Abstract

Noninvasive compression-induced anterior cruciate ligament rupture (ACL-R) is an easy and reproducible model for studying post-traumatic osteoarthritis (PTOA) in mice. However, equipment typically used for ACL-R is expensive, immobile, and not available to all researchers. In this study, we compared PTOA progression in mice injured with a low-cost custom ACL-rupture device (CARD) to mice injured with a standard system (ElectroForce 3200). We quantified anterior–posterior (AP) joint laxity immediately following injury, epiphyseal trabecular bone microstructure, and osteophyte volume at 2 and 6 weeks post injury using micro-computed tomography, and osteoarthritis progression and synovitis at 2 and 6 weeks post injury using whole-joint histology. We observed no significant differences in outcomes in mice injured with the CARD system compared to mice injured with the Electroforce (ELF) system. However, AP joint laxity data and week 2 micro-CT and histology outcomes suggested that injuries may have been slightly more severe and PTOA progressed slightly faster in mice injured with the CARD system compared to the ELF system. Altogether, these data confirm that ACL-R can be successfully and reproducibly performed with the CARD system and that osteoarthritis (OA) progression is mostly comparable to that of mice injured with the ELF system, though potentially slightly faster. The CARD system is low cost and portable, and we are making the plans and instructions freely available to all interested investigators in the hopes that they will find this system useful for their studies of OA in mice.

Introduction

Osteoarthritis (OA) is the most common degenerative joint disease and is the leading cause of disability in the U.S. [1], currently affecting over 32 million U.S. adults [2]. Specifically, OA of the knee is the most prevalent, affecting over 15 million people in the U.S. [3]. Individuals who suffered previous trauma to the knee joint such as anterior cruciate ligament (ACL) rupture are nearly four times more likely to develop OA of the knee than uninjured people [4]. About half of individuals diagnosed with ACL rupture or meniscus tear develop post-traumatic osteoarthritis (PTOA) within 10–20 yrs [5].

Animal models of OA are essential tools for studying the etiology of the disease and potential therapies for slowing or preventing OA progression. Mouse models in particular are critically important due to the abundance of genetic models and the compressed and predictable timeline of disease progression. The recent development of noninvasive mouse models of PTOA is an important step for mechanistic studies of OA, since these models initiate OA with externally applied mechanical loads [6], closely resembling the initiation of human PTOA since no surgical procedures or intra-articular injections are used. Our lab has published several studies using tibial compression overload to cause ACL rupture (ACL-R) [717]. This is a simple and quick method that produces a consistent and reproducible joint injury that initiates a rapidly progressing OA in the mouse knee joint. However, this injury method typically requires access to a materials testing system (Electroforce 3200 or comparable). These systems may be prohibitively expensive for some investigators. Additionally, these systems are physically large and heavy, so they cannot easily be brought into vivaria or barrier facilities. These limitations make the ACL-R model unfeasible for some studies of PTOA etiology or treatment.

To address these limitations, a group of Mechanical Engineering students at Montana State University designed a low-cost and portable custom-made ACL rupture device (CARD) for inducing joint injuries in mice. This system weighs ∼8 kg allowing for more portability, and is a fraction of the cost of the materials testing systems typically used for this application. In this study, we validated the CARD system relative to a standard system for ACL-R of mice, including assessment of post injury joint laxity, subchondral bone changes, osteophyte formation, synovitis, and PTOA progression. Successful validation of this device would make ACL-R in mice more financially accessible and more feasible for some studies due to the portability of the system.

Methods

Design of the Custom-Made Anterior Cruciate Ligament Rupture Device.

Design requirements for the CARD system included sterilization, force, velocity, stopping ability, data acquisition, and cost. The apparatus was designed to be portable and user friendly, sterilizable with ethanol or other cleaning agents, with force and displacement limits of 14 N and 2.25 mm, respectively. The system was designed to apply a displacement rate of 200 mm/s, which we have previously shown to cause a midsubstance ACL rupture without an avulsion [8]. Finally, the apparatus was designed to cost less than $5000 (at the time of design). Images and technical drawings of the CARD system are shown in Fig. 1.

