Poster AA1 - Disuse Reduces Achilles Tendon Mechanics in DO Mice, But Via Different Mechanisms in Males and Females

Introduction: Hierarchical collagen fibers are the main source of strength and function in tendons and ligaments.1 It has been well-established that mechanical cues are necessary for the health and maintenance of these fibers.1,2 Further, disuse or reductions in mechanical loading significantly reduces tendon mechanics;2-4 however the exact mechanism is largely unknown. A better understanding of cellular response to unloading in tendons is necessary to prevent the deleterious effects of inactivity, prevent tendon degeneration during long-term space travel, and inform better rehabilitation protocols during recoveries from lower-limb injury.2,3,5 Further, there is a need to investigate whether tendon response to disuse is different between males and females. The majority of studies on tendon response to disuse—which are few compared to the number of studies researching disuse in muscle and bone—are conducted on male inbred mice or rats, which lack the genetic and gender variability of humans, limiting the potential impact of these studies on human tissues.5 Diversity outbred (DO) mice are developed by crossbreeding eight genetically diverse inbred founder strains of mice—C57Bl/6J, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ and WSB/EiJ—resulting in a population of genetically variable mice that more closely match human responses to tendon disuse.4 The objective of this study was to investigate the response of Achilles tendons to disuse via single limb immobilization in genetically variable DO mice to better understand tenocyte response to unloading. Further, we were interested in exploring whether there were sex differences in tenocyte response to disuse.

Materials and

Methods: All procedures were approved by the VCU IACUC. Thirty-two male and female 16-week old DO mice had their right hind-limbs immobilized in casts for 3 weeks as part of a larger study evaluating the effects of disuse on bone and muscle. Left hind-limbs were uncast to serve as contralateral controls. After 3 weeks, mice were sacrificed and the Achilles tendons from both hind-limbs were removed. Tendons were randomly divided between analysis techniques: for each sex, N=9 were analyzed for organization with confocal reflectance microscopy,6 N=10 were frozen for mechanical analysis, and N=13 were analyzed for gene expression and composition. Mechanical properties were determined by immersing tendons in PBS and running a stress relaxation test at 5% strain for 10 minutes, followed by a ramp-to-failure at 0.5% strain/s to determine elastic mechanics.7 Mechanics are reported as the ratio of individual mechanics in male or female cast tendons to the average of uncast tendons of the same sex. Gene expression was analyzed via RT-PCR for COL1A1, COL3A1, lysyl oxidase (LOX), Aggrecan (ACAN), scleraxis (SCX), MMP3, and MMP13, normalized to GAPDH and reported as 2-∆∆CT comparing expression in cast to uncast tendons. Tendons for compositional analysis were weighed wet, lyophilized, and weighed dry to determine percent water. DNA, glycosaminoglycan (GAG), and collagen content were determined by Picogreen, DMMB, and hydroxyproline assays, respectively. Data was normalized to wet weight and reported as the ratio of cast to uncast tendons for each mouse. Significance was determined using one-way and paired two-way ANOVA with Tukey’s post-hoc(p < 0.05).

Results, Conclusions, and Discussions: Similar to previous studies,2 confocal reflectance revealed no apparent differences in collagen organization, regardless of disuse or sex. However, unloaded/casted tendons had significantly decreased stress relaxation and toe-region mechanics compared to loaded/uncast tendons for both male and female mice, representing a decrease in viscoelastic and elastic mechanics (Fig 1A-B). These findings largely correlate with previous studies performed on inbred animal models,2 however it is not well understood why mechanics decrease when collagen organization remains unchanged.
Interestingly, males and females differentially regulated gene expression in tendons in response to disuse. Female unloaded/cast tendons showed significantly upregulated expression of COL1A1, COL3A1, LOX, ACAN, SCX, MMP3, and MMP13 compared to contralateral loaded/uncast tendons, while males did not upregulate any of these genes. Further, casting in females resulted in significantly higher gene expression in all genes compared to males, except MMP3 (Fig 2A). Similarly, compositional differences were also found between DO male and female tendons; female mice showed a trend of increasing DNA and GAG content in cast tendons, while male mice significantly decreased collagen content in cast tendons (Fig 2B). Overall, this suggests that 3 weeks of disuse in the Achilles tendons of genetically variable DO mice leads to differential disruptions in tendon composition based on the sex of the mouse. Female mice appeared to have a pathological-anabolic response while male mice demonstrated a more catabolic response in unloaded tendons. However, despite sex-specific changes in composition, both sexes showed similar reductions in tendon mechanics as a response to disuse. LOX crosslink accumulating is being further analyzed to investigate whether disuse affects collagen crosslinking or contributes to the reductions in mechanics in cast tendons.2,4
This study is one of the first to evaluate the effects of disuse on the Achilles tendons of genetically variable mice, and we found significant sex differences between male and female mice in response to unloading. A better understanding of how tendons in males and females respond to disuse could help protect tendons against the deleterious effects of prolonged inactivity like bedrest, prevent degeneration in long-term space travel, and help improve rehabilitation protocols after tendon injury.2,3,5

Acknowledgements (Optional):

References: 1. Galloway+J Bone Surg Am 2013; 2. Magnusson+J of Physiology 2018; 3. Roffino+Life Sci Space Res 2021; 4. Almeida-Silveira+Eur J Appl Physiol 2000; 5. Liphart+NPJ Microgravity 2023; 6. Puetzer+Biomat 2021; 7. Ansorge+Ann Biomed Eng 2011.