A Preclinical Model to Study the Influence of Graft Force on the Healing of the Anterior Cruciate Ligament Graft.

The purpose of this study is to establish a small animal anterior cruciate ligament (ACL) reconstruction research model where ACL graft force can be varied to create different graft force patterns with controlled knee motion. Cadaveric (n = 10) and in vivo (n = 10) rat knees underwent ACL resection followed by reconstruction using a soft tissue autograft. Five cadaveric and five in vivo knees received a nonisometric, high-force femoral graft tunnel position. Five cadaveric and five in vivo knees received a more isometric, low-force graft tunnel position. ACL graft force (N) was then recorded as the knee was ranged from extension to 90 degrees using a custom knee flexion device. Our results demonstrate that distinct ACL graft force patterns were generated for the high-force and low-force femoral graft tunnels. For high-force ACL grafts, ACL graft forces increased as the knee was flexed both in cadaveric and in vivo knees. At 90 degrees of knee flexion, high-force ACL grafts had significantly greater mean graft force when compared with baseline (cadaver: 7.76 ± 0.54 N at 90 degrees vs. 4.94 ± 0.14 N at 0 degree, p = 0.004; in vivo: 7.29 ± 0.42 N at 90 degrees vs. 4.74 ± 0.13 N at 0 degree, p = 0.007). In contrast, the graft forces for low-force ACL grafts did not change with knee flexion (cadaver: 4.94 ± 0.11 N at 90 degrees vs. 4.72 ± 0.14 N at 0 degree, p = 0.41; in vivo: 4.78 ± 0.26 N at 90 degrees vs. 4.77 ± 0.06 N at 0 degree, p = 1). Compared with nonisometric ACL grafts, the graft force for grafts placed in an isometric position had significantly lower ACL graft forces at 15, 30, 45, 60, 70, and 90 degrees in both cadaveric and in vivo knees. In conclusion, we have developed a novel ACL reconstruction model that can reproducibly produce two ACL graft force patterns. This model would permit further research on how ACL graft forces may affect subsequent graft healing, maturation, and function.

Use of a new model allowing controlled uniaxial loading to evaluate tendon healing in a bone tunnel.

The optimal mechanical loading regimen for the healing of a tendon graft in a bone tunnel is unknown. We developed a rat model that directly tensions a healing tendon graft, without the use of confounding joint motion. Fifty cycles of either 0, 3, or 6 N of tension were applied to groups daily for 3 or 6 weeks. At 3 weeks the low load (3 N) group had the highest failure load (p = 0.009), but by 6 weeks there were no differences in failure load among groups. At 3 weeks the high load (6 N) group had greater osteoclast activity compared to the immobilized (0 N) group (p < 0.05), and by 6 weeks there were significantly more osteoclasts in the high load group compared to the low load group (p = 0.01). Bone volume fraction was higher in the immobilized group compared to the 3 N load group at 3 weeks (p = 0.014) and 6 weeks (p = 0.007). At 6 weeks, the immobilized group had greater trabecular number compared to both loading groups (p < 0.05). In conclusion, low magnitude loading had a beneficial early effect but continued loading led to poorer new bone formation over time and no beneficial effect at 6 weeks, perhaps due to delayed maturation from cumulative loads. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:852-859, 2016.

Effect of immediate and delayed high-strain loading on tendon-to-bone healing after anterior cruciate ligament reconstruction.

BACKGROUND: We previously demonstrated, in a rat anterior cruciate ligament (ACL) graft reconstruction model, that the delayed application of low-magnitude-strain loading resulted in improved tendon-to-bone healing compared with that observed after immediate loading and after prolonged immobilization. The purpose of this study was to determine the effect of higher levels of strain loading on tendon-to-bone healing. METHODS: ACL reconstruction was carried out in a rat model in three randomly assigned groups: high-strain daily loading beginning on either (1) postoperative day one (immediate-loading group; n = 7) or (2) postoperative day four (delayed-loading group; n = 11) or (3) after prolonged immobilization (immobilized group; n = 8). Animals were killed two weeks after surgery and micro-computed tomography (micro-CT) and biomechanical testing of the bone-tendon-bone complex were carried out. RESULTS: The delayed-loading group had greater tissue mineral density than either the immediate-loading or immobilized group (mean [and standard deviation], 813.0 ± 24.9 mg/mL compared with 778.4 ± 32.6 mg/mL and 784.9 ± 26.4 mg/mL, respectively; p < 0.05). There was a trend toward greater bone volume per total volume fraction in both the immobilized and the delayed-loading group compared with the immediate-loading group (0.24 ± 0.03 and 0.23 ± 0.06 compared with 0.20 ± 0.05; p = 0.06). Trabecular thickness was greater in the immobilized group compared with the immediate-loading group (106.5 ± 23.0 µm compared with 72.6 ± 10.6 µm; p < 0.01). There were no significant differences in failure load or stiffness between the immobilized group and either high-strain cyclic-loading group. CONCLUSIONS: Immediate application of high-strain loading appears to have a detrimental effect on healing in this rat model. Any beneficial effects of delayed loading on the healing tendon-bone interface (after a brief period of immobilization) may be offset by the detrimental effects of excessive strain levels or by the detrimental effects of stress deprivation on the graft. CLINICAL RELEVANCE: The timing and magnitude of mechanical load on a healing rat ACL reconstruction graft may have important implications for postoperative rehabilitation. Avoidance of exercises that cause high graft strain in the early postoperative period may lead to improved tendon-to-bone healing in humans.

