Collagen organization regulates stretch‐initiated pain‐related neuronal signals in vitro: Implications for structure–function relationships in innervated ligaments

Abstract

Injury to the spinal facet capsule, an innervated ligament with heterogeneous collagen organization, produces pain. Although mechanical facet joint trauma activates embedded afferents, it is unclear if, and how, the varied extracellular microstructure of its ligament affects sensory transduction for pain from mechanical inputs. To investigate the effects of macroscopic deformations on afferents in collagen matrices with different organizations, an in vitro neuron-collagen construct (NCC) model was used. NCCs with either randomly organized or parallel aligned collagen fibers were used to mimic the varied microstructure in the facet capsular ligament. Embryonic rat dorsal root ganglia (DRG) were encapsulated in the NCCs; axonal outgrowth was uniform and in all directions in random NCCs, but parallel in aligned NCCs. NCCs underwent uniaxial stretch (0.25 ± 0.06 strain) corresponding to sub-failure facet capsule strains that induce pain. Macroscopic NCC mechanics were measured and axonal expression of phosphorylated extracellular signal-regulated kinase (pERK) and the neurotransmitter substance P (SP) was assayed at 1 day to assess neuronal activation and nociception. Stretch significantly upregulated pERK expression in both random and aligned gels (p < 0.001), with the increase in pERK being significantly higher (p = 0.013) in aligned than in random NCCs. That increase likely relates to the higher peak force (p = 0.025) and stronger axon alignment (p < 0.001) with stretch direction in the aligned NCCs. In contrast, SP expression was greater in stretched NCCs (p < 0.001) regardless of collagen organization. These findings suggest that collagen organization differentially modulates pain-related neuronal signaling and support structural heterogeneity of ligament tissue as mediating sensory function. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:770-777, 2018.

A Nociceptive Role for Integrin Signaling in Pain After Mechanical Injury to the Spinal Facet Capsular Ligament

“…65,67 NCCs were made using rat tail Type I collagen (2mg/ml; Corning Inc., Corning, NY) cast in 12-well plates at 37°C overnight. DRGs from all spinal levels were dissected using fine forceps from embryonic day 18 Sprague-Dawley rats after exposing the rat’s spine and removing the spinal cord.…”

“…In addition, 2mg/mL glucose (Sigma-Aldrich Corp.; St. Louis, MO), 10ng/mL 2.5S nerve growth factor (Thermo Fisher Scientific; Waltham, MA), 10mM FdU (Sigma-Aldrich Corp.; St. Louis, MO) and 10mM uridine (Sigma-Aldrich Corp.; St. Louis, MO) were suppemented. 11,67 The culture media was changed every 2–3 days.…”

“…NCCs were cut into a cruciform shape with each arm having the dimensions of 6.25mm×8mm, 55,67 and loaded onto a planar biaxial testing machine (574LE2; TestResources; Shakopee, MN) equipped with a bio-bath filled with PBS maintained at 37°C. NCCs were stretched equibiaxially to 1.5mm (to induce ~20% strain) at 0.3mm/s (~4%/s) to simulate sub-failure strains that induce pain in vivo.…”

“…NCCs were stretched equibiaxially to 1.5mm (to induce ~20% strain) at 0.3mm/s (~4%/s) to simulate sub-failure strains that induce pain in vivo. 16,38,39,67 The MPS in sub-regions of the NCCs were measured by tracking fiducial markers drawn on the NCC surface and the embedded visible DRGs using a high-speed camera (Phantom-v9.1; Vision Research Inc.; Wayne, NJ). Based on the marker and DRG positions before and after stretch, the average MPS in all non-overlapping four-node sub-regions across the surface of the NCC was computed in LS-DYNA 67 and compared between treated and untreated groups using a one-way ANOVA to ensure consistent loading.…”

“…16,38,39,67 The MPS in sub-regions of the NCCs were measured by tracking fiducial markers drawn on the NCC surface and the embedded visible DRGs using a high-speed camera (Phantom-v9.1; Vision Research Inc.; Wayne, NJ). Based on the marker and DRG positions before and after stretch, the average MPS in all non-overlapping four-node sub-regions across the surface of the NCC was computed in LS-DYNA 67 and compared between treated and untreated groups using a one-way ANOVA to ensure consistent loading. Immediately after mechanical loading, NCCs were released from the grips, washed with fresh PBS with 1% Pen-Strep (Thermo Fisher Scientific; Waltham, MA) and transferred to pre-warmed culture media supplemented by 1% Pen-Strep for 1 day, after which time they were fixed with 4% PFA.…”