Subchondral and epiphyseal bone remodeling following surgical

Osteoarthritis and Cartilage 24 (2016) 698e708

Subchondral and epiphyseal bone remodeling following surgical transection and noninvasive rupture of the anterior cruciate ligament as models of post-traumatic osteoarthritis T. Maerz y z x, M. Kurdziel y z, M.D. Newton y, P. Altman k, K. Anderson z k, H.W.T. Matthew x ¶, K.C. Baker y z * y Orthopaedic Research Laboratory, Beaumont Health System, Royal Oak, MI, United States z Department of Orthopaedic Surgery, Oakland University e William Beaumont School of Medicine, Rochester, MI, United States x Department of Biomedical Engineering, Wayne State University, Detroit, MI, United States k Department of Orthopaedic Surgery, Beaumont Health System, Royal Oak, MI, United States ¶ Department of Chemical Engineering & Materials Science, Wayne State University, Detroit, MI, United States

a r t i c l e i n f o

s u m m a r y

Article history: Received 10 August 2015 Accepted 6 November 2015

Objective: Animal models are frequently used to study post-traumatic osteoarthritis (PTOA). A common anterior cruciate ligament (ACL) injury model is surgical transection, which may introduce confounding factors from surgery. Noninvasive models could model human injury more closely. The purpose of this study was to compare subchondral and epiphyseal trabecular bone remodeling after surgical transection and noninvasive rupture of the ACL. Methods: Thirty-six rats were randomized to an uninjured control, surgical transection (Transection), or noninvasive rupture (Rupture). Animals were randomized to 4 or 10 week time points (n ¼ 6 per group). Micro computed tomography (mCT) imaging was performed with an isotropic voxel size of 12 mm. Subchondral and epiphyseal bone was segmented semi-automatically, and morphometric analysis was performed. Results: Transection caused a greater decrease in subchondral bone volume fraction (BV/TV) than Rupture in the femur and tibia. Rupture had greater subchondral bone tissue mineral density (TMD) at 4 and 10 weeks in the femur and tibia. Subchondral bone thickness (SCB.Th) was decreased in the femur in Transection only. Epiphyseal BV/TV was decreased in Transection only, and Rupture exhibited increased femoral epiphyseal TMD compared to both Control and Transection. Rupture exhibited greater femoral epiphyseal trabecular thickness (Tb.Th.) compared to Control and Transection at 4 weeks, and both Rupture and Transection had increased femoral epiphyseal Tb.Th. at 10 weeks. Epiphyseal trabecular number (Tb.N) was decreased in both injury groups at both time points. Femoral and tibial epiphyseal structure model index (SMI) increased in both groups. Conclusions: The two injury models cause differences in post-injury bone morphometry, and surgical transection may be introducing confounding factors that affect downstream bony remodeling. © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Keywords: Post-traumatic osteoarthritis Anterior cruciate ligament rupture Subchondral bone remodeling Epiphyseal bone remodeling Bone morphometry mCT

Introduction Post-traumatic osteoarthritis (PTOA) is the consequence of acute and/or chronic joint trauma1, and although several risk factors

* Address correspondence and reprint requests to: K.C. Baker, Orthopaedic Research Laboratory, Beaumont Health System, 3811 W Thirteen Mile Road, Royal Oak, MI 48073, United States. Tel: 1-248-551-9177; fax: 1-248-551-0191. E-mail address: [email protected] (K.C. Baker).

contributing to the progression of PTOA have been identified2, its pathophysiology has yet to be fully defined. Anterior cruciate ligament (ACL) rupture is a common sporting injury and a wellknown risk factor for the development of PTOA. Although reported PTOA incidence rates after ACL rupture vary in the literature, long-term clinical studies have demonstrated the risk for the development of PTOA after ACL rupture to be as high as 100%3e6. While surgical reconstruction alleviates pain and enables the patient's return to sport, it does not appear to lower the incidence of PTOA6e10.

http://dx.doi.org/10.1016/j.joca.2015.11.005 1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