The effect of mechanical load on tendon-to-bone healing in a rat model.

BACKGROUND: Joint motion is commonly prescribed after tendon repair surgeries such as rotator cuff repairs; however, the ideal rehabilitation program to optimize tendon-to-bone healing is unknown. HYPOTHESES: (1) Delayed loading would result in a mechanically stronger and better organized tendon-to-bone interface compared with prolonged immobilization or immediate loading. (2) Low-magnitude load would lead to superior healing compared with high-magnitude load. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 192 rats underwent unilateral patellar tendon detachment and repair followed by placement of a custom external fixator. Rats were assigned to immobilization, immediate postoperative loading, or delayed-onset loading (4- or 10-day delay). Loading was controlled using a specially designed motorized device to apply constant strain until 3 N (low load) or 6 N (high load) of axial tensile force was reached through the healing bone-tendon complex for 50 cycles per day. Rats were sacrificed at 4, 10, 21, or 28 days postoperatively for histomorphometric, immunohistochemical, radiographic, molecular, and biomechanical analyses. RESULTS: The load to failure was significantly higher in the immobilized group compared with the immediate and delayed loading groups (P < .05). Compared with loaded specimens, the immobilized specimens had significantly less fibrocartilage (at 4, 10, and 28 days), significantly better collagen fiber organization (at 4, 10, and 21 days), decreased expression of matrix metalloproteinase-13 (at 10, 21, and 28 days), and significantly fewer apoptotic cells (at 21 and 28 days). Micro-computed tomographic analyses showed that the 3-N immediate load group had significantly less total volume (P = .012), bone volume (P = .012), and bone mineral density (P = .023) for cortical bone, and the immobilized group had significantly more specimens with new bone formation at the enthesis (100%; P = .001). CONCLUSION: Immobilization results in a stronger tendon-bone complex, with less scar tissue and a more organized tendon-bone interface compared with all loading regimens in this study. CLINICAL RELEVANCE: Given the relatively high rate of failure after rotator cuff and other tendon-to-bone repairs, identification of optimal rehabilitation programs postoperatively is an important research goal.

A novel device to apply controlled flexion and extension to the rat knee following anterior cruciate ligament reconstruction.

We designed and validated a novel device for applying flexion-extension cycles to a rat knee in an in vivo model of anterior cruciate ligament reconstruction (ACL-R). Our device is intended to simulate rehabilitation motion and exercise post ACL-R to optimize physical rehabilitation treatments for the improved healing of tendon graft ligament reconstructions. The device was validated for repeatability of the knee kinematic motion by measuring the force versus angular rotation response from repeated trials using cadaver rats. The average maximum force required for rotating an ACL reconstructed rat knee through 100 degrees of flexion-extension was 0.4 N with 95% variability for all trials within ±0.1 N.

Effect of short-duration low-magnitude cyclic loading versus immobilization on tendon-bone healing after ACL reconstruction in a rat model.

BACKGROUND: Successful anterior cruciate ligament reconstruction with use of soft-tissue grafts requires healing between tendon and bone. Little is known about the effect of mechanical load on the cellular and molecular cascade of tendon-to-bone healing. Understanding these mechanical influences has critical implications for postoperative rehabilitation following anterior cruciate ligament reconstruction. The purpose of this study was to test the hypothesis that, compared with perioperative immobilization, short-duration low-magnitude cyclic axial loading would result in impaired tendon-to-bone healing after anterior cruciate ligament reconstruction in a rat model. METHODS: Fifty-two male Sprague-Dawley rats underwent anterior cruciate ligament reconstruction with use of a flexor digitorum longus autograft. The patellar tendon, capsule, and ligamentous structures were circumferentially released, and an external fixator parallel to the anterior cruciate ligament graft was placed across the knee. Mechanical loading, consisting of cyclic displacement of the femur and tibia constrained to axial translation parallel to the graft, was applied daily. The rats were randomly assigned to immobilization or daily loading, for fourteen or twenty-eight days. Biomechanical, micro-computed tomographic, and histomorphometric analysis was performed on the bone-tendon-bone complexes. RESULTS: The load measured across the knees during cyclic displacement increased over time (p < 0.05). Load-to-failure testing of the isolated femur-anterior cruciate ligament graft-tibia specimens revealed no significant differences between groups at two or four weeks. By two weeks postoperatively, a greater number of ED1+ inflammatory macrophages (phagocytic cells involved in the initial injury response) were seen at the tendon-bone interface after loading in the cyclically loaded group than in the immobilized group (p = 0.01). Compared with the baseline values, the number of trabeculae was significantly lower after loading for four weeks (p = 0.02). CONCLUSIONS: Short-duration low-magnitude cyclic axial loading of the anterior cruciate ligament graft in the postoperative period is not detrimental to the strength of the healing tendon-bone interface but appears to be associated with greater inflammation and less bone formation in the tunnel in this rat model.

