Duration of postoperative immobilization affects MMP activity at the healing graft-bone interface: Evaluation in a mouse ACL reconstruction model

Abstract

Excessive MMP activity may impair tendon-to-bone healing. However, little is known about the effect of joint motion on MMP activity after ACL reconstruction. The aim of this study was to determine the effect of different durations of knee immobilization on MMP activity in a mouse ACL reconstruction model using a fluorescent MMP probe which detects MMP 2, 3, 9, and 13 and near-infra red in vivo imaging. Sixty C57BL male mice underwent ACL reconstruction. Post-operatively, the animals were treated with free cage activity (Group 1), or with the use of an external fixator to restrict knee motion and weight bearing for 5 days (Group 2), 14 days (Group 3), and 28 days (Group 4). At days 3, 7, 16, 23, and 30, five mice underwent IVIS imaging. At days 3, 7, 16, and 30, histological analysis was also performed. Probe signal intensity in the whole limb peaked at day 7, followed by a decrease at day 16, and maintenance up to day 30. There was no significant difference among groups at any time point based on IVIS, but histologic localization of MMP probe signal showed significantly less activity in Group 2 and Group 3 compared to Group 4 in the bone tunnel at day 30. We demonstrated that short-term immobilization led to less MMP activity around the bone tunnel compared with prolonged immobilization. A short period of immobilization after ACL reconstruction might enhance graft-bone interface healing by mitigating excess MMP expression. These findings have implications for post-operative rehabilitation protocols following ACL reconstruction. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:325-334, 2019.

Keywords: ACL reconstruction; MMP; graft-bone interface healing; immobilization.

Figure 1.

Study design. Sixty C57BL male mice (age: 12 weeks) underwent ACL reconstruction in the right knee using ipsilateral FDL tendon graft. Post-operatively the animals were treated with no immobilization (Group 1), or with the use of an external fixator to immobilize the operated knee joint for 5 days (Group 2), 14 days (Group 3), and 28 days (Group 4). At day 3, 7, 16, 23, and 30, five mice underwent IVIS imaging using MMP Sense 680 probe, which localizes MMP-2, −3, −9, and −13. One mouse without the probe was imaged at each time point to serve as control. At day 3, 7, and 16, two mice with the probe and one mouse without the probe in each group were euthanized, and at 30 days five mice with the probe and one mouse without the probe in each group were euthanized for histological analysis.

Figure 2.

The difference in autofluorescence in external fixators. (a) New external fixator used in this study; (b) Original external fixator used in the previous our study. Scale bar = 1 cm. (c and d) IVIS images with color bar (yellow = higher). Scale bar =1 cm. White arrows and white dotted line indicate external fixator. IVIS images demonstrated that our new external fixator had lower autofluorescence.

Figure 3.

Surgical procedures and post-operative X-ray. (A) Surgical procedures of ACL reconstruction and an external fixator. (a) Harvest ipsilateral FDL tendon. (b) Tie a 6–0 prolene suture at proximal end of the tendon. (c) Apply a clip to distal end of the tendon. White arrow indicates clip. (d) Prepared graft (e) Drill bone tunnel in the femur with a 23-gauge needle. (f) Transect ACL. (g) Drill bone tunnel in the tibia with a 23-gauge needle. (h and i) Pass the graft through tunnels using the attached 6–0 prolene needle. White arrow indicates suspensory fixation utilizing the clip. (j) Fixation of the graft using a 5–0 wire. White arrow head indicates the wire. (k) Insert two 30-gauge needles in the femur. (l) Two femoral pins after percutaneous placement. (m) Insert two 30-gauge needles in the tibia. (n) The pins were placed percutaneously. (o) The medial side of the pins were bent and then pulled back flush with the medial femoral bone. (p) The external fixator was connected to the four pins. (B) X-ray. (a) AP and (b) lateral X ray after ACL reconstruction using FDL tendon. (c) AP and (d) lateral X-ray after placement of external fixator following ACL reconstruction. Scale bar = 2 mm.

Figure 4.

Quantitative analysis of IVIS images. Methodology of setting the region of interest (ROI) for quantifying fluorescent signals in each knee. (A) The ROI was set on the entire limb from heel to inguinal line (indicated by blue line). The background was set at the center of the abdomen (indicated by red circle). (B) The ROI was focused on the knee joint, registered to superimposed X-ray image (indicated by blue line). The background was set to the center of the abdomen (indicated by red circle).

Figure 5.

Quantitative analysis of histology images. (A) Quantification methodology of MMP probe positive area in the fluorescent microscope pictures. (a) HE picture. (b) Fluorescent gray scale image of the same section as HE. A 0.64mm circle (blue circle; the diameter of 23G needle is 0.64 mm) was drawn in the center of the bone tunnel. The graft was manually delineated (yellow line) referring to HE picture. (C) The threshold was set to cover MMP probe positive areas. The area between these two delineations represented the graft-bone interface. The percent of MMP probe positive area in the graft-bone interface and graft were calculated using Image J. Scale bar = 200 μm.

Figure 6.

Representative IVIS images of each group from days 3 to 30. (A) Right side was operated. The control group was not injected with probe. The normal group did not undergo surgery. Color bar indicates signal intensity (yellow = higher). Scale bar = 2.0 cm. Control animals showed much less signal than all study animals. Signal intensity peaked at day 7, followed by a decrease to day 16, and maintenance up to day 30 compared with normal animals. (B) Mean radiant efficiency values of each group from MMP sense 680 probe at each time point. The ROI was set on the entire limb. (C) The ROI was focused on the knee joint. All data are presented as mean ± standard deviation (n = 5). p-value was calculated by two-way ANOVA with post hoc Tukey’s test. Comparison with normal *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All groups showed significant difference between day 3, day 7, and normal. In most of groups signal intensity at day 7 was higher than later time points (day 16, day 23, and day 30). No significant difference was found among groups at all time points.

Figure 7.

Representative transverse section of fluorescent microscope and hematoxylin eosin (HE) pictures of each group at each time point. (A) Day 3. (B) Day 7. (C) Day 16. (D) Day 30. Left column is fluorescent image (MMP 680 probe fluorescence is red). Right column is HE stained section. A indicates anterior aspect of femoral condyle. Scale bar = 200 μm. White arrows indicate bone tunnel and graft. The control group was not injected with probe. Control specimens showed much less signal than study specimens. Probe signal intensity seemed to peak at day 16 (Fig. 6C) time points in all groups. MMP probe fluorescent positive area at graft-bone interface (E) and graft at day 30 (F). All data are presented as mean ± standard deviation (n = 5). p-value was calculated by one-way ANOVA with post hoc Tukey’s test. There was a significant difference in MMP probe positive area at the graft-bone interface between Groups 2, 3, and 4 (p < 0.05). (G) Representative transverse section of magnified graft and bone tunnel pictures of each group at day 30. Scale bar = 200 μm. (H) Tendon Bone Tunnel Healing (TBTH) scoring system. Group 2 showed better score than Group 1 and Group 4 (p < 0.05).

Figure 8.

Immunohistochemistry of MMP. Representative pictures at Day 30. G: Graft B: Bone Scale bar=50μm.