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Research Papers

Multiscale Mechanical Evaluation of Human Supraspinatus Tendon Under Shear Loading After Glycosaminoglycan Reduction

[+] Author and Article Information
Fei Fang

Department of Mechanical Engineering
and Materials Science,
Washington University in St. Louis,
1 Brookings Drive,
Campus Box 1185,
St. Louis, MO 63130
e-mail: fangfei@wustl.edu

Spencer P. Lake

Department of Mechanical Engineering
and Materials Science,
Washington University in St. Louis,
1 Brookings Drive,
Campus Box 1185,
St. Louis, MO 63130;
Department of Biomedical Engineering,
Washington University in St. Louis,
1 Brookings Drive,
Campus Box 1185,
St. Louis, MO 63130;
Department of Orthopaedic Surgery,
Washington University in St. Louis,
1 Brookings Drive,
Campus Box 1185,
St. Louis, MO 63130
e-mail: lake.s@wustl.edu

1Corresponding author.

Manuscript received November 30, 2016; final manuscript received April 26, 2017; published online June 6, 2017. Assoc. Editor: Eric A Kennedy.

J Biomech Eng 139(7), 071013 (Jun 06, 2017) (8 pages) Paper No: BIO-16-1488; doi: 10.1115/1.4036602 History: Received November 30, 2016; Revised April 26, 2017

Proteoglycans (PGs) are broadly distributed within many soft tissues and, among other roles, often contribute to mechanical properties. Although PGs, consisting of a core protein and glycosaminoglycan (GAG) sidechains, were once hypothesized to regulate stress/strain transfer between collagen fibrils and help support load in tendon, several studies have reported no changes to tensile mechanics after GAG depletion. Since GAGs are known to help sustain nontensile loading in other tissues, we hypothesized that GAGs might help support shear loading in human supraspinatus tendon (SST), a commonly injured tendon which functions in a complex multiaxial loading environment. Therefore, the objective of this study was to determine whether GAGs contribute to the response of SST to shear, specifically in terms of multiscale mechanical properties and mechanisms of microscale matrix deformation. Results showed that chondroitinase ABC (ChABC) treatment digested GAGs in SST while not disrupting collagen fibers. Peak and equilibrium shear stresses decreased only slightly after ChABC treatment and were not significantly different from pretreatment values. Reduced stress ratios were computed and shown to be slightly greater after ChABC treatment compared to phosphate-buffered saline (PBS) incubation without enzyme, suggesting that these relatively small changes in stress values were not due strictly to tissue swelling. Microscale deformations were also not different after ChABC treatment. This study demonstrates that GAGs possibly play a minor role in contributing to the mechanical behavior of SST in shear, but are not a key tissue constituent to regulate shear mechanics.

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Figures

Grahic Jump Location
Fig. 1

Normalized GAG amounts in SST samples treated for 6 h with different concentrations of ChABC buffer (a) or with 0.2 units/ml ChABC buffer for different durations of incubation (b). (c) GAG amount within DDFT after ChABC treatment exhibited a gradient distribution along the sample width; inset displays how clamps (left and right blocks) and attached sample (middle block) are secured for incubation and the subsequent mechanical test, where the normalized width is shown as 0 (top) to 1 (bottom) and is indicated as the x axis in the plot.

Grahic Jump Location
Fig. 2

Microscopy images from three locations of the same sample before and after application of 0.24 shear strain. Different locations within one sample from a single SST region showed heterogeneous organization of collagen fibers and different modes of deformation: no obvious deformation at location 1; fiber sliding at location 2; fiber reorganization at location 3. Rotation angle, θ, was calculated as the angle between the horizontal axis of images and deformed photobleached grids. Grid intersection points (dots added for visualization purpose only) were used to calculate local-matrix-based strain, while white arrows show shear loading direction.

Grahic Jump Location
Fig. 3

Typical histological images of paired control and treated SST samples, which were stained by H&E and Alcian blue to show collagenous ECM ((a)–(c)) and GAGs ((d)–(i)), respectively. After ChABC treatment, the ECM was not visibly disrupted. GAG amount decreased differentially by location within each sample (demonstrated by varying intensity of staining in (e) and (f) compared to (d)), and GAG-rich pericellular matrix was observed to have been particularly degraded. Images (a)–(f) were acquired by 10× objective and images (g)–(i) by 40× objective. Treated-top and treated-middle are sections from the top and middle of treated samples, respectively. Arrows in images (g)–(i) denote cell nuclei.

Grahic Jump Location
Fig. 4

Peak (a) and equilibrium (b) stresses for control (untreated) samples increased at larger strain steps, but were not significantly different across three SST regions; inset shows three regions (M = medial; A = anterior; P = posterior) in SSTs (mean ± SD)

Grahic Jump Location
Fig. 5

Averaged stress–time curves of SST samples from anterior (A), posterior (P), and medial (M) regions after PBS incubation and ChABC treatment, normalized by corresponding peak stresses from the 0.24 strain step of mechanical test conducted before incubation or treatment. Samples subjected to ChABC treatment showed larger decreases in stress than samples following PBS incubation. Short solid and dashed lines show normalized peak stresses for each group.

Grahic Jump Location
Fig. 6

Group peak stresses (a), equilibrium stresses (b), and stress relaxation percent (c) showed moderate changes at increasing strain steps, but were similar before and after ChABC treatment (mean ± SD)

Grahic Jump Location
Fig. 7

Larger absolute values of peak (a) and equilibrium (b) reduced ratios after ChABC treatment compared to values following PBS incubation indicate that stresses decreased more after ChABC treatment (mean ± SD)

Grahic Jump Location
Fig. 8

Samples before and after ChABC treatment exhibit similar measures of microscale deformation, namely local-matrix-based strain (a) and rotation (b) (mean ± SD)

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