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Published in final edited form as: J Orthop Res. 2019 Jun 25;37(10):2249–2257. doi: 10.1002/jor.24381

Cartilage Damage is Related to ACL Stiffness in a Porcine Model of ACL Repair

Jillian E Beveridge 1, Benedikt L Proffen 2, N Padmini Karamchedu 1, Kaitlyn E Chin 1, Jakob T Sieker 2, Gary J Badger 3, Ata M Kiapour 2, Martha M Murray 2, Braden C Fleming 1
PMCID: PMC6739195  NIHMSID: NIHMS1028497  PMID: 31125133

Abstract

Inferior anterior cruciate ligament (ACL) structural properties may inadequately restrain tibiofemoral joint motion following surgery, contributing to the increased risk of post-traumatic osteoarthritis. Using both a direct measure of ACL linear stiffness and an in vivo magnetic resonance imaging (MRI) T2*-based prediction model, we hypothesized that cartilage damage and ACL stiffness would increase over time, and that an inverse relationship between cartilage damage and ACL stiffness would emerge at a later stage of healing. After either 6, 12, or 24 weeks (w) of healing after ACL repair, ACL linear stiffness was determined from the force-displacement relationship during tensile testing ex vivo and predicted in vivo from the MRI T2*-based multiple linear regression model in 24 Yucatan minipigs. Tibiofemoral cartilage was graded post-mortem. There was no relationship between cartilage damage and ACL stiffness at 6w (R2=0.04; p=0.65), 12w (R2=0.02; p=0.77), or when the data from all animals were pooled (R2=0.02; p=0.47). A significant inverse relationship between cartilage damage and ACL stiffness based on both ex vivo measurement (R2=0.90; p<0.001) and in vivo MRI prediction (R2=0.78; p=0.004) of ACL stiffness emerged at 24w. This result suggests that 90% of the variability in gross cartilage changes is associated with the repaired ACL linear stiffness at 6 months of healing.

Clinical Significance: Techniques that provide a higher stiffness to the repaired ACL may be required to mitigate the post-traumatic osteoarthritis commonly seen after ACL injury, and MRI T2* can be used as a noninvasive estimation of ligament stiffness.

Keywords: ACL, stiffness, porcine, knee, cartilage, osteoarthritis, MRI

INTRODUCTION

Surgical reconstruction, using either an autograft or allograft, remains the current gold standard treatment for anterior cruciate ligament (ACL) rupture. Despite its widespread use and clinical success, the procedure does not recapitulate the geometry or structural properties of the native ACL.1,2 In turn, these shortcomings could contribute to the finding that ACL reconstruction does not restore the normal joint kinematics,3 and may, in part, explain why the long-term risk of developing post-traumatic osteoarthritis (PTOA) continues to be a concern after ACL surgery.4

Because it can take up to a decade after ACL injury for PTOA to present in humans,4 subtle abnormal mechanics during repetitive low load activities could potentially play a role in disease progression.5 Animal models have demonstrated that the graft linear stiffness decreases following implantation and does not return to that of the normal joint.6,7 Therefore, reduced graft linear stiffness may modulate abnormal contact mechanics during activities that challenge the graft but does not cause overt failure.

In humans, direct measurement of ACL stiffness is not possible. However, increased magnetic resonance (MR) imaging signal intensity is indicative of greater tissue hydration and reduced organization,8 both of which suggest a more compliant tissue (i.e., lower linear stiffness).9 Coupled with the known residual kinematic abnormalities following ACL reconstruction, inferior graft stiffness may also contribute to residual abnormal joint kinematics. A significant barrier to understanding the trajectory of PTOA and determining a patient’s prognosis is that we do not yet know how closely the surgical procedure must come to restoring the native ACL function. In response to these shortcomings, a primary ACL repair procedure10,11 and a MR imaging technique12–14 has been developed to restore the native ACL geometry and to estimate its structural properties in vivo, respectively.

