collagen fibril organization in the grafts. EM analysis of collagen fiber diameter demonstrated significantly lower average fibril diameter at all post-operative time points compared to age-matched native control ACLs (p<0.001).
Conclusions:
In conclusion, our rabbit model of B-ACL-B allograft ACL reconstruction demonstrates restoration of the normal macroscopic anatomy of the ACL, with progressive graft incorporation and remodeling. The B-ACL-B allograft, rather than a tendon graft, may provide the closest possible structural and biomechanical match to the native ACL, resulting in improved functional outcomes and may serve as a future option for ACL reconstruction. The results of this study provide important preliminary information to allow further study of this approach to ACL reconstruction and may serve as a baseline for future investigations.
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Illustration of reconstruction plan for the B-Acl-B allograft
Histolgy of interface and ligament.
Results of TEM and MPM Analysis.