T. Maerz et al. / Osteoarthritis and Cartilage 24 (2016) 698e708

ACL rupture induces a biologic tissue response in articular cartilage, subchondral bone, epiphyseal bone, the meniscus, the synovium, and other ligaments of the knee6. In addition to the acute joint trauma from ACL injury, chronic joint destabilization causes adverse, non-anatomic tissue loading, which is a major proposed contributor to chronic inflammation and the perpetuation of OA1. Bony remodeling, including loss of subchondral and epiphyseal bone and decreased epiphyseal bone mineral density (BMD), is known to occur following ACL injury11e14 and as part of the chronic OA cascade11e17. However, clinical studies outlining longitudinal morphological changes in subchondral and epiphyseal bone following ACL injury are, to date, lacking. Animal models of PTOA are widespread, and most models are based on surgical destabilization or chemically/enzymaticallyinduced degeneration. In the rat, the most prevalent PTOA model involves surgical transection of the ACL to induce tibiofemoral instability, which has been shown to induce osteoarthritis (OA)like symptoms including loss of cartilage proteoglycan content18,19, osteophyte formation20, altered biomechanical properties of articular cartilage19, loss/thinning of articular cartilage18, and chondrocyte death21. The ACL transection model has been employed extensively to study bony changes after injury, including subchondral bone loss22, trabecular thinning22, alterations in BMD23, and osteophyte formation20,24. However, given the inherently invasive nature of this surgical model, it is unclear whether these effects are representative of bony changes in humans after traumatic ACL rupture. As such, noninvasive injury models may provide a more accurate method to study degenerative changes of the tibiofemoral joint after ACL injury. Few studies assessing bony changes after noninvasive ACL injury have, however, been published. Christiansen et al. utilized a tibial compression overload model in mice and demonstrated bone loss, trabecular thinning, and decreases in BMD of the femoral and tibial epiphysis25,26. Recently, the tibial compression model has been implemented and biomechanically characterized in the rat27, but no studies assessing bony changes after this injury are, to date, available. Onur et al. have utilized cyclic tibiofemoral compression to noninvasively induce ACL rupture and demonstrated osteophyte formation and subchondral sclerosis, but no quantitative morphometric analysis of subchondral or trabecular bone was performed28. Xue et al. developed a rotational model to noninvasively induce ACL rupture in the rat to study matrix metalloproteinase (MMP) expression following injury, but this study also did not perform quantitative assessment of bony remodeling29. It is unclear whether surgical animal models of ACL injury accurately mimic the bony response of human ACL rupture, and, to date, no study has compared bony remodeling between noninvasive and surgical ACL injury models. To this end, the purpose of this study was to quantify and compare subchondral and epiphyseal bone morphometry following an open-joint ACL injury model (i.e., surgical ACL transection) and a closed-joint, mechanically-induced ACL injury model (i.e., noninvasive ACL rupture). We hypothesized that due to the traumatic nature of the rupture injury, noninvasive rupture will result in a more severe onset of degenerative morphometry, namely a greater degree of subchondral bone loss, trabecular thinning, and loss of inherent bone tissue mineral density (TMD) compared to surgical ACL transection. Methods Treatment groups and procedures Under an institutional animal care and use committee (IACUC)approved protocol, thirty-six female Lewis Rats (14 weeks of age, Charles River Laboratories, Wilmington, MA, USA) were

