Meniscus Remodeling after ACL Injury is Region-Specific in a Skeletally Immature Porcine Model

Introduction: The number of anterior cruciate ligament (ACL) injuries are increasing in pediatric patients annually [1]. These ACL injuries are commonly associated with meniscal tears and/or injuries. The lateral meniscus (LMEN) is more commonly torn at the time of injury, whereas the medial meniscus (MMEN) is more commonly injured over time in an ACL-deficient knee [2,3]. Studies have shown some clinicians favor non-surgical treatment of pediatric ACL injuries, however, the risk of degeneration in the joint is increased [2,3,4]. Additionally, studies have suggested that meniscal injuries may be isolated to the posterior region after ACL tears, suggesting that regional changes to the meniscus after ACL injury should be investigated [5]. Our previous studies showed menisci volume increased after ACL transection (ACLT), however, regional volumes of the meniscus were not investigated [6]. Therefore, the objective of this study is to determine regional changes in MRI-based size and signal parameters, histology, and biochemistry of the menisci after an ACLT using a juvenile porcine model.

Materials and

Methods: All animal protocols were approved by IACUC. Seven female juvenile (3 month) Yorkshire cross-breed pigs underwent a unilateral ACL transection arthroscopically and a sham incision was made on the contralateral joint. After 12 weeks, both hind limbs were imaged using the 3.0-T Siemens MAGNETOM Skyra MRI system (T2 SWI sequence, voxel size 0.5x0.5x0.8mm) (Fig 1A). Meniscal measurements were done for both the medial and lateral menisci. These measurements included middle height and width. The medial and lateral menisci were then segmented manually from the MRI scans. The segmented menisci were also separated by the posterior, middle and anterior regions respectively. All volumes (total, posterior, middle, and anterior) were recorded. Using the meniscal segmentations, T2* relaxation maps were created by fitting a monoexponential decay function on a voxel-by-voxel based on 6 echo times using MATLAB. Biochemical analysis was performed on the inner and outer areas of the middle region of both the medial and lateral menisci. A dimethylmethylene blue (DMMB) assay was performed to investigate the glycosaminoglycan (GAG) content of the meniscus before and after injury. Furthermore, compositional changes were histologically evaluated using a safranin O and fast green stain, where safranin O stains GAGs. In normal tissue, the inner two thirds mainly consist of GAGs and the outer third is primarily fibrous tissue. Meniscus volume, height, meniscus T2* values and meniscus GAG content were all compared between the ACLT and sham-operated joints using paired t-tests with Bonferroni-Dunn method as correction for multiple comparisons. Overall significance was set at α=0.05.

Results, Conclusions, and Discussions: At 12 weeks post-ACLT, total volume of both MMEN and LMEN increased by 27% and 22% relative to contralateral sham-operated controls (Fig 1B), respectively. The volume significantly increased in the middle region of the MMEN and LMEN and the posterior region of the MMEN (Fig 1D-F). The MMEN posterior horn height, MMEN middle width and LMEN middle height all significantly increased (Fig 1G-I). T2* values significantly decreased compared to the sham-operated controls in the entire lateral meniscus and regionally in the posterior. However, the T2* values in the posterior MMEN remained unchanged (Fig 2). Visually, the GAG content remains similar in LMEN, but decreases in MMEN after ACLT. Similar mean changes were observed via biochemistry, but with substantial variability (Fig 3).
ACLT caused significant changes to the meniscus within 12 weeks of injury, specifically in the posterior and middle meniscal regions. Although meniscus remodeling has not been a primary metric for degeneration, studies have shown hypertrophy to be indicative of osteoarthritis [7]. Our findings align with this, where meniscus size increased after ACLT. Furthermore, our study determined specific regions to better understand how the meniscus is impacted and where degeneration begins post ACLT. Higher T2* values show greater disorganization in collagen fibers [8]. Our results show a decrease in T2* values, which could be a result of early tissue remodeling in response to altered loading. Overall, this study helps to determine where early meniscal changes occur after ACLT to better understand degeneration and help aid in prevention of long-term changes.

Acknowledgements (Optional): We would like to acknowledge the NCSU College of Veterinary Medicine and Laboratory Animal Resource for their help.

References: [1] Zbrojkiewicz, D., Med J Aust, 2018., [2] James, E. et al., Am J Sports Med., 2021., [3] Feucht, Matthias. et al., J Orthop Res, 2015., [4] Kawashima, I. et al., Orthop J Sports Med, 2020., [5] Guermazi, A. et al., Radiology, 2013., [6] Howe, D. et al., biorxiv. 516262, 2022., [7] Jung, K. et al., Rheumatol Int., 2010., [8] Soellner, S.T. et al., Osteoarthritis and Cartilage, 2017.