Gene Expression in Response to High Frequency, Low Magnitude Loading on the Anterior Cruciate Ligament
Authors: Domire Zachary J. , Diefenbach Brian J. , Graber Theodore G. 
Published: May 2022

IntroductionPrevention of Anterior Cruciate Ligament (ACL) injuries is important, as ACL injuries are associated with the development of knee osteoarthritis, increased pain, functional limitations, and decreased quality of living. Despite the focus on numerous ACL injury prevention mechanisms, ACL injury rates are still on the rise. Investigating a way to strengthen the ACL may be an alternative method to prevent ACL tears. Traditionally, tissue strengthening would occur through a high magnitude load, applied a limited number of times. However, as ligaments are designed to restrict unhealthy joint movement, high magnitude loading of ligaments is likely non‐desirable, making high frequency, low magnitude loading an intriguing possibility. Previous studies have shown bone’s ability to adapt and strengthen in response to high frequency, low magnitude loading. Therefore, we hypothesized ligaments, the connective tissue connecting bones together, would also exhibit similar adaptive properties. However, there is limited research on a typical in vivo ligament response to mechanical loading.ObjectiveThe purpose of this study was to identify what genes were altered in expression in response to high frequency, low magnitude loading.MethodsSeven New Zealand white rabbits were loaded utilizing a custom device built to apply a high frequency, low magnitude load to the rabbit’s shank. While anesthetized, rabbits were placed into the loading device with the left leg secured in a cast and attached to the load cell with a non‐strain cord (Figure 1). To ensure the loading occurred along the longitudinal axis of the ACL, the hip was extended, knee positioned at 30° knee flexion, with the device pulling on the tibia at 35‐40° to apply tibial anterior translation. Loads were applied oscillating between 2‐12 N, at 15 Hz for 20 minutes. Four hours post loading, both of the rabbit’s ACLs were harvested (loaded ACL & internal control). Three rabbits served as external controls and received no loading. Total RNA was extracted from the ACLs using a Qiagen RNAeasy kit following manufacturer’s instructions. Next Generation Sequencing RNAseq detected gene expression differences between the loaded ACL and internal control ACL, and the loaded ACL and the external control ACL using a Sleuth Wald test during data analysis.ResultsIn response to mechanical loading, there were three genes differentially expressed between the loaded and the internal control ACL, none of which were annotated within the rabbit genome. Between the loaded and external control ACL, there were 121 genes differentially expressed. Grouping these genes by function, there were 15 related to mechanotransduction, five to metabolic pathways, four to interleukin response and tissue repair, three to actin cytoskeleton regulation, two relating to protein transport, and one to collagen synthesis.ConclusionsThis is the first data demonstrating a ligament adaptive response to in vivohigh frequency, low magnitude loading. Additionally, these results shed light on the possible mechanotransductive response pathway in ligament. Future studies should characterize optimal loading parameters (i.e., loading frequency, magnitude, and duration), and investigate the long‐term effects of mechanical loading on a ligament.