Fig. 1
Technical drawing and pictures of the CARD
Fig. 1
Technical drawing and pictures of the CARD
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The final CARD system design uses a frame made of 80/20® 40 × 40 mm aluminum tubing and 90-deg corners 40-4302 fasteners. The three pieces of the system that contact the mouse during loading (the mouse platform, knee holster, and ankle holster) were three-dimensional printed. The knee and ankle holsters were based on the fixtures used in the Electroforce (ELF) system. The ankle holster was attached to the end of the force applicator and the knee holster was attached to the end of a strain gauge load cell (Uxcell, Hong Kong, China). The force applicator is a voice coil actuator (VCA) from BEI Kimco (LAS13-56, Vista, CA). This VCA had a hard stop built in to ensure that the VCA cannot push the ankle holster farther than 2.25 mm. The VCA is held onto the frame with a three-dimensional printed sleeve, that is, attached to the frame with a thumb screw such that the sleeve, with the VCA on it, can manually be moved up and down the frame to apply the preload. The electronics to control the VCA and record the data including a microcontroller, a motion controller, and a power supply were built onto one of the frame legs. For a fully detailed system description and files/supplies list for constructing the CARD system, please contact the corresponding author or visit.

Animals.

A total of 30 male C57BL/6J mice, 12 weeks old at the time of injury, were obtained from Jackson Laboratory (Bar Harbor, ME). Half of the mice (n = 15) were randomly assigned to undergo ACL-R using the current gold standard loading system (ElectroForce 3200, TA Instruments, New Castle, DE), and the remaining mice (n = 15) were assigned to undergo ACL-R using the CARD system. Each experimental group was divided into three time points: euthanasia immediately following injury (n = 5 mice/group), or 2 weeks (n = 5) or 6 weeks (n = 5) after injury. Euthanasia was performed via carbon dioxide asphyxiation followed by cervical dislocation. Knee joints from mice that were euthanized immediately after injury underwent anterior–posterior (AP) joint laxity as described below. Knee joints from mice that were euthanized 2 or 6 weeks after injury were analyzed with micro-computed tomography to evaluate epiphyseal trabecular bone and osteophyte formation. Knee joints were also analyzed with whole-joint histology to measure synovitis and PTOA progression.

Noninvasive Anterior Cruciate Ligament Rupture by Tibial Compressive Overload.

Tibial compression overload was performed using the ElectroForce 3200 (ELF) as previously described [8]. Briefly, mice were anesthetized with isoflurane inhalation, then the right lower leg was positioned between an upper platen that held the flexed ankle at approximately 30 deg of dorsiflexion and a lower platen that held the flexed knee. A preload of 1–2 N was manually applied by raising the bottom platen and locking it in place, after which a single compressive load was applied to a target displacement of −1.7 mm at a target loading rate of 200 mm/s.

Tibial compression overload using the CARD system was performed using similar methods. The mouse was anesthetized using isoflurane inhalation, and was then loaded in a prone position with the right knee in the knee cup. The VCA with the ankle holster was then manually lowered to the level of the mouse's foot to stabilize the ankle, leg, and knee joint. Once the mouse leg was stabilized and the VCA was locked in place, the program was initiated and the VCA automatically adjusted the ankle holster until a 2 N preload was applied to the leg. Once a preload of 2 N was reached, the VCA compressed the lower leg at a target loading rate of 200 mm/s. The VCA compressed the lower leg until one of three conditions is met: (1) the force load detected by the load cell drops indicating an ACL rupture, (2) the 14 N maximum load is met, or (3) a displacement limit of 2.25 mm is met. The latter two endpoints ensure that tibial compression is stopped before tibial fracture or damage to other joint structures occurs. After injury, all mice were given a subcutaneous injection of buprenorphine (0.5 mg/kg body weight) for analgesia. Mice were allowed normal cage activity until euthanasia.

Assessment of Anterior–Posterior Joint Laxity.

Anterior– posterior joint laxity was analyzed as previously described [8,18] for both the injured and contralateral uninjured knees immediately following noninvasive ACL-R for both ELF-injured mice (n = 5) and CARD-injured mice (n = 5). Briefly, the distal tibia and proximal femur were fixed in brass tubes with polymethyl methacrylate and knees were tested at 60 deg of flexion with five loading cycles to a target force of  ± 1.5 N at a rate of 0.5 mm/s. The force was applied perpendicular to the longitudinal axis of the tibia, and the tibia was allowed to translate and rotate about its longitudinal axis during loading. Total AP joint laxity was computed based on the difference between displacement at +0.8 N and −0.8 N.