Using a preclinical porcine model, it has been shown that the linear stiffness of the repaired ACL is variable after repair, and that the variability in structural properties relates closely to the ACL’s histologic properties.15 It has been further shown that the total area of cartilage lesions at one year post-surgery is related to the structural properties of the repaired ACL.16 A recently developed MR-based imaging technique using a T2* multi-echo sequence has been used to predict the structural properties of the healing ACL in vivo as early as 6 weeks post-repair;12,13 however, it is unknown at what time after ACL surgery the relationship between ACL stiffness and cartilage damage might emerge.

Prior studies have shown that the biological milieu of healing ligament changes rapidly within the first 20 weeks of healing,17,18 after which the ligament enters a prolonged phase of collagen reorganization and remodeling.19 Our non-invasive T2* prediction model corroborated these histological observations, whereby the quantity of disorganized ACL tissue with higher T2* relaxation times within the first 6–12 weeks post-repair was a significant predictor of structural properties, but by 24 weeks, the repaired ACL was largely (>50%) composed of organized tissue.12 Therefore, it is likely that rapid functional healing may stabilize after 24 weeks, and the outcome measures collected at this stage of wound healing may be better predictors of long-term biomechanical function. This timing of when to examine ligament remodeling may be critical to the clinician’s return-to-sport decision making and gauging a patient’s risk for subsequent joint degeneration.

The purpose of this study was twofold: (1) to investigate the relationship between ACL linear stiffness and cartilage damage within the 24-week sub-acute post-operative period in a porcine model of ACL repair; and (2) to test whether the same relationship could be detected between cartilage damage and ACL stiffness predicted by our MRI T2*-based regression model. We hypothesized 1) that ACL stiffness and cartilage damage would increase over time while the median ACL T2* relaxation time would decrease, and 2) that cartilage damage would be inversely related to actual and predicted ACL stiffness by 24 weeks post-surgery.

METHODS

Subjects and Surgical Procedure

Twenty-four 15±1 month-old (12 castrated males, 12 females; Sinclair Bio Resources, MO) Yucatan minipigs were randomized to receive primary suture repair with (n=12; 6 female) or without (n=12; 6 female) a scaffold to enhance healing after ACL transection.20 Using a priori data,14 this sample size was based on the ability to detect an increase in R2 from 74% to 86% with over 95% power at α=0.05 when modeling the relationship between T2* metrics and ACL structural properties using multiple linear regression analyses.

Animals were housed individually in pens (minimum pen size 22.4 ft2), which were located adjacent to one another, on a 12/12 hour light/dark cycle, fed twice daily with a lab-based diet, had free access to drinking water, and were monitored daily by veterinary staff. Environments were enriched with toys on a regular basis. Animals were free to engage in unrestricted activity within the pens, which was not monitored quantitatively.

The minipigs were deemed healthy by veterinary staff prior to the start of the study, and all procedures were approved by the Institutional Animal Care and Use Committee. At the time of surgery, animals were sedated using telozol with xylazine, then intubated and maintained under general anaesthesia using isofluorane. The ACL was transected at the junction of the proximal and middle thirds of the ligament. Immediately following transection, animals received primary suture repair either with, or without, the scaffold.20 Other than the scaffold, the two surgical procedures were equivalent and were implemented to create a greater spread in the ligament stiffness and cartilage health data.12,21 Animals were allowed unrestricted weight bearing following the surgery.

