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Research Papers

Tissue Strain Reorganizes Collagen With a Switchlike Response That Regulates Neuronal Extracellular Signal-Regulated Kinase Phosphorylation In Vitro: Implications for Ligamentous Injury and Mechanotransduction

[+] Author and Article Information
Sijia Zhang

Department of Bioengineering,
University of Pennsylvania,
240 Skirkanich Hall,
210 South 33rd Street,
Philadelphia, PA 19104
e-mail: sijiaz@seas.upenn.edu

Xuan Cao

Department of Materials
Science and Engineering,
University of Pennsylvania,
3231 Walnut Street,
Philadelphia, PA 19104
e-mail: xuancao@seas.upenn.edu

Alec M. Stablow

Department of Bioengineering,
University of Pennsylvania,
240 Skirkanich Hall,
210 South 33rd Street,
Philadelphia, PA 19104
e-mail: stablowa@seas.upenn.edu

Vivek B. Shenoy

Department of Bioengineering,
University of Pennsylvania,
3231 Walnut Street,
Philadelphia, PA 19104;
Department of Materials
Science and Engineering,
University of Pennsylvania,
3231 Walnut Street,
Philadelphia, PA 19104;
Department of Mechanical Engineering and
Applied Mechanics,
University of Pennsylvania,
3231 Walnut Street,
Philadelphia, PA 19104
e-mail: vshenoy@seas.upenn.edu

Beth A. Winkelstein

Mem. ASME
Department of Bioengineering,
University of Pennsylvania,
240 Skirkanich Hall,
210 South 33rd Street,
Philadelphia, PA 19104;
Department of Neurosurgery,
University of Pennsylvania,
240 Skirkanich Hall,
210 South 33rd Street,
Philadelphia, PA 19104
e-mail: winkelst@seas.upenn.edu

1Corresponding author.

Manuscript received August 5, 2015; final manuscript received October 21, 2015; published online January 27, 2016. Editor: Victor H. Barocas.

J Biomech Eng 138(2), 021013 (Jan 27, 2016) (12 pages) Paper No: BIO-15-1392; doi: 10.1115/1.4031975 History: Received August 05, 2015; Revised October 21, 2015

Excessive loading of ligaments can activate the neural afferents that innervate the collagenous tissue, leading to a host of pathologies including pain. An integrated experimental and modeling approach was used to define the responses of neurons and the surrounding collagen fibers to the ligamentous matrix loading and to begin to understand how macroscopic deformation is translated to neuronal loading and signaling. A neuron-collagen construct (NCC) developed to mimic innervation of collagenous tissue underwent tension to strains simulating nonpainful (8%) or painful ligament loading (16%). Both neuronal phosphorylation of extracellular signal-regulated kinase (ERK), which is related to neuroplasticity (R2 ≥ 0.041; p ≤ 0.0171) and neuronal aspect ratio (AR) (R2 ≥ 0.250; p < 0.0001), were significantly correlated with tissue-level strains. As NCC strains increased during a slowly applied loading (1%/s), a “switchlike” fiber realignment response was detected with collagen reorganization occurring only above a transition point of 11.3% strain. A finite-element based discrete fiber network (DFN) model predicted that at bulk strains above the transition point, heterogeneous fiber strains were both tensile and compressive and increased, with strains in some fibers along the loading direction exceeding the applied bulk strain. The transition point identified for changes in collagen fiber realignment was consistent with the measured strain threshold (11.7% with a 95% confidence interval of 10.2–13.4%) for elevating ERK phosphorylation after loading. As with collagen fiber realignment, the greatest degree of neuronal reorientation toward the loading direction was observed at the NCC distraction corresponding to painful loading. Because activation of neuronal ERK occurred only at strains that produced evident collagen fiber realignment, findings suggest that tissue strain-induced changes in the micromechanical environment, especially altered local collagen fiber kinematics, may be associated with mechanotransduction signaling in neurons.

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Figures

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Fig. 2

ERK phosphorylation increases with increasing imposed distraction magnitude. (a) Representative confocal images showing neuronal structure labeled for βIII-tubulin (blue), pERK expression (red), and their colocalization (pink) demonstrate increased ERK phosphorylation at 8 mm than at 4 mm but with no difference for loading rate; the scale bar applies to all panels. (b) Quantification of pERK expression in loaded constructs normalized to unloaded controls indicates significant increases in NCCs distracted to 8 mm compared to 4 mm at both the 0.5 mm/s (*p < 0.0001) and 3.5 mm/s (#p = 0.002) distraction rates.