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randomized to one of three treatment groups: Control, noninvasive ACL rupture (Rupture), or surgical ACL Transection (Transection) (n ¼ 12 per group). Animals within each group were then randomized to one of two time points: 4 weeks or 10 weeks (n ¼ 6 per group). This sample size was determined based on previous studies assessing bone morphometry following joint injury. Randomizations were performed using computer software. On the morning of all procedures, animals in both the Rupture group and in the Transection group were administered 5 mg/kg subcutaneous Carprofen, a non-steroidal anti-inflammatory drug. Anesthesia was induced by intraperitoneal ketamine and xylazine and maintained with 0.5e1.5% inhaled isoflurane during the duration of the procedure. Postoperative analgesia was achieved via subcutaneous buprenorphine injection. Animals in the Rupture group underwent a noninvasive ACL rupture using a biomechanical loading protocol with a custom fixture on a materials testing system (Insight 150, MTS, Eden Prairie, MN, USA), as previously described27. In brief, the animal was placed prone on a thermally-regulated bed, and the right knee was flexed to 100 on a stage with a 3 mm deep trough restricting medial/lateral translation. The paw was mounted in 30 of dorsiflexion in a fixture constraining all motions except flexion/extension [Fig. 1(A)]. Following preload, 10 cycles of preconditioning, and a secondary preload to 15 N, a rapid vertical displacement of 3 mm at 8 mm/s was applied to the paw fixture. The rapid axial tibial displacement causes anterior tibial subluxation, tibial internal rotation, failure of the ACL, followed by tibial external rotation, as previously characterized27. ACL failure occurs at ca. 60e70 N of axial force and 2.0e2.5 mm of displacement, resulting in both anterior laxity and varus laxity. Complete ACL rupture was confirmed following biomechanical loading using an anterior drawer test, whereby anterior joint laxity is confirmed by the application of an anterior tibial force [Fig. 1(B, C)]. In the Transection group, complete surgical transection of the ACL was performed as previously described21,30,31. In brief, following anesthetic administration as described above, the right knee was shaved and prepared with betadine and alcohol. The animals were positioned supine on a warm water recirculator to aid in thermoregulation. A midline knee incision was made using a No. 15 scalpel blade. A medial parapatellar arthrotomy was made, and the patella was subluxated laterally. The knee was then hyperflexed to aid in visualization of the ACL and to maintain patellar subluxation. The ACL was then transected mid-substance using a Size 0 micro scalpel (Biomedical Research Instruments, Silver Spring, MD, USA), taking care to avoid contact with any cartilaginous surfaces. Anterior and posterior drawer testing was subsequently performed to confirm complete ACL rupture and posterior cruciate ligament integrity. The knee was lavaged with sterile saline. The arthrotomy was then closed using 6-0 Prolene (Ethicon 8697G, Somerville, NJ, USA) interrupted sutures and the skin was closed using 5-0 undyed Vicryl (Ethicon J493G) buried interrupted sutures. Animals in the Control group were administered identical analgesia and anesthesia but received no injury or surgical treatment. Since the purpose of this study was to compare the overall effect of ACL transection to a noninvasive, closed-joint ACL injury, a sham injury (i.e., skin incision, arthrotomy, patellar dislocation, and suturing) was not utilized in our Control group. The use of a surgical sham control would not have been appropriate for comparison to the Rupture group, and an age-matched, anesthesia- and analgesiaonly control was chosen to be most appropriate for accurate comparison to both injury groups. All animals were allowed normal diet and ad libitum cage activity in a light/dark cycle facility. Animals were housed individually for the first postoperative week and then in groups of 4e6 animals until sacrifice at either 4 or 10 weeks via CO2 asphyxia.

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Fig. 1. Noninvasive ACL rupture was induced using the tibial compression model with which a rapid 3 mm displacement is applied to the tibia via the dorsiflexed paw, as previously characterized27. During rupture loading, the knee is flexed to ~100 and held within a trough constraining only medial/lateral motion (A). Rupture was confirmed by an anterior drawer test, whereby anterior joint laxity is confirmed by the application of an anterior tibial force (B, C).

Micro computed tomography (mCT) and histology Immediately following sacrifice, the affected limbs of each animal were fully dissected to expose the articular cartilage surfaces of the femur and tibia. Each limb was fully immersed in 10% neutralbuffered formalin (NBF) for 48 h and rehydrated in phosphatebuffered saline (PBS) for 24 h. To facilitate contrast-enhanced imaging of articular cartilage as part of a related study, equilibrium partitioning of an ionic contrast agent (EPIC)-mCT was employed, and all specimens were fully immersed in 20% ioxaglate (Hexabrix, Guerbet Group, France), pH ¼ 7.2 for 24 h. This contrast agent increases the radiodensity of bone marrow and soft tissues but not of bone. Specimens were then carefully dabbed with a moist towel and mounted rigidly in a humidified mCT specimen holder containing humidifying beads able to maintain an 80% humid atmosphere within the holder. mCT imaging of the distal femur and proximal tibia was performed at 70 kVp, 114 mA with an isotropic voxel resolution of 12 mm and a 250 ms integration time (mCT40, Scanco Medical, Brütissellen, Switzerland). Following imaging, specimens were rinsed in PBS to remove the contrast agent and dehydrated using an ethanol series up to 70% v/v ethanol, in which all specimens were stored until histologic processing. All samples were decalcified to completion in 10% formic acid and dehydrated in an increasing ethanol series. Four sagittal, 200 mm-spaced sections from the medial and lateral compartment of the femur and tibia were then stained with both Hematoxylin &

