Objective: Females experience ACL tears at disproportionately higher rates potentially due to the effects of the hormone relaxin. To precisely measure ACL biomechanical properties in rodent (rat) model in which circulating relaxin level could be manipulated, the present work sought to create a methodology to noninvasively measure the cross-sectional area (CSA) of the rat ACL and calculate its biomechanical properties. In doing so, three aims were investigated: (1) Develop camera settings to visualize the rat ACL, (2) Test intra- and inter-user agreement in CSA calculations, and (3) Test the reproducibility of the methodology to ensure biomechanical properties can be measured with sufficient precision.
Methods: In Aim 1, the effect of camera placement and precision of image pixels to mm conversion were determined. In Aim 2, differences in digitally-acquired ACL CSA measurements were evaluated for intraand inter-user repeatability alongside Bland-Altman tests to evaluate measurement bias and agreement. Finally, for Aim 3, two iterative pilot tests with a total of 42 female rats were conducted to refine the mechanical tensile testing procedure to quantify ACL failure load and stiffness.
Results: The effects of camera placement on ligament radii measures used to calculate CSA were lower than measured bilateral differences. Intra- and inter-user differences for pixel to mm conversion were below 10%. Intra- and inter-user differences in measured ACL CSA ranged from 1.3 - 9.5% and 0.2 - 5.7%, respectively. The bilateral differences of the final tensile testing protocol were 9.8 - 57.4% for failure load, 8.7 - 18.8% for stiffness, and 1.9 – 4.4% for ACL CSA.
Conclusion: A method to digitally measure rat ACL CSA repeatably and reproducibly was developed; however large bilateral differences for ultimate failure load and stiffness between limbs suggests limitations associated with fixturing will likely overshadow potential differences attributable to relaxin administration. Future work that tests a redesigned fixture using the approaches developed here will be required.