Micro-Computed Tomography Analysis of Epiphyseal Trabecular Bone.

Knee joints from mice euthanized 2 and 6 weeks post injury (n = 5 mice/time point/experimental group) were scanned using micro-computed tomography (SCANCO μCT 35, Brüttisellen, Switzerland) to analyze epiphyseal trabecular bone microstructure. Scans were performed according to rodent bone structure analysis guidelines (X-ray tube potential = 55 kVp, intensity = 114 mA, 10 μm isotropic nominal voxel size, integration time = 900 ms) [19]. The trabecular bone volume of interest included all trabecular bone distal to the epiphyseal growth plate of the distal femur. Trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and other microstructural outcomes were measured using the manufacturer's analysis software.

Micro-Computed Tomography Analysis of Osteophyte Volume.

Osteophyte analysis was performed on knee joints from 6 week post injury mice as previously described [11]. Briefly, contours were drawn to include the patella, fabella, meniscal mineralized tissue, and non-native bone growth on and around the distal femur and proximal tibia for both knee joints. The difference between bone volume of the injured knee versus the uninjured contralateral knee was calculated to quantify osteophyte volume in the injured knee.

Histological Assessment of Synovitis and Osteoarthritis Progression.

Whole-joint histology was used to evaluate overall joint degeneration (OA scoring) and synovitis at both the 2-week time point (n = 5 mice/group) and 6-week time point (n = 5 mice/group). After euthanasia, knee joints were fixed in 4% paraformaldehyde for 5–7 days, and were then decalcified using 0.5 M EDTA and processed for standard paraffin embedding. Sagittal sections were cut across the medial and lateral aspects of the joint separated by 100 μm (approximately 8–10 section per joint), and were stained with toluidine blue. Three blinded readers independently graded OA and synovitis for both the medial and lateral compartments, and scores were averaged across all three readers. Synovitis was scored using a six-point system consisting of the sum of a synovial lining score on a scale of 0–3 and a synovial stroma cellularity score on a scale of 0–3 as described by Krenn et al. [20]. OA scoring for joint degeneration was scored on a scale of 0–10 with an emphasis on articular cartilage degeneration and/or tibial degeneration. A score of 0–4 indicates the absence of gross tibial degeneration with 0 being a healthy joint and a 4 being a joint with mostly intact articular cartilage that lacks toluidine blue staining. A score of 5–10 indicates a joint with tibial cartilage degeneration, with a 5 being a joint with some tibial degeneration but with more than 90% stained cartilage on the femoral condyle, and 10 being a joint with extensive tibial degeneration and less than 10% stained cartilage on the femoral condyle.

Statistical Analysis.

All data were analyzed at each time point using paired t-tests to compare right versus left limbs of mice (joint laxity test and μCT analysis) and unpaired t-tests to compare ELF versus CARD groups (all outcomes) using prism 9 software (GraphPad by Dotmatics, Boston, MA). All data are presented as mean ± standard deviation. Significance was defined a priori as p < 0.05 for all tests.

Results

Noninvasive Anterior Cruciate Ligament Rupture by Tibial Compressive Overload.

Successful ACL-R was confirmed in mouse knees using μCT imaging and whole-joint histology. Of the 15 mice loaded with the CARD system, 13/15 were successfully injured. The ACL was intact for the remaining two mice (both from the week 6 group), so they were removed from further analyses. All of the mice loaded with the ELF system were successfully injured.

Analysis of injury data from the ELF and CARD systems showed that the displacement rate was consistent between the two systems, though neither achieved the target displacement rate of 200 mm/s (Fig. 2). Failure (injury) compressive load was similar between the two systems, though the mean failure load for the CARD system was significantly higher than that of the ELF system (16.4 ± 2.0 N versus 14.2 ± 1.0 N). However, average total displacement during injury loading was considerably greater with the CARD system (2.33 ± 0.15 mm) than with the ELF system (0.84 ± 0.04 mm).

Fig. 2
Comparison of displacement rate, failure (injury) load, and total displacement during injury for the ELF and CARD systems. Displacement rates were equivalent for the two systems, though neither system achieved the target displacement rate (200 mm/s). Failure load was slightly higher for the CARD system than the ELF system, but total displacement during injury was considerably greater with the CARD system, which may result in a greater degree of soft tissue injury during loading.
Fig. 2
Comparison of displacement rate, failure (injury) load, and total displacement during injury for the ELF and CARD systems. Displacement rates were equivalent for the two systems, though neither system achieved the target displacement rate (200 mm/s). Failure load was slightly higher for the CARD system than the ELF system, but total displacement during injury was considerably greater with the CARD system, which may result in a greater degree of soft tissue injury during loading.
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Assessment of Anterior–Posterior Joint Laxity.