Eosin (H&E) as well as Safranin-O/Fast Green (Saf-O). Microscopic imaging was performed at 20 using an automatic slide imaging system (Aperio, Leica Biosystems, Buffalo Grove, IL, USA). Qualitative evaluation of articular cartilage was performed by three blinded investigators using the OARSI Modified Mankin score32. Results of all three raters were averaged to calculate a composite score for each animal, and composite scores of each animal within a group were averaged to obtain a group average at each time point. Image segmentation and analysis

mCT images were converted to DICOM, filtered using a noisereducing Gaussian filter (s ¼ 0.8), and a custom MATLAB (r2014a, The Mathworks, Natick, MA, USA) program was employed to segment femoral and tibial subchondral and epiphyseal bone. In order to analyze all subchondral bone, the entire articular cartilage surface was first manually segmented on serial raw images [Fig. 2(A, E)] as previously shown33. In brief, due to the drastic differences in the attenuation between air, contrast-enhanced articular cartilage, and bone, outlining around articular cartilage to include both air and subchondral bone allows for accurate segmentation [Fig. 2(B, F)]. A three-dimensional (3D) region-growing algorithm (Christian Wuerslin, University of Tuebingen and University of Stuttgart, Germany) was employed to segment cartilage voxels. Small, malsegmented objects representing subchondral bone marrow were removed using a purification algorithm, which


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Fig. 2. Segmentation process of femoral and tibial subchondral bone. Femoral and tibial articular cartilage was contrast enhanced via EPIC-mCT (A, E) to enable manual outlining (B, F, grey ROI) and segmentation of articular cartilage (B, F, black ROI) using a 3D region-growing algorithm. Subchondral bone was then segmented automatically (C, G, black ROI) using a dilation and thresholding algorithm to yield femoral (D) and tibial (H) subchondral bone volumes.

dictates that only one large volume (i.e., articular cartilage) can remain following processing. Subchondral bone was then segmented automatically using the already-segmented articular cartilage volume. A custom program segmented subchondral bone 22 voxels (i.e., 264 mm) deep to articular cartilage in the femur and 30 voxels (i.e., 360 mm) deep to articular cartilage in the tibia [Fig. 2(C, G)], parameters which were optimized in preliminary experiments. A high-pass intensity threshold of 3500 HU removed air, articular cartilage, bone marrow, and other soft tissue to yield only subchondral bone [Fig. 2(D, H)]. Mean Subchondral bone plate thickness (SCB.Th.Mean) was quantified using a cortical thickness analysis program which employs a sphere-fitting, 3D directdistance transform, previously described for calculation of subchondral bone thickness (SCB.Th)25. Bone volume fraction (BV/TV) and bone TMD were also calculated. The femur was analyzed as the whole femur and as its individual medial, lateral, and trochlear compartments, which were segmented manually using the femoral intercondylar notch as the dividing landmark. The tibia was analyzed as the whole tibia and its individual medial and lateral compartments, which were segmented manually using the intercondylar tibial eminence as the dividing landmark. Femoral and tibial epiphyseal bone was segmented by outlining the epiphysis on serial raw images [Fig. 3(A, D)]. A high-pass intensity threshold of 3500 HU was used to isolate only bone voxels, excluding air, bone marrow, and soft tissues. Subchondral and cortical bone was removed via erosion to yield a volume of interest only containing trabecular bone [Fig. 3(B, E)]. The MatlabeImageJ interface Miji34,35 was used to analyze binarized, purified trabecular volumes [Fig. 3(C, F)] using the boneJ plug-in36, which performs trabecular morphometric analysis according to established standards37. BV/TV, bone TMD, trabecular thickness (Tb.Th.), trabecular number (Tb.N), trabecular spacing (Tb.Sp), structure model index (SMI), and connectivity density (Conn.D) were quantified. Statistical analyses All statistical analyses were performed in SPSS (v22, IBM, Armonk, NY). The normality and equal variance assumptions were assessed using the ShapiroeWilk test and Levene's test, respectively. Differences in all variables as a function of treatment group and time point were assessed using two-way analysis of variance (ANOVA). Multiple comparisons were performed with a Sidak P-

