Poster AA11 - Mechanical and Structural Response Following Increased Tendon Loading in Synergistic Ablation Model

Introduction: Tendon homeostasis is maintained by a balance between the magnitude and duration of mechanical loading. Moderate increases in load magnitude can improve tendon material properties1, whereas larger overload can lead to degeneration and eventual rupture2. The threshold from healthy adaptation to degeneration is unknown, therefore, animal models of altered loading are needed. To address this need, we recently overloaded the plantaris tendon by 5-fold in an adult rat (7 mo old) using a synergist ablation (SynAb) model, which resulted in degenerative changes including an increase in CSA, cell density, fibril diameter, and a decrease in modulus.3 While the adult tendon response to overload was degenerative, it was unknown whether a younger rat, more typically used in tendon altered loading research, was also degenerative. Therefore, the objective of this study was to evaluate the tensile failure and fatigue mechanics following tendon overload in juvenile (3 mo old) rats. Tendon mechanics were evaluated after 8-weeks of overload in the SynAb model; additionally, MRI was used to evaluate 3D tissue structure and histology to evaluate tissue cellularity and fiber structure.

Materials and

Methods: Three month-old female Long Evans rats received bilateral SynAb surgery (Achilles tendon transected to overload plantaris tendon), sham surgery, or were age-matched intact control. Activity, measured by distance traveled, was evaluated over an 8 hr dark cycle for 3 consecutive days before surgery and biweekly post-surgery until sacrifice at 8 weeks. The two plantaris tendons from each rat were assigned to different assays (e.g., mechanical testing or histology). For mechanical testing, the plantaris tendon was removed and CSA was measured. To evaluate tensile failure mechanics, the tendon (n=10/group) was preloaded, preconditioned from 1% to 5% strain for 5 cycles, and loaded to failure at 1%/s. Structural and material properties were calculated from the load-extension and stress-strain response.3,5 For fatigue mechanics, separate tendons (n=9/group) were cyclically loaded between 5 and 20 MPa (10%-40% of the mean ultimate tensile stress from tensile failure tests) at 2 Hz until failure or 50,000 cycles. Cycles to failure (N) was determined. The loading modulus (slope of the loading ramp in each cycle), peak strain, and hysteresis area were evaluated at 50% of fatigue life. For histology (n=8/group), plantaris tendon sections were stained with Hematoxylin and Eosin (H&E). Each leg (n=5/group) was imaged with a T1 FLASH 3D scan (9.4T MRI) to evaluate 3D tissue structure (resolution 0.1 x 0.1 x 0.1mm). The plantaris, Achilles, scar tissue, and calcaneus was manually segmented using Simpleware. A one-factor ANOVA (mechanics) and two-factor ANOVA (activity) with a Tukey’s post-hoc test was used to test for the effect of treatment.

Results, Conclusions, and Discussions: Activity, including total distance traveled and number of rearings (Fig 1) was not different between groups or any timepoints. The CSA in the SynAb was 12-16% higher in SynAb compared to the Intact and Sham (Fig 2A). Tendons from juvenile rats had an adaptive mechanical response to increased loading. Specifically, in tensile failure tests we observed a significant increase in ultimate load (Fig 2C). However, the material properties, ultimate tensile stress and modulus, did not change, suggesting that there may be more of the same quality of material being deposited in SynAb. In cyclic fatigue, there was an increase in cycles to failure in the SynAb suggesting that the tendon has increased resistance to fatigue loading and therefore supporting the observed adaptive mechanical response (Fig 2D,E). The increase in cell density in the SynAb model at 8-weeks is similar to our previous study in 7-month old rats (Fig 3A-C).3 From MRI, scar tissue formed adjacent to the plantaris, appearing to push the plantaris toward the posterior side of the ankle, which may have affected the load distribution and remodeling (Fig 3C-E). The SynAb tendon also increases in thickness closer to the muscle side, which could be to the loading axis change. The mechanical and structural adaptations to altered loading were different than previous work showing degenerative outcomes at 8-weeks, which may be due to the use of younger rats (3 mo versus 7 mo). At 3 months, rats are still growing, which may enhance their developmental adaptation over degenerative changes seen in older rats.7 Additionally, it is not clear from this study if overload will eventually lead to further adaptation or degeneration at later timepoints. A limitation of the SynAb model is the potential role of inflammation from the surgery itself and possible biological contributions from the adjacent scar tissue, which will be assessed in future studies. In conclusion, increased tendon load was adaptive in young rats, while is degenerative in older rats.The animal age should be considered when interpreting altered loading studies.

Acknowledgements (Optional):

References: [1] Silbernagel+ 2001, [2] Jung+ 2009, [3] Bloom+ 2023, [4] Peloquin+ 2023, [5] Lee+ 2017 [6] Gumucio+ 2014 [6] Pardes+2016
ACKNOWLEDGEMENTS: Funding for this research was provided by NIH/NIAMS R01 AR080059-01