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Fig. 3

Elemental pERK expression is positively related to elemental MPS. (a) Significant positive correlations exist between normalized elemental pERK expression and elemental MPS at both 0.5 mm/s (R2 = 0.200, *p < 0.0001) and 3.5 mm/s (R2 = 0.041, #p = 0.0171) distraction rates. (b) pERK expression is significantly (p < 0.0001) regressed against elemental MPS at both distraction rates. Solid dots () and open triangles (Δ) represent samples in which elevated pERK expression was detected (probability of 1) or not detected (probability of 0) at each strain level for 0.5 mm/s and 3.5 mm/s, respectively. The predicted 50th-percentile thresholds for ERK phosphorylation are 11.7% for 0.5 mm/s rate, 10.2% for 3.5 mm/s; the corresponding 95th-percentile thresholds are 24.9% and 23.7% at the 0.5 mm/s and 3.5 mm/s distraction rates, respectively.

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Fig. 4

Neuronal AR and orientation toward the loading direction depend on NCC displacement and loading rate. (a) Significant correlations are detected between normalized cell AR and MPS for both 0.5 mm/s (*p < 0.0001) and 3.5 mm/s (#p < 0.0001) rates of distraction. (b) Cell orientation angle (α) is measured as the angle between cell's long-axis (dotted line) and the direction of applied tension (arrow). (c) Distributions of cell orientation angles show that cell orientation angle is significantly smaller (p = 0.004) at the slow distraction rate (0.5 mm/s) than at the fast rate (3.5 mm/s).

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Fig. 1

Representative images of the experimental test setup demonstrating (a) the mechanical testing system integrated with the elements of the QPLI system, (b) an NCC in an unloaded reference state and at 8 mm of distraction, (c) the corresponding force–displacement response, and (d) strain map showing both the magnitude and directions of MPS at 8 mm displacement. (e) A schematic overview showing the DFN model as constructed using the confocal images of collagen in elements of the unloaded control NCCs, as well as the boundary conditions simulating experimental conditions for the NCCs.

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Fig. 5

For the 0.5 mm/s loading rate, fiber realignment exhibits a switchlike response. (a) Representative collagen fiber alignment maps in two adjacent elements before (reference) and at maximal distraction (distracted) show different fiber realignment responses depending on both the distraction magnitude and rate. The direction of the orientation vector indicates the average fiber alignment direction measured through the NCC thickness and its length represents the alignment strength in that direction. Fiber reorientation toward the loading direction is apparent by the changes in the length of the orientation vectors and their directions toward the vertical direction, with the most realignment along the lateral edges (indicated by vertically orientated vectors). (b) Quantification of the normalized circular variance measures the degree of fiber realignment in the distracted configuration relative to the unloaded reference and indicates significantly more collagen fiber realignment at 8 mm of distraction at 0.5 mm/s than at either 4 mm at the same rate (*p = 0.009) or 8 mm at the higher distraction rate (+p = 0.040). (c) The mean circular variance of collagen fiber orientation angles during loading at 0.5 mm/s exhibits a biphasic response with increasing applied strain (MPS), with a transition point of 11.3% strain estimated. The insets show representative distributions of collagen fiber alignment angles before (0%) and after (16%) evident fiber realignment occurs.

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Fig. 6

DFN modeling predicts that collagen fiber realignment and fiber strain increase with bulk strain. (a) The distribution of fiber angle indicates more realignment toward the loading direction (±90 deg) at 16% strain, which is different from the uniform distributions that are observed at 0% and 7% strain. Plotting the Chi-squared statistic against the applied bulk strain shows that the fiber angle distribution becomes less uniform with increasing strain above 10%. (b) Visualization of the heterogeneous fiber axial strains during network loading shows more fibers under large tensile strain as more fibers realign toward the loading direction at 16% bulk strain compared to 0% and 7% strains. (c) Distributions of fiber axial strains show that most fibers have strains lower than the applied bulk strain and both compressive and tensile fiber strains increase with increasing bulk strain.

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