value correction to yield a family-wise error rate of 0.05 (i.e., Pcritical ¼ 1 (1 0.05)1/3 ¼ 0.0169). Corrected P values were then transformed in SPSS according to the Sidak correction such that 0.0169 ¼ 0.05, and P values lower than 0.05 were considered significant. Correlations were calculated using the Pearson productmoment correlation coefficient. Results Histologic score All animals tolerated the post-procedure time course, and each analysis includes n ¼ 6 animals per experimental group per time point. Both injury models induced marked joint degeneration, evident both histologically and on mCT images (Fig. 4). OARSI histologic score was significantly increased over Control (4 weeks: 2.10 ± 0.42 [95% confidence interval (CI): 1.76e2.44]; 10 weeks: 3.21 ± 1.20 [95% CI: 2.25e4.17]) at both time points in both Rupture (4 weeks: 12.19 ± 1.6 [95% CI: 10.87e13.51], P < 0.001; 10 weeks: 11.38 ± 0.92 [95% CI: 10.64e12.12], P < 0.001) and Transection (4 weeks: 10.78 ± 0.93 [95% CI: 10.04e11.52], P < 0.001; 10 weeks: 11.33 ± 1.2 [95% CI: 10.37e12.29], P < 0.001). Extensive articular cartilage degeneration is evident in both injury groups, and the medial condyle exhibits drastic cartilage thinning with hypocellularity, surface fissures, and isolated zones of cartilage thickening with hypercellularity. Subchondral bone remodeling Femoral and tibial subchondral bone remodeling was observed as a function of both injury type and time point. Both Transection and Rupture induced significant decreases in femoral subchondral BV/TV at both 4 and 10 weeks [Fig. 5(A)], and this decrease was significantly greater in Transection compared to Rupture at 4 weeks. At 10 weeks, Rupture exhibited significant decrease in whole-femur and trochlear BV/TV whereas Transection had significant decreases in whole-femur, medial-femur, and trochlear BV/ TV. Whole-tibia subchondral BV/TV [Fig. 5(B)] was significantly decreased compared to Control in Transection only at 4 weeks, and Transection had a significantly greater overall decrease in tibial subchondral BV/TV compared to Rupture. At 10 weeks, both injury groups had significant decreases in whole-tibia and medial-tibia

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Fig. 3. Segmentation process of femoral and tibial epiphyseal bone. Raw images of the femur (A) and tibia (D) were manually outlined to segment epiphyseal bone. Subchondral and cortical bone was removed from the volume of interest using voxel erosion (B, E, black ROI) to yield trabecular epiphyseal bone volumes (C, F).

subchondral BV/TV, but only Transection had a significant decrease in lateral-tibia BV/TV. Femoral and tibial subchondral TMD was significantly higher in Rupture compared to Transection at both time points [Fig. 5(C, D)], and Rupture exhibited significantly higher TMD compared to Control in the lateral femoral compartment at both 4 and 10 weeks. Compared to Control, Transection had significantly lower TMD in the femoral trochlear compartment and medial tibial compartment at 4 weeks and in the whole tibia and medial tibia at 10 weeks. Femoral SCB.Th [Fig. 5(E)] was significantly decreased at 4 weeks in all femoral compartments in Transection only, and Transection exhibited significantly thinner femoral SCB.Th compared to Rupture. At 10 weeks, femoral SCB.Th was significantly decreased in Rupture in the trochlear compartment only, but all femoral compartments exhibited significant decreases in the Transection group. Tibial SCB.Th [Fig. 5(F)] was decreased only in the medial compartment in Transection at 4 weeks, and Rupture did not exhibit any decreases in tibial SCB.Th. Epiphyseal bone remodeling As in subchondral bone, Transection exhibited a greater overall loss of epiphyseal trabecular BV/TV compared to Rupture in both the femur and tibia, most notably at 4 weeks. Whole-femur epiphyseal BV/TV [Fig. 6(A)] was significantly decreased at 4 weeks in Transection only, but both injury groups had significant decreases in epiphyseal trochlear BV/TV. At 10 weeks, neither group had significant decreases in whole-femur epiphyseal BV/TV, but only Transection had a decrease in epiphyseal trochlear BV/TV. Tibial epiphyseal BV/TV [Fig. 6(B)] was significantly decreased in Transection in all compartments at both 4 and 10 weeks, but Rupture did not demonstrate any decreases in tibial epiphyseal BV/TV. Femoral epiphyseal TMD [Fig. 6(C)] was significantly lower in the Transection group compared to the Rupture group, a finding consistent with results in subchondral bone. At 4 weeks, Rupture had significantly higher whole-femur, lateral-femur, and trochlear epiphyseal TMD compared to Transection, and Transection exhibited a significant decrease in trochlear TMD compared to Control. At 10 weeks, Rupture exhibited a significant increase in whole-femur,

