Short-Term Evaluation of Bone-ACL-Bone Complex Allograft in ACL Reconstruction in a Rabbit Model

Abstract

The study is to evaluate incorporation of a bone-anterior cruciate ligament-bone (B-ACL-B) allograft in anterior cruciate ligament (ACL) reconstruction in a rabbit model. A total of 61 New Zealand white rabbits were used, with 23 donor rabbits for harvesting B-ACL-B allografts and 38 recipient rabbits undergoing unilateral ACL reconstruction with B-ACL-B allograft. Animals were euthanized for biomechanical testing, micro-computed tomography examination, histological analysis, multi-photon microscopy and transmission electron microscopy testing at 2, 4 and 8 weeks after surgery. Gross inspection and radiographs confirmed the intact ACL allograft in the proper anatomic position. Progressive healing occurred between the bone block and the bone tunnel as demonstrated by a gradual increase in average bone volume fraction and total mineral density at 4 and 8 weeks. Histological analysis showed new bone formation at the bone block-tunnel interface, with maintenance of the native ACL enthesis. Ultrastructural analysis demonstrated the maintenance of overall collagen matrix alignment, while there was repopulation with smaller diameter collagen fibrils. There was no significant difference between 4 and 8 weeks in mean failure force (p = 0.39) or stiffness (p = 0.15) for the B-ACL-B allografts. This study demonstrates the restoration of the normal anatomy of the ACL and progressive graft incorporation and remodeling using a B-ACL-B allograft for ACL reconstruction in the rabbit knee.

Keywords: allograft; animal model; anterior cruciate ligament reconstruction; bone–ACL–bone; healing.

Figure 1

Study design and surgical groups. ACLR: Anterior cruciate ligament reconstruction. μCT: micro-computed tomography, Histo: histology, Biomech: biomechanics, MPM: multi-photon microscopy, TEM: transmission electron microscopy.

Figure 2

(A) Illustration of reconstruction plan for the bone-anterior cruciate ligament-bone (B-ACL-B) allograft. (B) Gross appearance after allograft implantation. (C) Typical appearance of harvested B-ACL-B allograft with passing sutures.

Figure 3

(A) Biomechanical setup. (B) Measurement of new bone formation between bone block and tunnel wall. The region between the yellow circle (diameter: 3 mm, same as bone block diameter) and the red circle (diameter: 3.5 mm, same as tunnel diameter) represents the interface between the bone block and the tunnel wall. (C) Measurement of cellularity in the central area and periphery of ligament. Magnification: 4×. Yellow boxes represent 3 consecutive regions of interest of 300 × 300 μm established centrally and peripherally along the mid-portion of the ACL respectively. (D) Measurement of cellularity in the interface between bone block and tunnel wall. Magnification: 10×. Yellow box represents a region of interest of 300 × 300 μm set at the bone block–tunnel interface.

Figure 4

(A–D) Gross inspection of the specimen at all time points demonstrated intact anterior cruciate ligament allograft without rupture. (E,F) Radiographic imaging showing satisfactory fixation without bone block pull-out or fracture.

Figure 5

(A) Failure force. (B) Stiffness. (C) Bone volume fraction (BVF) in the femoral tunnels. (D) BVF in the tibial tunnels. (E) Cell density results at the tibial tunnel–bone block interface. (F) Cell density results at femoral tunnel–bone block interface. (G) Cell density of ACL central substance. (H) Cell density of ACL periphery. (*: p < 0.05, “++”: p < 0.01, ***: p < 0.001).

Figure 6

Native anterior cruciate ligament (ACL) –bone insertion site. New bone formation at the bone block–tunnel interface, and around and within the bone block. Hematoxylin and eosin staining of ACL–bone insertion site demonstrates maintenance of the histologic microstructure and composition of the native ACL insertion to bone at 2, 4 and 8 weeks. Yellow dot lines show the bone block–tunnel interface and the region of new bone. The regions in black boxes are shown in the following photos with magnification of 20×. BB: bone block, TW: tunnel wall.

Figure 7

Bone block remodeling at 2 weeks. BB: bone block, TW: tunnel wall. Arrow: osteoclasts. There was a densely cellular connective tissue around the bone fragments, with osteoclast-mediated scalloping on the perimeter and proliferation showing areas of bone resorption. The regions in black boxes are shown in the photos below with magnification of 20×.

Figure 8

Anterior cruciate ligament (ACL) mid-substance histology. (A–E) Hematoxylin and eosin (H&E) staining, 4×. (F–J) H&E staining, 20×. H&E staining shows cellular infiltration into the periphery of the ACL, resulting in increased cell density at 8 weeks compared to fresh and frozen control ACL and 2 weeks group. (K–O) Polarized microscopy shows decreased birefringence at 8 weeks in the peripheral aspect of the graft in the areas of cellular infiltration, consistent with loss of collagen fibril organization due to matrix remodeling. The regions in black boxes are shown in the photos below with magnification of 20×.

Figure 9

(A,B) Representative photos of multi-photon microscopy (MPM) of normal control anterior cruciate ligament (ACL) (A) and an 8 week specimen (B). 5× Whole ACL with punch out photos at 10X magnification. The regions in red boxes are showed in the photos above with magnification of 10×. (C) MPM quantitative results. *: p < 0.05, **: p < 0.01, ns: no significance.

Figure 10

(A–D) Representative photos of transmission electron microscopy (TEM) of normal control anterior cruciate ligament (ACL) (Figure 10A) and surgical specimens at 2, 4, and 8 weeks (Figure 10B, Figure 10C and Figure 10D, respectively). (E) TEM quantitative results. ****: p < 0.0001.