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

Abstract

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 (R 2 ! 0.041; p 0.0171) and neuronal aspect ratio (AR) (R 2 ! 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.

The Physiological Basis of Cervical Facet-Mediated Persistent Pain: Basic Science and Clinical Challenges

“…Imposing bulk strains greater than 11% to a gel increases phosphorylation of extracellularregulated kinase (ERK), an indicator of neuron activation, and causes collagen reorganization. 115 This finding demonstrates that strains sufficiently large to initiate a neuronal response can also change the microstructure of the neuron's environment. These in vitro results suggest that the strain thresholds for neuronal activation (ERK phosphorylation) and local tissue injury (collagen matrix reorganization) may be the same.…”

“…Although basic science studies collectively have defined thresholds for neuronal activation and the induction of pain, those studies are based on work in vivo in the rat and/or goat, or even in artificial constructs simulating the ligament. 14,19,26,27,57,115 Nonetheless, because a common, nondimensional biomechanical metric (strain) has been used across all of those studies, the findings are agnostic of species and/or scale, and it may therefore be possible to extend such findings to the human. The bigger challenges of understanding injury risk and predicting injury in humans are complicated by very complex and integrated systems at play in pain.…”

“…These in vitro results suggest that the strain thresholds for neuronal activation (ERK phosphorylation) and local tissue injury (collagen matrix reorganization) may be the same. 115 In addition to immediate neuronal responses, facet capsular loading that induces behavioral hypersensitivity in the rat is also associated with many sustained modifications in the nociceptive signaling from the primary afferents that are evident in the DRG. Protein expression of the nociceptive neurotransmitter, substance P, is increased in the DRG after painful facet joint stretch by day 7, and that change is absent in nonpainful stretch.…”

Stretch-induced network reconfiguration of collagen fibres in the human facet capsular ligament

“…Fibre strains in computationally modelled fibrous networks during uniaxial tensile loading can vary spatially and exceed the applied bulk strain depending on the fibre orientation [49]. The cascade of fibre failure, load redistribution and reorientation of intact fibres that leads to anomalous realignment at the microscale can be complex and non-uniform, particularly considering the structural heterogeneity of the FCL.…”

“…However, the use of network analysis techniques is not limited to natural materials; it could be extended to characterize microstructural reorganization in hydrogels for tissue engineering applications [49,56,57] and to examine dynamic reconfiguration of complex polymer structures [58]. Community detection techniques are highly applicable to studying properties of non-biological materials [24][25][26] and their use may be expanded in the future to quantitatively investigate physical interactions between material components at intermediate length scales and over time [59][60][61].…”