medial-femur, and lateral-femur epiphyseal TMD compared to Control, and all compartments had significantly higher TMD compared to Transection. No differences in tibial epiphyseal TMD were observed between the two injury groups or compared to Control at either time point [Fig. 6(D)]. Changes in trabecular morphology were observed in both injury groups. Whole-femur epiphyseal trabecular thickening [Fig. 7(A)] was observed in the Rupture group at 4 and 10 weeks, whereas Transection did not exhibit trabecular thickening until 10 weeks. At 4 weeks, Rupture had significantly higher whole-femur, lateralfemur, and trochlear Tb.Th. compared to Transection, but there were no differences between the two groups at 10 weeks. No changes in Tb.Th. were observed as a function of injury group or time point in the tibia [Fig. 7(B)]. A significant decrease in Tb.N was observed in both the femur and tibia in both injury groups at both time points [Fig. 7(C, D)]. Both Rupture and Transection exhibited significant decreases whole-femur, medial-femur, and lateral-femur Tb.N compared to Control at both 4 and 10 weeks. No decrease in trochlear Tb.N was observed in either injury group. Whole-tibia Tb.N was significantly decreased in both injury groups at both time points. Medial-tibia Tb.N was decreased at 4 weeks in the Rupture group only, but both injury groups exhibited a decrease in medialtibia Tb.N at 10 weeks. No change in lateral-tibia Tb.N was observed in either group at either time point. No significant changes in femoral or tibial Tb.Sp were observed in either injury group [Fig. 7(E, F)]. Femoral and tibial epiphyseal SMI increased in both injury groups at both time points [Fig. 8(A, B)], demonstrating a change to a more rod-like rather than plate-like trabecular geometry. Wholefemur, medial-femur, and trochlear epiphyseal SMI were significantly higher in both injury groups at both time points, but there was no change in lateral-femur SMI. There was no difference in femoral SMI between Rupture and Transection. Transection exhibited an increase in whole-tibia and medial-tibia SMI at 4 weeks and in all tibial compartments at 10 weeks. Rupture displayed a significant increase in medial-tibia SMI at 4 weeks and in whole-tibia and medial-tibia SMI at 10 weeks. There were no differences in tibial SMI between the two injury groups directly. Only slight changes in Conn.D were observed as a result of ACL injury


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Fig. 4. Histology and mCT images of the medial compartment of representative Control (left column), Transection (center column), and Rupture (right column) animals at 4 weeks. In the femur, compared to Control animals (A, D), an overall loss of trabecular and subchondral bone volume is evident in both Rupture (B, E) and Transection (C, F), though more pronounced in Transection. Only Transection exhibited notable decreases in femoral (SCB.Th). In the tibia, compared to Control animals (G, J), only Transection (I, L) exhibited a marked decrease in trabecular bone volume, whereas both Rupture (H, K) and Transection (I, L) showed a decrease in subchondral bone volume.

[Fig. 8(C, D)], and although neither group exhibited significant changes compared to Control, Transection exhibited significantly higher whole-femur and lateral-femur Conn.D compared to Rupture at 4 weeks. Discussion Subchondral and epiphyseal trabecular remodeling following injury are hypothesized contributors to articular cartilage degeneration by altering the biomechanical and biochemical environment to which articular cartilage is chronically exposed. As animal models are frequently used to simulate the plethora of processes occurring after joint trauma, accurate representation of these processes is crucial to translate animal-based findings to clinical application. The surgical ACL transection model is commonly employed in rodents, but the surgical nature of this model may introduce confounding biological factors that could skew the accuracy of the model. The purpose of this study was to compare the ACL transection model to a noninvasive model of ACL rupture. Our results indicate that while similarities are observed between the two models, numerous differences in subchondral and epiphyseal bone remodeling were found. Transection caused greater subchondral bone thinning and a greater loss of subchondral BV/TV,

disproving our hypothesis that Rupture would result in greater bone loss. Rupture exhibited higher subchondral TMD, higher epiphyseal TMD, and higher epiphyseal Tb.Th., also disproving our hypotheses. Volumetric bone loss and loss of BMD are known phenomena following ACL injury11e14. While the exact mechanism of bone loss following ACL injury is not yet known, the role of the injuryinduced release of inflammatory factors has been partially described. Pro-inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and IL-6 known to be released in the joint following ACL injury29,38,39 perpetuate bone resorption directly or indirectly by promoting osteoclastogenesis40e42. Furthermore, the change in joint loading following injury, namely the pain-mediated reduction in physical activity and, postoperative immobilization, are known contributors to bone loss and loss of BMD43,44. Our data demonstrates that both injury models induce a decrease in subchondral BV/TV, and this decrease was greater in the Transection group. Epiphyseal bone loss was observed only in the Transection group, and these findings likely represent the difference in the level of injury severity between the two animal models. Although our study cannot definitively outline the etiology of the higher bone loss observed in the Transection group, a possible mechanism is an increased concentration of pro-