In Vivo MR Imaging

In a cross-sectional study design, animals were randomly allocated to one of three groups based on post-operative healing duration of 6, 12, or 24 weeks (w) (n=8 per group with an equal number of suture repairs vs. enhanced repairs and males/females in each group). The baseline weights (mean±standard deviation) of the 6-, 12- and 24-week groups were 54.2±4.9 kg, 53.1±2.8 kg and 52.5±4.2 kg, respectively. For in vivo MR imaging, animals were sedated and maintained under general anesthesia using the same drug regimen described for the surgical procedures. The knees were imaged with a 3T scanner (Prisma; Siemens, Erlangen, Germany) using a four-channel flexible coil (Flexcoil; Siemens), and a 3D gradient multi-echo sequence, the details of which have been described previously.12,13 As a result of sedation, all animals were non-weight bearing for at least 60 minutes prior to imaging. Care was taken to place the knee coil and orient the limb such that the knee flexion angle and the orientation of the ACL relative to the magnetic field (i.e., B0) was similar across animals. Data demonstrating reproducibility are provided in the online supplement. The MR images for seven of the eight 6w pigs were acquired using a 6-echo sequence and a 384×384 acquisition matrix (TEs=2.48, 6.86, 11.24, 15.62, 20.00 and 24.38); a 4-echo sequence and 512×512 acquisition matrix (TEs=2.8, 7.88, 12.96, 18.04) was used for all other animals. The matrix resolution was increased to enhance visualization of the ACL border. Other MR sequence parameters (FOV=160×160mm; ST/gap=0.8mm/0mm; TR=29ms; FA=12°) were consistent for all scans. We have shown that the differences in the T2* fit of these in vivo data as a result of differing echo number and resolution were minimal, but that MR T2* metrics were sensitive to in vivo versus in situ or ex vivo conditions.13 This latter finding led us to conduct all scanning in vivo to better approximate future clinical applications. Animals were euthanized immediately after imaging with an injection of phenytoin/pentobarbital solution, and the hind limbs were harvested and frozen.

ACL Structural Properties

Prior to mechanical testing, the hind limbs were thawed to room temperature and dissected leaving only the femur-ACL-tibial complex intact. The proximal end of the femur and distal end of the tibia were potted in urethane resin-filled PVC pipe, mounted in a custom frame such that the long axis of the ACL was aligned with the direction of the applied tensile load, and then tensile tested to failure at a rate of 20mm/min with a servohydraulic material testing system (MTS 810; Prairie Eden, MN).22 Linear stiffness (N/mm) was calculated from the load-displacement data.22

Cartilage Grading

Medial and lateral tibial plateaus and femoral condyles were examined for cartilage damage by a single observer with significant experience performing gross scoring in this model (author BLP). We have previously determined that the ICC in the macroscopic scoring system between examiners was .96.23The articular cartilage of these four weight-bearing regions was graded from 0 (no damage) to 4 (exposed bone >10%) (Table 1).24 Scores were then summed within the surgical and contralateral knees, and the contralateral scores were subtracted from the surgical scores; positive cartilage scores indicate the degree that the surgical limb cartilage exhibited greater damage than the contralateral limb. The maximum possible cartilage damage score was 16.

Table 1.

Scoring for assessment of cartilage gross morphology.24

Grade Description
0 No observable gross changes are present
1 Intact surface with color changes
2 Surface fibrillation
3 Exposed subchondral bone
4 Subchondral bone is exposed in >10% of the lesion, and cartilage is fragmented at lesion borders
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ACL T2* Estimation & Prediction Model

To calculate voxel T2* relaxation times, ACLs were segmented manually (Mimics v16, Belgium) from the MR T2* image stack by a single observer, and then a monoexponential decay function25 was fitted to the signal decay on a voxel-wise basis to all voxels contained within the segmented ACL mask as described previously.14,26 Organized and disorganized ACL sub-volumes (in mm3) were calculated by summing the volume of all voxels with a T2* relaxation time between 0–12.5ms (denoted as Vol1), or 37.6–50ms (denoted as Vol4), respectively.12,14 All T2* calculations were performed using custom-written software with Matlab (v2015b, Natick, MA).

