Online Resource Appendix 1. Supplementary Protocols for Animal Surgery, Vivact Imaging
Online Resource Appendix 1.Supplementary protocols for animal surgery, vivaCT imaging, biomechanical test, histology and immunohistochemistry
Animal surgery
To perform ACL reconstruction, animals were sedated by intramuscular injection of 10% ketamine/2% xylazine (Kethalar, 0.3mL: 0.2mL) and maintained sedation with 10% ketamine injected intravenously (Sigma Chemical Co, St Louis, MO, USA). The ipsilateral flexor digitorum longus tendon (20mm in length and 1mm in diameter) was harvested through a longitudinal medial incision. The ACL was excised and completely removed. A tibial tunnel and a femoral tunnel of 1.1mm in diameter and about 7mm in length were created from the footprint of the original ACL to the medial side of the tibia and lateral-anterioral femoral condyle, respectively, with an angle of 55o to the articular surface. The graft was inserted and tight fitted into the bone tunnels, fixed on the femoral and tibial tunnel exits with suture tied over the neighboring periosteum. The graft was sutured to the femoral tunnel exit first. Then a tension of 0.7N was applied by hanging an object of constant weight of 72g at 30°of knee flexion while suturing the graft to the tibial tunnel exit. No suture was found within the bone tunnel. Soft tissue was closed in layers. Lachman test was performed both after ACL excision and after ACL reconstruction. Only one randomly-selected limb was operated. 0.1 ml of Temgesic was injected intra-muscularly when the rat regained consciousness. The animals were allowed to have free cage movement immediately after operation. Except redness and swelling at the reconstructed site, no other postoperative complications were observed after the surgery.
vivaCT imaging and data analysis
After thawing the femur-graft-tibia complex and removing the surrounding muscles, the region covering the entry and exit of bone tunnel was scanned with vertical displacement of 30μm for about 220 and 350 consecutive sections, respectively, for the femoral and tibial tunnels at the resolution of 35m. The whole process took 20 minutes and the sample was kept moist during scanning. The sample was then subjected to biomechanical test immediately after scanning. The consecutive sections were 3-dimensionally (3D) reconstructed and rotated to align the bone tunnel vertically using the built-in software. To measure the mass and density of the mineralized tissue inside the bone tunnel, a circular region of interest (ROI) of 1.2mm in diameter, covering the bone tunnel and closest to the initial tunnel diameter that was allowed by the vivaCT built-in software and confirmed to be appropriate by the low bone volume/total volume fraction (BV/TV) of the ROI of the samples harvested at day 0, was used to represent the perimeter of the bone tunnel. The same circular ROI was selected for different sections along the length of the bone tunnel and these ROIs were 3D reconstructed after thresholding to remove the signals from the soft tissue (e.g. tendon graft) (Figure 1B). To measure the peri-tunnel bone mass and density, a 0.1cm x 0.1cm square ROI was chosen next to the bone tunnel at the trabecular bone region. The selected square ROIs in consecutive sections were 3D reconstructed after thresholding (Figure 1C). Bone mineral density (BMD) as defined by vivaCT (unit: mg HA/ccm (milligram of hydroxyapatite per cubic centimeter)) and BV/TV were calculated for the selected volume covering the whole femoral tunnel, the epiphyseal and the metaphyseal segments of tibial tunnel (Figure 1A). Due to its shorter length and located only at the epiphyseal region, the femoral tunnel was not further divided into 2 segments in vivaCT analysis. About 50 sections were analyzed in each segment and were very consistent among different animals.