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Fig. 5. Subchondral bone morphometric parameters in Control, Rupture, and Transection. Both Rupture and Transection exhibit loss of subchondral BV/TV in the femur (A) and tibia (B). Transection exhibited higher loss of femoral and tibial subchondral BV/TV (A, B). Rupture had significantly higher TMD in the femur (C) and tibia (D) compared to Transection at both 4 and 10 weeks. Femoral subchondral bone plate thickness (SCB.Th.Mean) was decreased significantly only in Transection (E) and there were significant differences in SCB.Th.Mean in all femoral compartments between Rupture and Transection at 4 weeks (E). Only subtle changes were observed in tibial SCB.Th.Mean (F). * indicates significant difference to Control. > indicates significant difference between Rupture and Transection. Error bars represent 95% confidence interval.

Fig. 6. Epiphyseal bone BV/TV and TMD in Control, Rupture, and Transection. Only Transection exhibited significant decreases in epiphyseal BV/TV in both the femur (A) and tibia (B). Femoral epiphyseal TMD (C) was significantly lower in Transection at both 4 and 10 weeks compared to Rupture, and at 10 weeks, Rupture had higher femoral epiphyseal TMD compared to Control. No differences in tibial epiphyseal TMD were observed between groups at either time point (D). * indicates significant difference to Control. > indicates significant difference between Rupture and Transection. Error bars represent 95% confidence interval.


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Fig. 7. Epiphyseal bone Tb.Th.Mean, Tb.N, and Tb.Sp.Mean in Control, Rupture, and Transection. Rupture had significantly higher femoral Tb.Th.Mean compared to Control and Transection at 4 weeks, and both injury groups exhibited higher Tb.Th.Mean compared to Control at 10 weeks (A). No significant differences in tibial Tb.Th.Mean were observed between groups (B). Compared to Control, Tb.N was significantly decreased in the femur (C) and tibia (D) of both injury groups at both time points. Neither injury group exhibited significant changes in Tb.Sp.Mean in either the femur (E) or tibia (F). * indicates significant difference to Control. > indicates significant difference between Rupture and Transection. Error bars represent 95% confidence interval.

Fig. 8. Epiphyseal bone SMI and Conn.D in Control, Rupture, and Transection. Both Rupture and Transection exhibited increases in femoral (A) and tibial (B) SMI at 4 and 10 weeks. No significant changes in Conn.D were observed compared to Control in either injury group in the femur (C) or tibia (D), but Transection exhibited significantly higher Conn.D compared to Rupture in the femur at 4 weeks (C). * indicates significant difference to Control. > indicates significant difference between Rupture and Transection. Error bars represent 95% confidence interval.

