Abstract
Objectives:
Pediatric anterior cruciate ligament (ACL) injuries are on the rise and result in high rates of post-traumatic osteoarthritis (PTOA). The pro-inflammatory biomarker monocyte chemoattractant protein-1 (MCP-1) is elevated in serum and synovial fluid (SF) in skeletally mature ACL injured patients as well as patients with OA, making it a potential target for treatments to prevent PTOA. Understanding the time course of MCP-1 after ACL injury and how it relates to cartilage health in skeletally immature subjects is important for identifying OA risk and developing novel treatment targets for OA prevention. Therefore, the objective of this study was to evaluate the time course of MCP-1 levels in serum and synovial fluid (SF) after ACL injury in a skeletally immature porcine model and assess how MCP-1 levels relate to T2* magnetic resonance imaging (MRI) measurements, indicative of cartilage matrix organization. We hypothesized that higher levels of MCP-1 in serum and synovial fluid post-injury would correspond to higher cartilage T2* values 12 weeks after ACL injury.
Methods:
All experimental protocols were approved by the NC State IACUC. Juvenile (3 month old) female Yorkshire cross-breed pigs (n=7) underwent unilateral arthroscopic ACL transection (ACLT) and a sham operation in the contralateral joint (Fig. 1A). Prior to surgery and at 2, 4, and 12 weeks post- operatively, injured stifle SF and serum samples were collected (pre-op synovial fluid only obtained from 4 animals). MRI scans were performed prior to surgery and at 12 weeks post-operatively. MCP-1 levels in serum and SF samples were evaluated longitudinally using a commercial enzyme-linked immunosorbent assay. Cartilage matrix organization was evaluated longitudinally using T2* MRI. Specifically, both knee joints were imaged using a 3T Siemens MAGNETOM Skyra MRI system (T2 SWI sequence, FA=15°, TR=50ms, TE: 3.4, 7, 13.47, 21, 29, 37.07ms, voxel size: 0.5x0.5x0.8mm). T2* relaxation maps were created in MATLAB by fitting mono-exponential curves on a voxel-by-voxel basis. Weight-bearing cartilage was segmented from the medial and lateral compartments of the femur, and the median T2* value of each compartment was calculated. Paired MCP-1 levels in the serum and injured stifle SF across all time points were compared using linear regression. Multiple paired t-tests with Holm-Sidak correction were performed to compare cartilage T2* values between ACLT and sham- operated joints at each time point. The interlimb percent difference in femoral cartilage T2* at 12 weeks was calculated and compared to MCP-1 levels in serum and SF using linear regressions and several methods to assess MCP-1 levels (Fig. 2). Statistical significance was set at α=0.05 overall for each analysis.
Results:
Generally, MCP-1 levels in serum and ACLT stifle SF were elevated after ACLT (Fig. 1). The serum MCP-1 response over time differed between animals. Specifically, serum MCP-1 levels peaked at 2 weeks in 2 animals, peaked at 4 weeks in 3 animals, and decreased after ACLT in 2 animals (Fig 1A).
Contrastingly, ACLT stifle SF MCP-1 responses were more consistent between animals; six peaked at 2 week and returned near baseline levels by 12 weeks (Fig 1B). The linear regression between paired measurements of MCP-1 concentrations in the serum and ACLT stifle SF were associated (p=0.053), yet had high variability (r2=0.15, Fig. 1C). Interlimb differences in cartilage matrix organization were largest and most consistent at 12 weeks in the medial femoral cartilage compartment, where median T2* times were 8% longer in the medial femoral cartilage compartment of the ACLT limb compared to sham limb, yet this was not statistically significant due to variability between animals (p>0.05, Fig. 2). In the lateral femoral cartilage, the median T2* times were 3-4% longer at 12 weeks (p>0.05) and were more variable between subjects compared to the medial femoral cartilage. Associations between the interlimb percent difference in medial femoral cartilage T2* and serum MCP-1 fit well (r2=0.25-0.69), with Methods 2, 3, and 5 achieving statistical significance (p=0.02, p=0.03, p=0.03), but resulted in poor fits for associations with SF MCP-1 (r2=0.07-0.45) using a variety of MCP-1 measurement methods (Fig 2C). Similar associations between lateral femoral cartilage T2* and serum MCP- resulted in poor fits (r2=0.01-0.24), and associations with SF MCP-1 resulted in negative slopes (Methods 2-5, r2=0.00-0.58).
Conclusions:
This preliminary study showed that inflammatory biomarker MCP-1 levels in serum displayed a subject-dependent response, which correlated with later femoral cartilage compositional changes after ACLT in the skeletally immature porcine model. This partially supported our hypothesis. The poor correlation between serum and SF MCP-1 indicate different local and systemic responses to ACLT. The stronger correlations between serum MCP-1 and cartilage T2* values could point to the importance of the systemic inflammatory response to OA progression. The increases in cartilage T2* values and serum MCP-1 levels after ACL injury in this study are consistent with those reported in adult humans. This study includes several limitations, including the use of an animal model to study human disease, the small sample size, and the relatively short time-course. However, the results demonstrate feasibility of using a large animal porcine model to longitudinally study the link between early MCP-1 serum levels and long-term cartilage health after ACL injury. Establishing the pig as a preclinical model to study longitudinal inflammation and OA progression after ACL injury will enable development of effective treatments before translating to humans.