As expected, ACL-R resulted in notable increases in AP joint laxity for mice loaded with either the CARD or ELF systems (Fig. 3). Mice loaded with the CARD system had AP joint laxity of 1.74 ± 0.24 mm in the injured leg compared to 0.82 ± 0.15 mm in the uninjured contralateral leg (+112%). Mice loaded with the ELF system had AP joint laxity of 1.61 ± 0.10 mm in the injured leg compared to 0.93 ± 0.13 mm in the uninjured contralateral leg (+72%). No significant differences were observed between the CARD and ELF systems.

Fig. 3
AP laxity testing of mouse joints. ACL-R increased AP joint laxity for mice loaded with the CARD system (+112%) and mice loaded with the ELF system (+72%) compared to their uninjured contralateral joints. No significant differences were observed in AP joint laxity between mice injured with the CARD and ELF systems. * indicates significant difference between injured and contralateral joints (p < 0.05). No significant differences were observed between joints injured with the ELF versus CARD systems.
Fig. 3
AP laxity testing of mouse joints. ACL-R increased AP joint laxity for mice loaded with the CARD system (+112%) and mice loaded with the ELF system (+72%) compared to their uninjured contralateral joints. No significant differences were observed in AP joint laxity between mice injured with the CARD and ELF systems. * indicates significant difference between injured and contralateral joints (p < 0.05). No significant differences were observed between joints injured with the ELF versus CARD systems.
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Micro-Computed Tomography Analysis of Epiphyseal Trabecular Bone and Osteophyte Volume.

Noninvasive ACL injury resulted in a loss of epiphyseal trabecular bone in the injured joint of mice injured with both the ELF system and the CARD system at both week 2 and week 6 post injury (Fig. 4). At week 2 post injury, injured knees from mice loaded with the ELF system had an 11% decrease in BV/TV and a 9% decrease in Tb.Th compared to the uninjured contralateral leg, while injured knees from mice loaded with the CARD system had an 20% decrease in BV/TV and a 15% decrease in Tb.Th compared to the uninjured contralateral leg. At week 6 post injury, injured knees from mice loaded with the ELF system had an 18% decrease in BV/TV and an 11% decrease in Tb.Th compared to the uninjured contralateral leg, while injured knees from mice loaded with the CARD system had an 18% decrease in BV/TV and a 9% decrease in Tb.Th compared to the uninjured contralateral leg. No significant differences were observed between ELF and CARD experimental groups for any μCT outcomes.

Fig. 4
Micro-computed tomography analysis of epiphyseal trabecular bone microstructure and osteophyte formation. ACL-R resulted in decreased epiphyseal trabecular bone in the injured joint of mice injured with both the ELF system and the CARD system at both week 2 and week 6 post injury and considerable formation of osteophytes by week 6 post injury. * indicates that injured was significantly different than contralateral (p < 0.05). No significant differences were observed between ELF and CARD experimental groups for any μCT outcomes.
Fig. 4
Micro-computed tomography analysis of epiphyseal trabecular bone microstructure and osteophyte formation. ACL-R resulted in decreased epiphyseal trabecular bone in the injured joint of mice injured with both the ELF system and the CARD system at both week 2 and week 6 post injury and considerable formation of osteophytes by week 6 post injury. * indicates that injured was significantly different than contralateral (p < 0.05). No significant differences were observed between ELF and CARD experimental groups for any μCT outcomes.
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As expected, ACL-R resulted in considerable formation of osteophytes by week 6 post injury (Fig. 4). Injured knees from mice loaded with the ELF system had mineralized osteophyte volumes of 1.86 ± 0.76 mm3, while injured knees from mice loaded with the CARD system had mineralized osteophyte volumes of 1.61 ± 0.07 mm3. No significant differences were observed between ELF and CARD experimental groups.

Histological Assessment of Osteoarthritis Progression.