Effect of early and delayed mechanical loading on tendon-to-bone healing after anterior cruciate ligament reconstruction.

BACKGROUND: Modulation of the mechanical environment may profoundly affect the healing tendon graft-bone interface. The purpose of this study was to determine how controlled axial loading after anterior cruciate ligament reconstruction affects tendon-to-bone healing. Our hypothesis was that controlled cyclic axial loading after a period of immobilization would improve tendon-to-bone healing compared with that associated with immediate axial loading or prolonged immobilization. METHODS: One hundred and fifty-six male Sprague-Dawley rats underwent anterior cruciate ligament reconstruction with use of a flexor digitorum longus autograft. A custom-designed fixture was used to apply an external fixator across the knee parallel to the anterior cruciate ligament graft. Animals were randomly assigned to be treated with immobilization (n = 36) or controlled knee distraction along the long axis of the graft to achieve approximately 2% axial strain beginning (1) immediately postoperatively (n = 36), (2) on postoperative day 4 ("early delayed loading," n = 42), or (3) on postoperative day 10 ("late delayed loading," n = 42). The animals were killed at fourteen or twenty-eight days postoperatively for biomechanical testing, micro-computed tomography, and histomorphometric analysis of the bone-tendon-bone complex. Data were analyzed with use of a two-way analysis of variance followed by a post hoc Tukey test with p < 0.05 defined as significant. RESULTS: Delayed initiation of cyclic axial loading on postoperative day 10 resulted in a load to failure of the femur-anterior cruciate ligament-tibia complex at two weeks that was significantly greater than that resulting from immediate loading or prolonged immobilization of the knee (mean and standard deviation, 9.6 ± 3.3 N versus 4.4 ± 2.3 N and 4.4 ± 1.5 N, respectively; p < 0.01). The new-bone formation observed in the tibial tunnels of the delayed-loading groups was significantly increased compared with that in the immediate-loading and immobilization groups at both two and four weeks postoperatively (1.47 ± 0.11 mm(3) [postoperative-day-10 group] versus 0.89 ± 0.30 mm(3) and 0.85 ± 0.19 mm(3), respectively, at two weeks; p < 0.003). There were significantly fewer ED1+ inflammatory macrophages and significantly more ED2+ resident macrophages at the healing tendon-bone interface in both delayed-loading groups compared with the counts in the immediate-loading and immobilization groups at two and four weeks (2.97 ± 0.7 [postoperative day 10] versus 1.14 ± 0.47 and 1.71 ± 1.5 ED2+ cells, respectively, per high-power field at two weeks; p < 0.02). The numbers of osteoclasts in the delayed-loading groups were significantly lower than those in the immediate-loading and immobilization groups at two and four weeks postoperatively (0.35 ± 0.15 [postoperative-day-10 group] versus 1.02 ± 0.08 and 1.44 ± 0.2 cells, respectively, per high-power field at two weeks; p < 0.01), and the delayed-loading groups also had significantly reduced interface tissue vascularity compared with the other groups (p < 0.003). CONCLUSIONS: Delayed application of cyclic axial load after anterior cruciate ligament reconstruction resulted in improved mechanical and biological parameters of tendon-to-bone healing compared with those associated with immediate loading or prolonged postoperative immobilization of the knee.

A Novel In Vivo Joint Loading System to Investigate the Effect of Daily Mechanical Load on a Healing Anterior Cruciate Ligament Reconstruction.

We designed and validated a novel knee joint fixation/distraction system to study tendon-to-bone healing in an in vivo rat model of anterior cruciate ligament (ACL) reconstruction. The system uses an external fixator to apply a cyclic distraction of the knee joint while monitoring the resultant force developed across the joint, thus providing a temporal indication of structural changes during the healing process of the bone-tendon-bone reconstruction. The validation was performed using an optical kinematic tracking system to determine the local displacement of the knee. The average system compliance was determined to be 42.4 +/- 8.8 mum/N with a coefficient of variation of 20.7%. The compliance was used to obtain a best fit correction factor which brought the total root mean square error of knee joint distraction to within 179 mum (16.1%) of the applied distraction. We performed a pilot study using 15 rats that had ACL reconstructions using a flexor digitorum longus tendon autograft and found that the animals tolerated the indwelling fixator and daily anesthesia over a 10 day loading protocol. Our knee joint fixation/distraction system provides a valuable tool to study how mechanical stimuli affect in vivo bone-tendon-bone healing.