The multiple linear regression model used to predict ACL structural properties was based on the changes in the proportion of organized to disorganized collagen contained within the healing ACL over time,12 with shorter T2* relaxation times corresponding to more organized collagen.27 The following equations were used to predict ACL stiffness at 6, 12 and 24 weeks post-operatively:

Stiffness6w = −8.58 + 1.91(Vol1) + 0.70(Vol4) −0.10(Vol1 x Vol4)

Stiffness12w = −59.73 + 10.59(Vol1) + 11.41(Vol4) −1.90(Vol1 x Vol4)

Stiffness24w = 68.05 – 9.62(Vol1) − 15.74(Vol4) + 2.38(Vol1 x Vol4)

The model coefficients were based on log-transformed data to account for the skewed distribution of sub-volumes. A detailed description of the regression models is available in Beveridge et al.12

Statistical Analyses

Differences in post-operative ACL median T2* relaxation time, stiffness, and cartilage damage were tested using a one-way analysis of variance with Tukey’s adjustments for multiple comparisons between time points. Linear regression analyses were used to test the relationship between the severity of cartilage damage and ACL stiffness at each time point and across time points. All statistical calculations were performed using GraphPad Prism (v.7.1, San Diego, CA). Results were considered significant if P<0.05.

RESULTS

All animals completed the study. Except for one animal, no gross degenerative cartilage changes were noted at the time of ACL surgery. All contralateral cartilage scores were zero (no damage) except for one 12w animal (score=2), and one 24w animal (score=1). Cartilage scores increased with post-operative time (p=0.016). Post-hoc analyses revealed that 12w and 24w scores were significantly greater than 6w scores (mean, [95% CI]): 2.3, [1.08, 4.17] at 12w, 4.1, [2.37, 5.8] at 24w, versus 1.4, [0.49, 2.26] at 6w (Figure 1.A). The mean scores for each post-operative time point represented approximately 9%, 16%, and 26% of the highest possible cartilage score.

Figure 1.

Figure 1.

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Cartilage scores (A), ACL median T2* relaxation time (B) and stiffness (C) at 6, 12, and 24 weeks (w) post-ACL repair. Bars indicate the group mean and 95% CIs. *p<0.05.

Median ACL T2* decreased with healing time (p=0.02), with 24w relaxation times (12.4 ms, [11.4, 13.3]) being significantly shorter than 6w times (17.2 ms, [13.8, 20.6])(p=0.02). 12w median T2* relaxation times (14.4 ms, [11.5, 17.3]) were between 6 and 24w values (Figure 1.B).

ACL stiffness increased significantly with healing time (p=0.001), with 12w (62.1 N/mm, [40.1, 84.1]) and 24w (86.6 N/mm, [65.3, 108]) stiffness being significantly greater than 6w stiffness (27.1 N/mm, [17.5, 36.8]) (Figure 1.C). These mean stiffness values correspond to 13%, 28% and 37% of contralateral values at 6, 12, and 24w, respectively.

There were no relationships between cartilage score and ACL stiffness at 6 weeks (R2=0.04; p=0.65. Figure 2.A), 12 weeks (R2=0.02; p=0.77. Figure 2.B), or when the data from all animals were pooled (R2=0.02; p=0.47). However, there was a strong and significant inverse relationship between cartilage damage and ACL stiffness at 24 weeks (R2=0.90; p<0.001. Figure 2.C). Examples of the cartilage damage at 24 weeks and their relative distribution along the regression line are shown in Figure 3.

Figure 2.

Figure 2.

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Linear regression at 6 weeks (A. n=8), 12 weeks (B. n=8), and 24 weeks (C. n=8). Dotted lines indicate 95% CIs. n.s. = not significant.

Figure 3.

Figure 3.

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Examples of the corresponding cartilage scores for three animals that spanned the spectrum of ACL stiffness values and corresponding cartilage scores (A-C, circled in red in the scatter plot on the left). The black arrows point to the regions of articular degradation that contributed to the cartilage score noted above each panel. Shown are distal femurs and lateral tibial plateaus.