Noninvasive ACL injury led to OA progression in the injured joints of mice loaded with both the ELF and CARD systems at 2 and 6 weeks post injury (Fig. 5). Similar to our previous studies, notable osteophyte/chondrophyte formation was observed at both time points, particularly at the anterior–medial femur, posterior–medial tibia, and the anterior horn of the medial meniscus. At 2 weeks post injury, OA scores for mice injured with the CARD system were increased compared to those injured with the ELF system. The medial and lateral OA scores for mice injured by the CARD system were 4.8 ± 1.7 and 3.1 ± 0.6, respectively, while the medial and lateral OA scores for the mice injured by the ELF system were 2.6 ± 2.7 and 1.3 ± 0.5, respectively. At 6 weeks post injury, the OA scores for the injured joints of mice loaded with the CARD system did not exhibit a large amount of progression relative to week 2, while the injured joints of mice loaded with the ELF system exhibited greater disease progression during this time, resulting in similar scores between groups at week 6. The medial and lateral OA scores for mice injured with the CARD system were 7.2 ± 0.6 and 3.7 ± 1.2, respectively, while the medial and lateral scores for mice injured with the ELF system were 6.8 ± 0.4 and 4.8 ± 2.6.

Fig. 5
Histological assessment of PTOA progression. ACL-R led to OA progression in the injured joints of mice loaded with both the ELF and CARD systems. At 2 weeks post injury, OA scores for mice injured with the CARD system were increased compared to those injured with the ELF system, though this was statistically significant only for the lateral aspect of the joint. * indicates significant difference between joints injured with the ELF versus CARD systems (p < 0.05).
Fig. 5
Histological assessment of PTOA progression. ACL-R led to OA progression in the injured joints of mice loaded with both the ELF and CARD systems. At 2 weeks post injury, OA scores for mice injured with the CARD system were increased compared to those injured with the ELF system, though this was statistically significant only for the lateral aspect of the joint. * indicates significant difference between joints injured with the ELF versus CARD systems (p < 0.05).
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Histological Assessment of Synovitis.

Noninvasive ACL injury led to synovitis in the injured joints of mice loaded with both the ELF and CARD systems at 2 and 6 weeks post injury (Fig. 6). At 2 weeks post injury, joints injured with the CARD system had greater synovitis than those injured with the ELF system, particularly at the lateral aspect, with greater synovial thickness and overall cellularity. Medial and lateral synovitis scores at 2 weeks post injury of joints loaded with the CARD system were 3.7 ± 1.1 and 2.9 ± 0.5, respectively, while the ELF system counterparts were 2.9 ± 0.9 and 1.9 ± 0.7, respectively, for the medial and lateral aspects of the joint. At 6 weeks post injury, there were no significant differences in synovitis between joints injured with the CARD and ELF systems. Medial and lateral synovitis scores of joints loaded with the CARD system were 3.2 ± 0.6 and 3.2 ± 0.6, respectively, while the ELF system counterparts were 3.9 ± 0.4 and 3.0 ± 0.8, respectively.

Fig. 6
Evaluation of synovitis in injured joints. ACL-R induced synovitis in the injured joints of mice loaded with both the ELF and CARD systems. At 2 weeks post injury, lateral synovitis scores for mice injured with the CARD system were increased compared to those injured with the ELF system. No significant differences were observed at the medial aspect of the joint or at 6 weeks post injury. The synovium is indicated with arrows. * indicates significant difference between joints injured with the ELF versus CARD systems (p < 0.05).
Fig. 6
Evaluation of synovitis in injured joints. ACL-R induced synovitis in the injured joints of mice loaded with both the ELF and CARD systems. At 2 weeks post injury, lateral synovitis scores for mice injured with the CARD system were increased compared to those injured with the ELF system. No significant differences were observed at the medial aspect of the joint or at 6 weeks post injury. The synovium is indicated with arrows. * indicates significant difference between joints injured with the ELF versus CARD systems (p < 0.05).
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Discussion

In this study, we validated a low-cost portable system for inducing ACL-R in mice. We found that this system was able to induce a joint injury and rate of PTOA in mice, that is, comparable to a conventional materials testing system (ElectroForce 3200). This novel CARD system may make the ACL-R method more accessible for research groups that do not have access to conventional loading systems and could potentially be used in barrier facilities or other remote locations due to the portability of the system. We are making the plans and instructions for the CARD system freely available2 in the hopes that others will find this system useful for their studies of OA in mice.