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inflammatory cytokines expressed in the joint as a function of the additional surgical trauma (i.e., arthrotomy, skin incisions) leading to a greater activation of osteoclastogenic pathways. Furthermore, the introduction of foreign materials such as surgical tools and suture may be exacerbating the inflammatory response in the Transection group. Our data indicates that the two injury models yield differences in bone TMD, the intrinsic mineral density of bone, following injury. We found that TMD was significantly higher in femoral and tibial subchondral bone and femoral epiphyseal bone in the Rupture group compared to the Transection group. Furthermore, at 10 weeks, femoral epiphyseal TMD was significantly higher compared to Control, though marginally. The mechanisms responsible for these differences remain unclear, and, to the authors' knowledge, no clinical study has assessed the change in TMD following ACL rupture. As most studies employ dual-energy X-ray absorptiometry (DEXA/DXA), BMD is the most abundantlyreported densitometric parameter. However, given the inherent coupling of BMD to overall bone volume, a true determination of TMD is not possible, and the loss of BMD observed in previous studies may be largely due to volumetric bone loss rather than densitometric decreases. Furthermore, most clinically-utilized DEXA systems lack the resolution to determine the density of individual trabeculae. Due to the presence of contrast agent in the bone marrow cavities of each sample, our BMD results were inherently skewed, and we therefore chose to focus our analyses on TMD, making any comparisons with existing literature difficult. Further studies are, therefore, necessary to elucidate the potential differences in bony remodeling mechanisms between the two animal models and to assess how these compare to clinical findings of changes in intrinsic bone density after ACL injury. Due to the requirement of high-resolution 3D imaging of bone microarchitecture, clinical literature on changes in trabecular morphology/geometry after ACL injury is sparse. In a clinical study employing fractal signature analysis (FSA) on high-resolution radiographs of the knee, Buckland-Wright et al. demonstrated thickening of horizontal trabeculae in the medial compartment of the knee following ACL rupture15. In a preclinical study with the most similar methodology to our study, Christiansen et al. demonstrated drastic acute decreases in femoral and tibial epiphyseal BV/TV, Tb.Th., and BMD at 7e14 days with partial recovery up to 56 days in a mouse model25. While our findings regarding BV/TV decreases agree with their study, our results conflict with regards to Tb.Th. At both 4 and 10 weeks, our data indicates significant increases in femoral Tb.Th. in the Rupture group whereas their data demonstrates decreased Tb.Th. This discrepancy may be due to differences in the mouse and rat response to injury in addition to differences in injury loading. Their study utilizes a load-controlled injury loading protocol during which 1 mm/s displacement is applied up to 12 N. According to graphical representation of their loading protocol, rupture occurs at ~1.7 mm. Our study utilizes a displacementcontrolled protocol during which 3 mm of displacement is applied at 8 mm/s and ACL rupture occurs at ~2.0e2.5 mm of displacement. Although the rat is considerably larger than the mouse, the overall difference in tibial displacement of ~0.3e0.8 mm between our two models is relatively small, and this may indicate that total joint distraction is much larger in the mouse model, which could result in a more severe resorptive response in bone. This study is not without limitations. As part of another study, we performed contrast-enhanced imaging of the articular cartilage of the samples used in this study. This caused diffusion of the contrast agent into the bone marrow cavity, precluding the use of BMD as a densitometric parameter. Our imaging parameters

yielded an isotropic voxel size of 12 mm, a sufficient resolution for subchondral and trabecular morphology, but volume averaging of small pores or microcracks remains an inherent limitation. We did not image the contralateral, uninjured limb of each animal, which could have provided a useful internal control. We made attempts to limit animal-to-animal variability by utilizing an inbred rat strain and using animals with identical age. In order to minimize subjectivity and operator error, we developed semi-automatic segmentation algorithms which utilize 3D dilation and erosion to segment subchondral and epiphyseal trabecular bone. Extensive optimization of this algorithm was performed to minimize malsegmentation, but it cannot be fully ruled out. While we have previously demonstrated that our noninvasive injury loading protocol yields a complete, repeatable, and isolated ACL injury27, we only employed a drawer test as a way to confirm ACL rupture, and future studies may benefit from the use of magnetic resonance imaging to confirm injury. To manage post-surgical pain, we administered a single dose of Carprofen, non-steroidal anti-inflammatory drug (NSAID), to all animals in our study. Chronic NSAID administration has been shown to inhibit the progression of OA in an ACL transection model45, but little data exists on a single, post-surgical dose, and we cannot isolate any potential effect of our single dose. Lastly, quadruped animals are known to adapt a threelegged stance and gait following joint destabilization to compensate for pain and instability46e48, similar to compensatory weightbearing changes observed in ACL-deficient patients49. Unloading of the affected limb has been shown to affect the progression of PTOA47, and as we did not undertake any quantification of gait patterns between our two injury models, we are unable to elucidate the effect of any potential gait differences on morphometric parameters in our data set. This study assessed differences in subchondral and epiphyseal trabecular bone remodeling after surgical ACL transection and noninvasive rupture in the rat. We found that surgical transection yields greater bone loss and subchondral bone thinning whereas noninvasive rupture induced increases in subchondral and epiphyseal TMD and a higher degree of femoral trabecular thickening. These results demonstrate that tissue remodeling is dependent on the mode of ACL injury. In the present study, surgical transection of the ACL leads to an exacerbated bone tissue response when compared to a closed, traumatic ligament rupture. We hypothesize that surgical invasion of the joint may lead to the increased expression of biologic factors involved in downstream bone remodeling, but follow-up studies are needed to confirm or refute this assumption.

Author contributions All authors have made substantial contributions to the conception and design of the study, acquisition of data, or analysis and interpretation of data, as well as manuscript preparation and final approval of the submitted article. KCB takes responsibility for the integrity of the work as a whole.

Conflict of interest The authors have no relevant conflicts of interest to disclose.

Role of the funding source The authors wish to acknowledge funding from the American Orthopaedic Society for Sports Medicine, which was employed to cover expenses associated with animal care and surgical procedures.


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