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Biomechanics

Biomechanics - Poster Session C

Poster D9 - Histological Analysis Shows Minimal Inflammation in Sub-Rupture Tendon Fatigue Model

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Tendon is a fibrous tissue that transmits forces from muscle to bone. Due to repetitive and intense loading, overuse tendon injuries are common and do not heal effectively. Accumulation of overuse sub-rupture injuries lacks inflammation, leaving the healing cascade untriggered and allowing injuries to worsen.[1] To study onset of chronic tendon injuries, our lab has developed a protocol to induce sub-rupture fatigue in mouse patellar tendon. [2] The animal is anesthetized, the tendon is exposed, and the patella and the tibia are clamped to allow cyclic loading of the tendon without directly gripping the tendon. We are examining the inflammatory response in tendons following sub-rupture fatigue injury since inflammation is crucial for initiating healing. Preliminary findings showed an increase in leukocytes, M1 macrophages, and Cx3Cr1-positive cells in the first week following injury, though these have not been examined at later timepoints.[3] This project aims to examine the tissue-level inflammatory response of our fatigue injury model at 14 days post-injury. We use Hematoxylin and Eosin (H&E) staining to perform bulk histological analysis on fatigue damaged and naïve tendons. H&E staining provides excellent visible contrast between cell nuclei and cytoplasm, allowing identification of specific inflammatory cell types, visualization collagen alignment, and location of peritendinous blood vessels. We hypothesize that there will be minimal difference in the inflammatory response between fatigue loaded and naïve tendon, explaining the observed untriggered healing response.

Materials and

Methods:  15–16-week-old male wild-type (C57BL/6) mice were anesthetized, and the left patellar tendon was subject to one of two injury protocols: (1) sub-rupture fatigue loading (n=3), or (2) sham fatigue surgery (n=2), where the patella and tibia are gripped but do not undergo cyclic loading. Animals resumed cage activity for two weeks following injury before being sacrificed. Patella-tendon-tibia complexes were harvested from both hind limbs, allowing for the uninjured contralateral limbs to serve as the naïve control group (n=3). Limbs were fixed, decalcified, embedded in paraffin, and 6 µm thick sagittal sections were obtained. Three sections from each sample were stained using H&E and were graded for inflammation. Each section was divided into three regions: tendon midsubstance, origin and insertion sites (entheses), and peritendinous tissue. To examine inflammatory severity, samples were graded by a blinded observer on the following criteria: (1) inflammatory cell presence, (2) increased vascularization, (3) collagen alignment, (4) cell roundness, and (5) cell density. Each criterion was graded on a 0-3 scale, with 0 indicating no abnormality and 3 indicating severe abnormalities. A detailed description of the grading criteria is compiled in Table 1. Slight adjustments were made to the scale based on the region of the tendon being assessed; for example, collagen alignment was not graded within the peritendinous tissue. Inflammation grading was then averaged for each animal and pooled by experimental group. Due to the small sample size of the sham group, naïve and sham samples were combined and compared to fatigue-injured using a Mann-Whitney Test.

Results, Conclusions, and Discussions: Results from inflammation grading are compiled and shown in Figure 1. As expected, there were no significant differences in most of the grading criteria used to assess inflammation between the fatigued and pooled naïve and sham groups. However, a significant increase in unaligned collagen (p=0.0179) and cell roundness (p=0.0357) was observed in fatigue-damaged tendons compared to the pooled naïve and sham groups. This observation is attributed to induced matrix damage rather than an inflammatory response. Consistent with our previous findings, induced tendon fatigue injury results in plastic deformation of collagen fibers, [4] leading to damage kinks and increased collagen misalignment in this group. The observed increase in the number of rounded cells is likely due to the abnormal loading environment experienced by cells in regions of plastic matrix deformation as tenocyte cell morphology has been shown to change as their loading shifts from tensile to shear or compressive. However, there was no observed effect of fatigue injury on cell density, inflammatory cell presence, or vascularity, suggesting that there is no significant difference in the overall inflammatory response between tendon overuse injuries and naïve tendon. In previous studies, we noted an upregulation in Cx3Cr1-positive cells at this time point.[3] However, analyzing samples using H&E grading has allowed us to assess the bulk inflammatory response following sub-rupture fatigue injury, supporting the notion that the inflammatory response is limited. Future work will expand upon this grading scale to more fully quantify cell density, inflammatory cell presence, and cell roundness, reducing the subjectivity associated with the current grading scale. Creating a more standardized quantitative grading system will further validate our assumption that our sub-rupture fatigue injury model does not lead to a significant inflammatory response.

References:
1. Andarawis-Puri, N et al., J Musculoskelet Neuronal Interact, 2011.
2. Fung, D et al., J Biomech, 2010.
3. Bell, R et al., Innate Inflammatory Response is Implicated in Onset of Tendinopathy [Abstract no. 0048]. Orthopaedic Research Society, 2022.
4. Neviaser, A et al., J Shoulder Elbow Surg, 2012.

Acknowledgements (Optional):  Andarawis-Puri Lab
- Nelly Andarawis-Puri
- Diane Stonestreet
- Ben Johnston
- Marguerite Pacheco
- Ellen Brooks
- Josette Vigil
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