A Biomechanical-Biochemical Hypothesis for the Role of Collagen in Injury
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Date
2022-07-25
Authors
Barrett, Jeffery
Journal Title
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Publisher
University of Waterloo
Abstract
Low back pain affects 80% of the population at some point in their lives and is the most common musculoskeletal complaint for workplace injuries. Further, it presents as episodic, with sufferers typically experiencing recurrent flares of symptoms. However, the mechanisms that underpin chronic, recurrent low back pain are still disputed, and many cases are paradoxically related to sedentary occupations. Therefore, this thesis explores whether the pathomechanics of low back pain may be partially explained in terms of extracellular matrix homeostasis with a particular focus on collagen. The balance of collagen synthesis, degradation, and mechanical disruption, mediated through an inflammatory response, is foundational for chronic degenerative diseases like tendonitis, osteoarthritis, and degenerative disc disease. Still, it is unclear whether the same pathways may be involved in recurrent low back pain. For this reason, this thesis formulates a mathematical model of this response, supported by three experimental studies that aim to describe the degradation and mechanical disruption properties of collagenous tissues.
Some experiments have suggested that the rate of collagen disruption in collagenous tissues may be related to the strain rates they experience. However, there has yet to be an experiment to quantify this effect. Thus, the first experimental study in this thesis aimed to quantify whether the rate of damage accumulation is directly proportional to the strain rate. Fifty rat-tail tendon specimens were strained to failure, on one axis of a biaxial biological tissue testing apparatus (Cellscale, Waterloo, Ontario), at one of five strain rates: 0.01, 0.05, 0.10, 0.15 or 0.20 s−1. Force and displacement, later normalized to nominal stress and strain, were least-squares fit to a computational model of collagen fibril recruitment employing a Tobolsky-Eyring rate law to describe the rate of bond-breaking among the fibrils. Overall, the direct proportion constant increased linearly with the magnitude of the applied strain rate, yet the exponential parameter did not. Ultimately, this result suggested that the rate of collagen disruption in collagenous tissues may indeed be linearly related to the magnitude of strain rate. In response to potential damage to the extracellular matrix, the resident fibroblasts of soft tissues secrete inflammatory cytokines and matrix-degrading enzymes. Previous studies have suggested that mechanical strain to collagen may inhibit these enzymes, suggesting a potential remodelling mechanism that spares frequently used fibrils. Further, the stress-strain curve of biological tissues exhibits a prominent toe-region as a higher proportion of collagen fibrils become engaged. However, this mechanism implies that the activity of collagenases would be proportionately inhibited throughout the toe region. Thus, this study explored whether this graded inhibition of collagenases would occur for tissues held at loads corresponding to specific proportions of collagen fibrils being uncrimped. Ninety-two rat tail tendon samples were mounted along one axis of a biaxial biological tissue testing apparatus (Cellscale, Waterloo, Ontario), immersed in a Ringer’s solution bath heated to 37◦C with or without the presence of collagenase from C. histolyticum. Their forceelongation curves were measured, and its derivative evaluated to determine instantaneous stiffness-elongation. Tendons were held at levels of force corresponding to 0, 25, 50, 75 and 100% of the linear region’s stiffness, corresponding to approximately that proportion of collagen being uncrimped, for two-hours. Following this exposure, tendons were strained to failure at 0.2 s−1, and the change in linear region modulus was evaluated. Linear regressions between applied strain and the change in tissue stiffness suggested that mechanical strain proportionately inhibited the activity of collagenases inside the toe region. These results supported the proposed fibril selection hypothesis and were consistent with the fibril recruitment model. Several experimental studies have documented inflammatory responses downstream of repetitive lifting and cyclic loading in rat-tail intervertebral discs. These inflammatory responses precede the synthesis of matrix-degrading enzymes, which may contribute to a substantive feedforward inflammatory loop. However, none of these experiments have aimed to detect an inflammatory response downstream of a static flexion-induced creep on the intervertebral disc. Therefore, the purpose of this experiment was to document an inflammatory response in the rat-tail intervertebral disc downstream of a statically applied moment. Sixty rats were assigned to one of four time points (0, 8, 24, and 72 hours), two loading conditions (15 or 75 Nmm), or control for nine total groups. A robotic arm (Yaskawa Inc., Japan) applied a static moment to the caudal motion segment between vertebrae 8 and 9 of the experimental groups, at the specified loading magnitude, for one hour. Before and immediately after this creep protocol, the robotic arm determined the passive moment-angle curve of the motion segment. After waiting the prescribed time for each timepoint group, their passive moment-angle curves were measured again, the animals sacrificed, and the annulus fibrosis harvested between Ca8-9. Western Blot analyses were conducted to measure IL-1β, MMP-1, MMP-8, and TIMP-1. Overall, despite a substantial initial lengthening of the neutral zone (12.0 to 25.9◦), the creep exposure had no longterm mechanical consequences. This response was similarly reflected in the physiological outcome measures, where there was no sign of an inflammatory or remodelling response downstream of the applied mechanical creep. Ultimately, these results suggested that cyclic loading, compression, or a higher magnitude exposure may be required to induce an inflammatory response downstream of mechanical loading.
The final contribution of this thesis was the development of a mathematical model of collagen engagement, damage, synthesis, and enzymatic degradation. This model was divided into a mechanical model, which dealt with the sequential fibril engagement in the toe region and failure according to the earlier Tobolsky-Eyring rate law. The physiological model focused on modelling the response of the fibroblast to damage-sensing. The mechanical model reproduced many salient features of viscoelastic materials, like creep, stress-relaxation, hysteresis and rate-dependence. Further, with the Tobolsky-Eyring rate law, the model could reproduce many features of fatigue, like the S-N curve or the results of creep-rupture experiments. The physiological model predicted a tri-phasic response, with an initial pro-inflammatory wave, followed by an anti-inflammatory wave and a remodelling phase, which was consistent with the description of the acute inflammatory response but could not be calibrated to experimental data. This combined model predicted an inflammatory response downstream of mechanical exposure in a threshold-constant relationship.
This thesis explored collagen homeostasis as a potential contributor to chronic low back pain pathomechanics. It successfully measured the strain-rate proportionality of collagen rupture and the strain inhibition of collagenases, both of which are related to the rates of degradation and mechanical disruption of extant fibrils. It also showed that mechanical creep in isolation might not be a sufficient inflammatory stimulus in the annulus fibrosis of the intervertebral disc. A pain-generating pathway stemming from a one-time creep exposure may not elicit a painful inflammatory response. The mathematical model developed based on this work predicted a threshold-constant adaptive response to mechanical loading, and the exposure to living rats may have been below this threshold. However, more experimental work is required to establish this effect rigorously. Ultimately, this thesis further elucidated the potential interplay between mechanical exposures and physiological responses.
Description
Keywords
matrix metalloproteinase, low back pain, cumulative load, extracellular matrix, homeostasis, partial differential equation