In vivo MR imaging revealed that animals with a larger ACL sub-volume comprised of short T2* relaxation times had greater ACL stiffness. The T2* maps for the same three animals in Figure 3 are shown in Figure 4.A–C. The corresponding inverse relationship between cartilage score and T2* model-predicted ACL stiffness was also significant (R2=0.78; p=0.004; Figure 5).

Figure 4.

Figure 4.

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Maps of ACL T2* relaxation times at a mid-sagittal slice is shown for the same three subjects that spanned the healing ACL stiffness values shown in Fig 3. The predicted stiffness values are in close agreement with the actual values. T2* relaxation times correspond to the color bars on the right, and are reported in milliseconds.

Figure 5.

Figure 5.

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Cartilage scores were related to model-predicted ACL stiffness at 24 weeks (n=8) from MR imaging.

DISCUSSION

The purpose of this study was to investigate whether a relationship existed between the healing ACL linear stiffness and cartilage damage within a 24-week postoperative period in a porcine model of ACL surgery, and to test whether the same relationship, if present, held true for stiffness values obtained from our in vivo MRI T2* prediction model. The results supported our hypotheses that ACL stiffness and cartilage damage would increase with healing duration while median ACL T2* relaxation time would decrease, and that cartilage damage would be inversely related to actual and MR-predicted ACL stiffness by 24 weeks post-surgery.

Results from the regression analyses (Figure 3) suggest that inferior 24-week ACL stiffness is associated with cartilage damage. Although we cannot ascertain from the current data whether the cartilage damage we observed was a direct result of a lower ACL stiffness, there is in vivo,28 in vitro,29 and in silico30 evidence to suggest that ACL-mediated abnormal contact mechanics contribute to cartilage damage. Studies in a sheep model of combined ACL and medial collateral ligament (MCL) deficiency have shown that the severity of gross cartilage damage observed 20 weeks after injury increases linearly with the degree of abnormal dynamic alignment31 and increased relative contact surface velocities.32 A sheep model of “idealized” ACL reconstruction – where the native ACL was used as the graft – has shown that the kinematics of some animals become more ACL-deficient-like by 20 weeks. Similarly to the ACL-deficient model, sub-regional cartilage scores correlated with the magnitude of the shift in internal tibial rotation and anterior translation.33 The authors of this previous work speculated that the ACL “graft” could have become more compliant given the kinematic similarities between the affected ACL-reconstructed and ACL-deficient animals.34

Although we did not perform ACL reconstruction in the current study, the variation in ACL stiffness after 24 weeks of healing, which also correlated with gross cartilage damage, adds support to the hypothesis that post-operative ACL (and/or graft) stiffness modulates stifle contact mechanics. Recent work by Halonen and colleagues further supports this view, whereby the authors used finite element modeling to demonstrate that ACL grafts with stiffness properties closest to those of the native ACL restored tibiofemoral contact mechanics, regardless of the reconstruction technique.35 That is, ACL graft stiffness had the greatest bearing on contact mechanics. In light of the results by Halonen et al and those of our current study, recapitulating native ACL stiffness may be required to mitigate long-term degenerative changes in the ACL-injured joint.

