MMPs Regulate Neuronal Substance P After a Painful Equibiaxial Stretch in a Co-Culture Collagen Gel Model Simulating Injury of an Innervated Ligament

Abstract

Chronic joint pain is a major health problem that can result from abnormal loading of the innervated ligamentous capsule that surrounds synovial joints. The matrix metalloproteinases-1 (MMP-1) and MMP-9 are hypothesized pain mediators from stretch-induced injuries since they increase in pathologic joint tissues and are implicated in biomechanical and nociceptive pathways that underlay painful joint injuries. There is also emerging evidence that MMP-1 and MMP-9 have mechanistic interactions with the nociceptive neuropeptide substance P. Yet, how a ligament stretch induces painful responses during sub-failure loading and whether MMP-1 or MMP-9 modulates nociception via substance P are unknown. We used a neuron–fibroblast co-culture collagen gel model of the capsular ligament to test whether a sub-failure equibiaxial stretch above the magnitude for initiating nociceptive responses in neurons also regulates MMP-1 and MMP-9. Pre-stretch treatment with the MMP inhibitor ilomastat also tested whether inhibiting MMPs attenuates the stretch-induced nociceptive responses. Because of the role of MMPs in collagen remodeling, collagen microstructural kinematics were measured in all tests. Co-culture gels were incubated for one week in either normal conditions, with five days of ilomastat treatment, or with five days of a vehicle control solution before a planar equibiaxial stretch that imposed strains at magnitudes that induce pain in vivo and increase nociceptive modulators in vitro. Force, displacement, and strain were measured, and polarized light imaging captured collagen fiber kinematics during loading. At 24 h after stretch, immunolabeling quantified substance P, MMP-1, and MMP-9 protein expression. The same sub-failure equibiaxial stretch was imposed on all co-cultures, inducing a significant re-organization of collagen fibers (p ≤ 0.031) indicative of fiber realignment. Stretch induces a doubling of substance P expression in normal and vehicle-treated co-cultures (p = 0.038) that is prevented with ilomastat treatment (p = 0.114). Although MMP-1 and MMP-9 expression are unaffected by the stretch in all co-culture groups, ilomastat treatment abolishes the correlative relationships between MMP-1 and substance P (p = 0.002; R2 = 0.13) and between MMP-1 and MMP-9 (p = 0.007; R2 = 0.11) that are detected without an inhibitor. Collectively, these findings implicate MMPs in a painful ligamentous injury and contribute to a growing body of work linking MMPs to nociceptive-related signaling pathways and/or pain.