This study quantified local fiber re-alignment and crimp behavior in a mature mouse SST model. Uncrimping of collagen fibers was confined to the toe-region of the mechanical test at both the insertion site and midsubstance locations supporting previous work in the developmental mouse SST () (Miller, 2012a
). Additionally, this result provides further support for the theory that the uncrimping of collagen fibers may explain the toe-region and contribute to the nonlinear behavior of tendons (Houssen et al.; Miller, 2012a
; Rigby, 1964
; Rigby et al., 1959
; Screen et al., 2004
; Viidik, 1972
; Woo et al., 2000
As expected, the insertion site re-aligned throughout the entire mechanical test (). Surprisingly, the midsubstance location did not re-align during the toe-region (). The insertion site is thought to experience more complex, multi-axial loads in vivo, resulting in a more disorganized collagen fiber distribution (Lake et al., 2009
; Miller, 2012b
; Miller et al., 2012
; Thomopoulos et al., 2003
), which may require additional collagen fiber re-alignment at the insertion site. Therefore, the midsubstance location may “pause” recruiting fibers following preconditioning to allow collagen fibers to uncrimp during the toe-region, thus giving the insertion site additional time to recruit fibers before transitioning into the linear-region. Alternatively, fibers initially recruited in the midsubstance during preconditioning may already be failing by the end of the toe-region as crimp histology identified regions of fiber damage at the transition point in some samples. Assuming these fibers were initially aligned in the direction of load or required a minimal amount of re-orientation, the fibers would be tensioned almost immediately and may fail earlier in the ramp-to-failure. Their early failure would require the recruitment of additional fibers in the linear-region at the midsubstance location. Further, at both locations, collagen fiber re-alignment occurred during preconditioning, supporting previous studies which identified a correlation between preconditioning and collagen fiber alignment (Miller, 2012b
; Miller et al., 2012
; Quinn and Winkelstein).
In addition, this study found no significant differences in crimp frequencies at the preload compared to points after preconditioning (5, 10 or 20 cycles). This suggests that the uncrimping of collagen fibers occurs primarily in the toe-region, regardless of the number of preconditioning cycles. However, histology and the reported average values of crimp frequency across all litters indicate that, while not significant, decreases in crimp frequency with increasing number of preconditioning cycles may be present at the midsubstance location in mature tendon (). This is supported by previous observations noting similar behavior and merits further study (Houssen et al.; Miller, 2012a
As expected, a more disorganized collagen fiber distribution and lower moduli values were identified at the insertion site compared to the midsubstance location ( & ). The local differences in organization and modulus support results in the human and mature rat SST (Lake et al., 2009
; Miller, 2012b
). Interestingly, unlike the mature rat SST (Miller, 2012b
), the midsubstance and insertion site in the mouse SST demonstrated different re-alignment behaviors (). This may suggest that the locations experience different loading conditions in vivo, which may initiate local remodeling, resulting in different fiber network configurations including alterations in collagen fiber alignment as well as cross-links, fiber-fiber and fiber-matrix interactions. These potential changes in collagen microstructure between locations may affect their ability to respond to load simultaneously or in the same manner.
While significant changes in alignment and mechanics were identified, no changes in crimp frequency were found with location, supporting previous results at 4, 10 and 28 days old (Miller, 2012a
). This suggests that crimp may not be affected by multi-axial loads seen at the insertion site and crimp may be a mechanism of tensile loading instead of compressive or multi-axial loading. The magnitude of crimp response was decreased at insertion compared to midsubstance, supporting previous results at 28 days old (Miller, 2012a
). Previous speculation that fiber re-alignment may be a more dominant or earlier mechanism at the insertion site than crimp is supported here (Miller, 2012a
; Sellaro et al., 2007
). It is possible that fibers at the midsubstance have finished their initial collagen fiber re-alignment and begin to uncrimp with increasing cycles of preconditioning. In this study, the entire tendon was flash frozen at 5 and 8% strain to represent points in the toe- and linear-regions (determined by the structural model (Peltz et al.)). While these are appropriate strains to represent each of the corresponding regions, it is possible that the insertion site and midsubstance locations are at different stages of the toe-region, which may provide explanation for these differences in structural behavior.
No significant differences were identified between crimp frequency at the reference configuration, or “in situ” condition, and after the preload. The lack of significant difference identified here indicates that the crimp frequency behavior demonstrated throughout the mechanical test may be applicable to the crimp behavior in vivo.
This study suggests that the uncrimping of collagen fibers may be an in vivo structural response to mechanical load and not solely an artifact of ex vivo mechanical testing and changing the residual stress experienced by the tendon. This study is not without limitations. First, the structural fiber recruitment model was utilized to determine average strain values for the entire tendon samples. While the average values are within the parameters to represent the toe- and linear-regions of both the insertion site and midsubstance locations, it is possible that the locations were at different “points” in the toe-region. However, the chosen method improves consistency within this experiment and allows for comparisons with other experiments, in addition to permitting conclusions to be drawn across collagen fiber alignment and crimp measurements. This study examined collagen fiber re-alignment and crimp behavior throughout only one mechanical testing protocol. Future studies are necessary to determine the effects of collagen fiber re-alignment and crimp behavior in response to different loading protocols and at additional strains in the toe- and linear-regions at both locations. Additionally, while sample were immediately flash-frozen following loading, we cannot guarantee that no stress relaxation occurs whatsoever while the sample was freezing. However, consistent, significant changes in crimp frequency were still identified across samples. Further, histology from this project was stained in multiple batches, though the analysis methods performed were consistent and repeatable. As previously described, the quantitative software normalizes each image by the average pixel intensity and establishes exclusion criteria based on the standard deviation of pixel fluctuations to exclude artifact and only include fluctuations representing fiber crimp (Miller, 2012a
). This method minimizes the potential effect of changes in stain intensity on the histological analyses. Finally, while the reference configuration samples were flash-frozen while still attached to the humerus and supraspinatus muscle, adjacent structures, such as the deltoid, the infraspinatus and subscapularis, were removed. The removal of these structures may have changed the supraspinatus tendon loading and subsequently, the in vivo crimp frequency. Additionally, the tone of the supraspinatus muscle may have changed post-sacrifice and altered the crimp frequency. Future studies are needed to further examine crimp frequency and fiber distribution of the supraspinatus tendon at its native state in the body.
In conclusion, this study quantified local mechanical properties as well as collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse SST model. Additionally, this study identified that collagen fiber realignment and crimp behavior were different at each location suggesting that the insertion site and midsubstance locations may respond to load differently and may have different microstructural and biochemical compositions. Future studies are needed to further characterize local compositional and microstructural properties to examine these differences in behavior and elucidate their potential effects on clinical treatments and diagnosis.