In addition to the inverse relationship between ACL stiffness and cartilage damage observed here, both cartilage scores and ACL stiffness increased with time post-surgery, while median ACL T2* relaxation time decreased (Figures 1 & 2). Taken together, the mean increase in ACL stiffness alongside the reduced median ACL T2* relaxation time over the 24 week study duration suggests that the ACL is healing following primary repair. We have described the biological and biomechanical trajectories of this procedure in detail previously,21,36 whereby the structural properties of the (enhanced) repaired ACL continue to increase with healing duration and are not different from those of conventional ACL reconstruction by one year post-surgery.20,37 If cartilage damage was the result of ACL stiffness alone, we would have expected to observe the relationship between ACL stiffness and cartilage scores across all time points – not just the 24w time point – as can be seen in Figure 2. Although there is strong evidence to support the paradigm that joint mechanics modulate long-term joint health, there is equally compelling evidence that the biological milieu within the acute and sub-acute injury phases could play a major role in early degenerative changes.38,39 And while some degree of inflammation is required for wound healing,40 chronic inflammation can lead to tissue catabolism, impaired soft tissue mechanical properties,41 and greater tissue damage with time.42 With respect to the Yucatan minipig specifically, we used RNA-sequencing to show that cartilage and synovium proteolytic pathways are altered ubiquitously within the first 4 weeks after stifle surgery, and that the corresponding histologic evidence of PTOA is likewise consistent across surgical interventions despite different degrees of joint instability (ACL transection vs. reconstruction or repair).43,44 Drawing on the results of these corollary studies, the mild degenerative cartilage changes observed at 6 and 12 weeks may be modulated predominantly by the biological milieu at these early post-surgical time points. Nevertheless, a more sensitive measure of articular cartilage change – such as histology or MR T2 mapping45 – may reveal that the relationship between cartilage damage and ACL stiffness is conserved at the microstructural scale. Although we do not know to what extent the cartilage damage observed at 24 weeks might continue to progress with time, previous work in our porcine model has shown that enhanced ACL repair results in less macroscopic articular damage after one year compared to conventional ACL reconstruction.16,20,37 It should be noted that the 24w cartilage damage scores were similar to those previously observed at the same post-surgical interval in ACL-repaired Yucatan minipigs,37 suggesting that the trajectory of long-term cartilage damage is likely to be similar.

As a secondary aim of the current study, we tested whether the cartilage scores at 24 weeks also correlated with their corresponding in vivo MR T2*-predicted ACL stiffness values. Although the prediction model uses T2* voxel information from the entire ACL volume, the shift towards shorter ACL T2* relaxation times across three subjects with varying ACL stiffness can be seen clearly from even a single mid-sagittal slice, as shown in Figure 4. Furthermore, the consistency in the coefficients of determination of the regression analyses based on actual versus predicted stiffness values suggests that the MRI T2* model provides some indication of cartilage damage risk. Of particular interest is that the relationship between cartilage damage and ACL stiffness emerges at 24 weeks, roughly at the same time clinicians begin to increase patient activity intensity and consider a return to sport.46

One of the advantages of using a preclinical model is that there is no concomitant injury at time zero. Therefore, the relationship between inferior ACL stiffness and cartilage damage that we observed was most likely related to the experimental condition. Nevertheless, it is worth acknowledging that the regression analyses cannot be interpreted as direct causality. It is possible that ultrastructural and compositional degenerative changes were present at the time of surgery, and were aggravated by the surgery irrespective of the changes in joint mechanics. We controlled for any pre-existing cartilage abnormalities by taking into account contralateral cartilage scores. In using this “normalization”, we assume that cartilage was symmetrical at time zero. We believe that this assumption was reasonable given that there was minimal contralateral damage observed at euthanasia and minimal experimental limb involvement at the time of surgery. Our ongoing study examining simultaneous longitudinal cartilage and ligament changes will likely yield more insight into the time-course and symmetry of changes in the intra-articular environment.

