Low-frequency high-magnitude mechanical strain of articular chondrocytes activates p38 MAPK and induces phenotypic changes associated with osteoarthritis and pain

Abstract

Osteoarthritis (OA) is a debilitating joint disorder resulting from an incompletely understood combination of mechanical, biological, and biochemical processes. OA is often accompanied by inflammation and pain, whereby cytokines associated with chronic OA can up-regulate expression of neurotrophic factors such as nerve growth factor (NGF). Several studies suggest a role for cytokines and NGF in OA pain, however the effects of changing mechanical properties in OA tissue on chondrocyte metabolism remain unclear. Here, we used high-extension silicone rubber membranes to examine if high mechanical strain (HMS) of primary articular chondrocytes increases inflammatory gene expression and promotes neurotrophic factor release. HMS cultured chondrocytes displayed up-regulated NGF, TNFα and ADAMTS4 gene expression while decreasing TLR2 expression, as compared to static controls. HMS culture increased p38 MAPK activity compared to static controls. Conditioned medium from HMS dynamic cultures, but not static cultures, induced significant neurite sprouting in PC12 cells. The increased neurite sprouting was accompanied by consistent increases in PC12 cell death. Low-frequency high-magnitude mechanical strain of primary articular chondrocytes in vitro drives factor secretion associated with degenerative joint disease and joint pain. This study provides evidence for a direct link between cellular strain, secretory factors, neo-innervation, and pain in OA pathology.

Figure 1

Cell stretching device and stretch protocol. Schematic representation of mechanical stretching device (A) used to apply low-frequency high-magnitude strains; (B) Graphical representation of the 8–16–8 and stretch protocol applied to primary chondrocytes.

Figure 2

Application of the 8–16–8 stretch protocol to primary articular chondrocytes. (A) Representative morphological images of static and HMS cultured primary articular chondrocytes. Scale bar: 200 μm; (B) Gene expression analysis immediately after stretch protocol ended. Error bars: ±SEM, n = 6 (TLR2, TLR4, NGF and TNF); n = 3 (MMP3, MMP13, ADAMTS4 and ADAMTS5). Student’s t-test. * indicates p < 0.05; ** indicates p < 0.01; # indicates p = 0.06.

Figure 3

Western blot analysis. (A) Images acquired from immunoblots probing cell lysates from 3 donor animals (Donor A–C) probing for phosphorylated p44/42 (ERK), total p44/42 (ERK), phosphorylated p38, and for α-tubulin as a loading control; (B) Densitometry analysis of pERK normalized to total ERK and p-p38 normalized toα-tubulin; (C) Mean fold-difference in high mechanical strain (HMS) induced p-ERK and p-p38 activity versus static culture chondrocyte controls. Error bars: ±SEM, n = 3. Student’s t-test. * indicates p < 0.05. ST, static; HMS, high mechanical strain.

Figure 4

Conditioned media from HMS-cultured chondrocytes promotes neurite outgrowth in PC12 cells. (A) Representative phase images showing conditioned media from static and HMS cultured chondrocytes applied to PC12 cells and neurite outgrowth was observed and compared to vehicle (−NGF) and NGF treated controls. White arrows in phase images indicate noticeable cell debris on dish bottoms. Scale bar: 200 μm; (B) Quantification of neurite outgrowth. Error bars: ±SEM, n = 6, Student’s t-test. ** indicates p < 0.01; *** indicates p < 0.001. All samples were compared to −NGF controls.

Figure 5

Conditioned media from HMS-cultured chondrocytes causes increased PC12 cell death. (A) Representative Live/Dead images showing conditioned media from static and HMS cultured chondrocytes applied to PC12 cells; viability was assessed and compared to vehicle (−NGF) and NGF treated controls. Green cells (Calcein AM) represent live cells, and red cells (Ethidium homodimer) indicate dead cells. Scale bar: 200 μm; (B) Quantification of viability. Error bars: ±SEM, n = 6, Student’s t-test. * indicates p < 0.05; ** indicates p < 0.01. All samples were compared to −NGF controls.