Through our use of the CARD system, we found that the system and software are not as user friendly as those of the ELF system. In particular, there was not the same level of real-time feedback on force and displacement. This lack of real-time feedback made it somewhat challenging to set the VCA, and may have contributed to the two failed injuries in the CARD group. These failed injuries also underline the importance of performing an anterior drawer test or comparable method after loading to confirm injury. The VCA was chosen as the most suitable method to apply compressive force in this system because it has an appropriate stroke length (12 mm), can reach the target displacement rate (200 mm/s) within the injury displacement (1.7 mm), and has an embedded hall effect sensor for measuring displacement. However, the ELF system still proved to be more user friendly for this application.

Additionally, we found that the loading parameters for the CARD system were not as easily modifiable as those of the ELF system, which may make it necessary for users to make changes in the software in some situations. For example, in studies involving mice with higher body mass, 14 N of compressive force may not be sufficient to induce ACL injury since the injury force has been shown to correlate with body mass [21]. There may also be a desire to modify individual components of the system such as the ankle holster and knee cup, though these changes will be dependent on the specific application.

Both systems were programed with the same target loading rate (200 mm/s) and the same target displacement (−1.7 mm). A target displacement was used for this study and our previous study [8] rather than a target compressive load because we found that the ELF system was less likely to overshoot a target displacement than a target compressive load at this high loading rate. However, neither system in this study achieved the target loading rate (both achieved a displacement rate of ∼130–135 mm/s), and there were notable differences in the total displacement during injury between the systems. The ELF system was able to successfully induce ACL injury in all mice with displacements well short of the target displacement, while the CARD system overshot the target displacement and was likely stopped by the built-in hard stop in the system. These findings underline the importance of validating these systems in each individual laboratory and adequately tuning the system to each specific application whenever possible.

Osteoarthritis progression following noninvasive knee injury with the CARD system was comparable to OA progression following injury with the ELF system. However, there were some indications that injury with the CARD system was slightly more severe and led to an accelerated rate of OA progression. In particular, OA progression and synovitis at 2 weeks post injury were greater for joints injured with the CARD system compared to those injured with the ELF system (though these were not different at week 6). This likely reflects the greater displacement during injury in the CARD system, which may have resulted in a greater degree of tissue damage in some knee joint structures in addition to ACL rupture. This may require further investigation and validation in a greater number of samples, though the internal consistency (i.e., variability) in outcomes with the CARD system was not greater than that of the ELF system injuries. In fact, variability in some outcomes from mice injured with the CARD system (e.g., osteophyte volume) was considerably less than that of mice injured with the ELF system. However, this finding may be coincidental due to the small number of samples in the week 6 CARD group. Our previous studies have found a larger variance in osteophyte volume at this time point, similar to what we observed for mice in the ELF group in this study. It is unclear if the lower variability observed with the CARD system would be maintained in subsequent studies with a larger number of mice.

This study has additional limitations that must be acknowledged. First, this study involved a relatively small number of mice (n = 5 mice/group/time point), and used only one strain, sex, and age of mice. Second, this study did not include functional analyses of gait or pain following injury. Third, all injuries were performed using both systems by an experienced user. It is unclear how easy and effective the CARD system will be for users who do not have previous experience with the ACL-R method. However, despite these limitations, this study described a custom-designed low-cost portable system for ACL-R and provided a validation of the CARD system relative to a commercially available system (ElectroForce 3200) and showed mostly comparable results in post injury joint laxity, subchondral bone changes, osteophyte formation, synovitis, and OA progression. This novel system could be tremendously useful for some studies of OA in mice and will provide increased accessibility and utility for the ACL-R method. For a fully detailed system description and files/supplies list for constructing the CARD system, please contact the corresponding author or visit the website2.

Acknowledgment

We would like to thank Benjamin Collins, Zachary Hein, Ryan Schwab, and Abigale Snortland, the designers of the CARD system. Thank you for your wonderful design, we hope it will be put to good use.

Funding Data

  • Dr. Christiansen receives funding from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR075013, R01 AR071459; Funder ID: 10.13039/100000069) and the Department of Defense (PR180268P1; Funder ID: 10.13039/100000005).

  • Dr. June receives funding from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR073964; Funder ID: 10.13039/100000069) and the National Science Foundation (CMMI 1554708 and CMMI 2140127; Funder ID: 10.13039/100000001).

Footnotes

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