While we chose to use a 3D gradient multi-echo sequence to quantify ACL T2* relaxation times, ultrashort echo (UTE) sequences could be a reasonable alternative; however, the large (3mm) slice thickness required for UTE MR protocols47,48 would likely negatively impact our ability to capture whole-ligament T2* in the (smaller) porcine ACL by introducing significant partial volume effects at the boundaries between ACL and adjacent tissues and/or synovial fluid. Because the structural properties of a tissue are governed by the total quantity and quality of tissue, we prioritized the ability to quantify T2* relaxation throughout the entire ACL volume rather than restricting the analysis to only a central region of the ligament. While UTE sequences have demonstrated sensitivity to changes in intact tendon and ligament ultrastructure with measured T2* relaxation times on the order of 3–14.8ms,47,48 we have previously shown that the median T2* relaxation of healing ACL after surgery falls within the range of 7–16ms,14 which can be nearly twice as long as the median T2* of intact ligament at 8.5±0.6ms.13 Furthermore, we have shown that these relaxation times correlate with semi-quantitative histology measures.49 The increase in median ACL T2* relaxation times in Figure 1.B. corroborates the findings of these earlier studies. We therefore believe that the gradient echo sequence described here is well-suited for capturing relevant changes in MR T2* relaxation times that are characteristic of healing ligament within six months after ACL repair. Nevertheless, the application of UTE sequences with smaller slice thicknesses that are sensitive to even shorter T2* values may be useful in characterizing the later stages of ligament healing, and could be an avenue of future investigation.

We must acknowledge several limitations of our study. In patients, reintroduction to limb loading and neuromuscular retraining are important elements of ACL rehabilitation protocols.46 Because neither of these were controlled in our animals, it is unknown how these factors may influence tissue healing and joint biomechanics in patients. Similarly, the timing of when a relationship between impaired ACL stiffness and cartilage damage emerges may be different in humans. Secondly, our results are based on ACL repair and not the current clinical gold standard of ACL reconstruction. Although primary healing and ligamentization share similar biological processes,18,50 differences are likely to exist at these early post-operative time points and direct translation of our results to ACL reconstruction should be done with caution. Third, ACL stiffness values at 24 weeks ranged from 16–48% of contralateral values over a continuous spectrum. Therefore, establishing clear criteria for “risk” from a linear relationship may be difficult to establish without larger group samples sizes and a longitudinal study design that would enable more sophisticated statistical analyses. Fourth, we were not able to evaluate the effects that sex and surgical repair technique may have played in ACL healing as the study was not powered to do so. Fifth, cartilage damage was quantified using only gross scoring measures and did not include other complementary measures such as histology or MR T2 mapping techniques. Lastly, the T2* linear regression prediction model was optimized from these same animals,12 and therefore represents a best-case-scenario estimation of the structural properties. In light of this limitation, the performance of the prediction model must be tested on a different set of animals, which we are doing in an ongoing longitudinal study.

Using a translational model of ACL repair, we tested the hypotheses that ACL stiffness would increase while median T2* relaxation time would decrease over time, and that cartilage damage would be inversely related to ACL stiffness after 24 weeks of healing. Our results supported the hypotheses, suggesting that restoring ACL stiffness may be necessary to mitigate cartilage damage progression following ACL surgery, and that the 24 week post-operative time frame may be an important time interval at which to assess biomechanical function.

Supplementary Material

Appendix

ACKNOWLEDGEMENTS

We gratefully acknowledge support from the National Institutes of Health [NIAMS 3R01-AR065462, NIAMS 2K99AR069004, and NIGMS 5P20-GM104937 (Bioengineering Core of the COBRE Centre for Skeletal Health and Repair)], the Lucy Lippitt Endowment, the RIH Orthopaedic Foundation, and the Boston Children’s Hospital Orthopaedic Surgery Foundation. We also thank the staff at the Brown University Magnetic Resonance and Animal Care Facilities for their technical assistance. We especially thank Scott McAllister for his help with animal husbandry and surgical procedures, Dr. Kimberly Waller for her assistance with surgery and oversight of the study logistics, and Gary Badger and Dr. Jason Machan for their assistance with the statistical analyses. Dr. Murray is an inventor on patents held by Boston Children’s Hospital related to the scaffold that was used in this study. Drs. Murray, Proffen and Boston Children’s Hospital have equity interests in MIACH Orthopaedics. Dr. Murray also holds research grants from the NIH, DoD and NFLPA through the Harvard Football Players Health Study. Drs. Murray and Fleming receive royalties from Springer Publishing, and Dr. Fleming receives a stipend from Sage Publishing for his work as an associate editor for a medical journal.

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