(A-20) Mechanical Properties of the Equine Superficial Digital Flexor Tendon Are Not Strongly Associated Across Macroscopic and Microscopic Scales

Introduction:: Tendon injuries account for approximately 50% of all musculoskeletal injuries in the United States1. Once the tendon has been injured, it undergoes a healing process that does not restore normal tendon properties2. This leaves a disorganized matrix that has a limited functionality compared to healthy tendon. Little is understood about cell-matrix interactions in this disorganized matrix2-3. To better understand these interactions, a tendon-on-a-chip model that replicates elements of the native matrix and mechanical environment needs to be developed. Such models can allow for the study of single cell-matrix mechanobiology response. However, the small size of the systems does not allow for typical mechanical testing for tendons such as tensile testing. Atomic force microscopy (AFM) has the possibility to be a promising solution to this issue since it allows for forces to be recorded on the micron scale4. The scale of the data obtained allows for local cell-matrix interactions to be studied; however, it is unknown if AFM can serve as a surrogate for macroscale tendon properties. The objective of this study was to compare tendon mechanical properties obtained through AFM to the tensile testing modulus for native equine superficial digital flexor tendons (SDFTs).


Materials and Methods:: The SDFT was obtained from 3 horses. The sheath was removed from the outside of the tendon. The tendon was divided into three sections: proximal, midsubstance and distal. The sections were embedded in Celluclay and sliced into 1 mm thick slices5. From each slice, longitudinal and transverse dog bones were taken relative to the long-axis of the tendon using a 10 mm Print-A-Punch and a 1 cm x 1 cm square was taken from the center of the slice for AFM6. Samples were tested using an Instron with pneumatic clamps to record the elongation and force the sample experienced under specific loading conditions. The longitudinal and transverse macroscopic samples were pulled to failure at a rate of 1 mm/s. ImageJ was used to measure the gauge length, width and thickness of the dog bone sample from images taken prior to testing. The elastic modulus was found by calculating the slope of the linear section of the stress-strain curve. The sections for AFM were cryosectioned into 10 µm slices. The moduli was found at four points on each sample using a colloidal tip on contact force mode masked and converted to stiffness values using the Hertz model. A one-way ANOVA with Tukey’s test (α=0.05) was used for statistical comparisons. Linear regression analysis was done between data for the AFM and longitudinal and transverse testing.


Results, Conclusions, and Discussions:: Longitudinal modulus values (88.92 ± 30.8 MPa) were found to be 3 orders of magnitude larger than AFM (0.061 ± 0.016 MPa), while transverse modulus values (2.98 ± 2.19 MPa) were approximately 1 order of magnitude larger (Fig. 1B). For longitudinal samples, the proximal section was found to have a mean modulus value about 1.7 times larger than the midsection and distal regions. Similar differences were found for AFM. However, for both longitudinal and AFM samples, the differences between regions were not statistically different (Fig. 1B-C). A weak positive correlation was found between the moduli obtained from AFM and longitudinal samples (R2 = 0.14, p = 0.01) and AFM and transverse samples (R2 = 0.17, p = 0.06) (Fig. 1E, Fig. 1F).

Overall, our study showed that mechanical properties for the SDFT obtained from AFM are not strongly associated with properties obtained from tensile testing. Although associations between macroscopic and microscopic testing were statistically significant, the high variability suggests that values obtained at the microscopic scale could not be used to predict values at the macroscopic scale or vice versa. Thus, tendon models cannot assume that modulus values of the local extracellular matrix directly relate to the macroscale tendon modulus. The difference found between the longitudinal and transverse directions further highlights the anisotropic properties of tendon, in which collagen fibers align along the longitudinal direction. Testing in the longitudinal and transverse directions gives information about the macroscale mechanical properties due to the collagen fibers and the non-collagenous matrix, respectively. Interestingly, the macroscale direction did not appear to impact associations to microscopic AFM testing. Previous studies found the longitudinal moduli to be approximately 1000 MPa; however, those studies looked at the whole tendon rather than thin samples, which may disrupt some of the collagen fibers or introduce clamping effects7-8. Future work will look at how the age and sex of the tissue affects the mechanics. Additional work will also be done to develop an engineered tendon model that mimics the native normal and diseased tendon modulus found on the microscopic scale.


Acknowledgements (Optional): :

References (Optional): :

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