Characterizing local collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse supraspinatus tendon model☆
Introduction
Collagen fiber re-alignment and uncrimping are two postulated mechanisms of tendon structural response to load. Crimp morphology is thought to be related to tendon mechanical behavior (Rigby, 1964, Rigby et al., 1959, Viidik, 1972, Woo et al., 2000). In previous work, a technique to quantify fiber crimp frequency at specific, quantifiable points throughout a mechanical test was presented and a decrease in crimp frequency was identified during the toe-region of the mechanical test in a developmental mouse supraspinatus tendon model at 4, 10, and 28 days old (SST) (Miller et al., 2012b). Similarly, a study examining collagen fiber re-alignment throughout postnatal development suggested that where fiber re-alignment occurs during mechanical testing may depend on tendon age and matrix maturity (Miller et al., 2012a). Local differences in crimp behavior and collagen fiber re-alignment as well as collagen organization and mechanical properties have been identified throughout postnatal developmental in the mouse SST (Miller et al., 2012b, Miller et al., 2012a). However, collagen fiber crimp, collagen fiber re-alignment and mechanical properties have not been characterized in a mature mouse SST model. While the rat SST is a well-established model for examining clinical problems in the shoulder, the mouse model has advantages including a wide range of genetically modified strains and an established developmental model to further elucidate structure-function relationships.
Recently, a correlation between collagen fiber re-alignment and preconditioning has been identified (Miller et al., 2012c, Quinn and Winkelstein,). Additionally, at late development, comparisons in crimp frequency between the preload and after preconditioning changed in response to increasing the number of preconditioning cycles, indicating that the number of preconditioning cycles applied may affect tendon structural response to load (Miller et al., 2012b). However, the effect of increasing the number of preconditioning cycles has not yet been examined in the mature SST. Further, it is not known how the residual stress of the tendon from the in vivo state to the excised test configuration affects local crimp frequency. Therefore, the objective of this study was to determine 1) where collagen fiber re-alignment and uncrimping occur throughout the mechanical test; 2) if collagen fiber re-alignment and crimp behavior are affected by increasing number of preconditioning cycles; and 3) if crimp behavior, re-alignment or mechanical properties vary by location in a mature mouse SST model. We hypothesize that 1) the largest shift in re-alignment will occur during preconditioning, but re-alignment will also occur in toe- and linear-regions with uncrimping occurring primarily in toe-region; 2) crimp frequency will decrease with increasing number of preconditioning cycles and 3) the insertion site will demonstrate a more disorganized collagen distribution, lower mechanical properties and higher crimp frequency compared to midsubstance location.
Section snippets
Sample preparation
This study was approved by the University of Pennsylvania IACUC. Postnatal mice in a C57/BL/6 background (Jackson Laboratory) were bred in house. All litters were reduced to 6 pups within 1 day of birth to reduce variance from litter size (Festing, 2006). Pups were weaned 21 days after birth and separated by sex. A sample was defined as a collective litter of 6 female pups from the same breeder pair. Ten tendons were used to examine structural changes throughout testing within a sample
Crimp
The 2-way ANOVA identified that the effect of time (throughout the mechanical test) was significant, while location (insertions versus midsubstance) was not found to be significant. No significant interactions were identified. Significant decreases in crimp frequency were identified at the toe-region with all preconditioning protocols at both locations (Fig. 1). No significant changes in crimp frequency were identified at additional points throughout the mechanical test or with increasing
Discussion
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 (Fig. 1) (Miller et al., 2012b). 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
Disclosure statement
The authors have no conflicts of interest and nothing to disclose.
Acknowledgments
The authors would like to acknowledge Jennica J. Tucker for assistance with breeding and the NIH/NIAMS supported Penn Center for Musculoskeletal Disorders for financial support.
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2022, Osteoarthritis and CartilageCitation Excerpt :In these earlier studies, crimping was measured in images obtained by PLM and SEM (thus in 2D and after substantial sample preparation), whereas our study shows that this can be done also in 3D image data sets from SR-PhC-μCT imaging of fresh frozen tissue. Crimping morphology has been studied in relation to mechanical loading for example by snap-freezing of tissue samples during loading34,35. Attempts has been done to integrate PLM with a mechanical testing devise for imaging of tendon36.
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All authors were fully involved in the study and preparation of this manuscript. This is an original submission and an abstract version of part of this work will be presented in a podium session at the 2012 annual meeting of the American Society of Mechanical Engineering Summer Bioengineering Conference.