Biomechanical test
After vivaCT scanning, the two bone ends of the femur-graft-tibia complex were placed in polytethylene tubes and potted in methyl-methacrylate. The samples were then stripped of all soft tissues, including the capsule and other knee ligaments. Cross pins were passed through the potting tube and the bone shaft to provide additional fixation.The whole construct was mounted onto a material testing machine (Hounsfield H25K-S, Hounsfield Test Equipment Ltd., Salfords, Redhill, UK) with the two tunnels oriented along the direction of tensile load. The sutures fixing the tendon to the bone tunnel were removed. To set the “zero” load state and ensure that the graft was now under tension, a displacement was applied with a 50N or 10N load cell slowly until the loading force increased (about 0.02N), and then the load cell was returned towards the initial position slightly. The constructwas then loaded at a displacement rate of 20mm/minute using a 50N or 10N load cell until failure after 5 cycles of preconditioning at a maximum displacement of 0.5mm, which was the smallest value possible for the machine, in moist condition. Failure of the construct was confirmed by the complete dissociation of the femoral and tibial halves of the complex which normally occurred at the displacement of 0.6-1.5mm and we stopped pulling at the displacement of about 20mm. The sample was kept moist during the whole experiment. The ultimate load to failure (N) and the failure mode were recorded. The slopes of the linear portion of the load (Y) - displacement (X) curve were calculated at the fixed displacement interval of 67.5µm using the formula (Y2-Y1) / (X2-X1) and the maximum slope was taken as the stiffness (N/mm) of the healing complex. Absolute values were comparedas all the specimens had the same tunnel diameter and similar graft diameter. The ultimate load and stiffness of normal ACL of another group of rats (n=9) equivalent to post-operative week 6 were used for comparison.
Histology and immunohistochemistry
The femur-graft-tibia complexes were fixed in 4% neutral buffered formalin, decalcified in 9% formic acid for about 4-6 weeks with the exact time determined by X-ray and embedded in paraffin.The femoral and tibial tunnel complexes were divided into two, with the intra-articular graft mid-substance preserved and attached to the tibial tunnel. 5μm-thick longitudinal sections parallel to the direction of the bone tunnel were cut and stained with Hematoxylin and Eosin (H&E) for the examination of healing at the tendon graft to bone tunnel interface and graft mid-substance under light microscopy. Sharpey’s fiber was examined by polarized light microscopy (Leica DMRXA2, Leica Microsystems Wetzlar GmbH, Germany). Only those sections that the entry and exit of bone tunnel could be identified were selected for histological assessment. One section was selected for histological assessment of each tunnel segment of each femur-graft-tibia complex.
The immunohistochemical staining of MMP1, MMP13 and CD68 (a monocyte-macrophage lineage marker that labels the potential osteoclast precursors and osteoclasts) was done using the Lab Vision kit (Thermo Fisher Scientific Inc., MI, USA). Briefly, after removal of paraffin and rehydration, endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 20 minutes at room temperature. Antigen retrieval was then performed with citrate buffer at 65oC for 20 minutes. After blocking with Ultra V block of the Lab Vision kit, the sections were stained with polyclonal rabbit antibody against rat/human/mouse MMP1 (NBP1-72209; 1:100; Novus Biologicals, CO, USA), mouse monoclonal antibody against rat/human MMP13 (ab3208; 1:100; Abcam, Cambridge, UK) or mouse monoclonal antibody against rat/mouse CD68 (ab31630; 1;100; Abcam, Cambridge, UK) in a humid chamber at 4oC overnight. The sections were then treated with primary antibody amplifier of the Lab Vision kit for 10 minutes at room temperature, followed by horse radish peroxidase (HRP) polymer Quanto from the same kit (which is a micropolymer conjugated with HRP) for another 10 minutes at room temperature. The spatial and temporal expression of the target proteins was then visualized by adding DAB Quanto Chromogen for 3 minutes at room temperature. Afterwards, the sections were rinsed, counterstained in hematoxylin, dehydrated with graded ethanol and xylene, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma Aldrich, St Louis, MO, USA). Primary antibody was replaced with blocking solution in the controls. For good reproducibility and comparability, all incubation times and conditions were strictly controlled. The sections were examined under light microscopy (Leica DMRXA2, Leica Microsystems Wetzlar GmbH, Germany).