AGEs induce ectopic endochondral ossification in intervertebral discs

Abstract

Ectopic calcifications in intervertebral discs (IVDs) are known characteristics of IVD degeneration that are not commonly reported but may be implicated in structural failure and dysfunctional IVD cell metabolic responses. This study investigated the novel hypothesis that ectopic calcifications in the IVD are associated with advanced glycation end products (AGEs) via hypertrophy and osteogenic differentiation. Histological analyses of human IVDs from several degeneration stages revealed areas of ectopic calcification within the nucleus pulposus and at the cartilage endplate. These ectopic calcifications were associated with cells positive for the AGE methylglyoxal-hydroimidazolone-1 (MG-H1). MG-H1 was also co-localised with Collagen 10 (COL10) and Osteopontin (OPN) suggesting osteogenic differentiation. Bovine nucleus pulposus and cartilaginous endplate cells in cell culture demonstrated that 200 mg/mL AGEs in low-glucose media increased ectopic calcifications after 4 d in culture and significantly increased COL10 and OPN expression. The receptor for AGE (RAGE) was involved in this differentiation process since its inhibition reduced COL10 and OPN expression. We conclude that AGE accumulation is associated with endochondral ossification in IVDs and likely acts via the AGE/RAGE axis to induce hypertrophy and osteogenic differentiation in IVD cells. We postulate that this ectopic calcification may play an important role in accelerated IVD degeneration including the initiation of structural defects. Since orally administered AGE and RAGE inhibitors are available, future investigations on AGE/RAGE and endochondral ossification may be a promising direction for developing non-invasive treatment against progression of IVD degeneration.

Fig. 1

Ectopic calcifications were located at ossification sites and close to fissures in human IVDs of various degeneration stages. Representative overview images of human IVDs of varying degenerative levels including (a) grade II, (b) grade III, (c) grade IV, and (d) grade V. The von Kossa staining identifies ossifications as dark black/brown staining and boxes mark region of interest in NP (blue box) and CEP (red box) regions. In NP regions (e-h), calcified deposits were located close to fissures. Arrows denote calcified deposits and asterisks denote fissures in NP tissues. In CEP regions (i-l), irregular ossified structures were also identified in all degenerative grades. Arrows denote calcified deposits and arrow heads denote intact CEP.

Fig. 2

Large COL10, OPN, MG-H1 and RAGE positive cells are often located in clusters and surrounded by granulated matrix. Human grade 3 IVD: Differential interference contrast image of (a) NP and (b) CEP tissue. Calcified deposits (von Kossa) are located pericellular to brown MG-H1 positive cells. Bright field images of (c,d) RAGE, (e,f) COL10 and (g,h) OPN positive NP cells (c,e,g) and CEP cells (d,f,h) co-stained with toluidine blue to visualise cell morphology (blue). (i,j) Negative controls (NP tissue). Arrow heads denote positive cells (brown chromogenic staining); arrows denote calcified particles (dark von Kossa stain); asterisks denote granulated structures.

Fig. 3

Co-localisation of COL10 and OPN with MG-H1 suggest a role of AGEs in endochondral ossification. Representative images of NP tissue of Grade 5 (COL10) and grade 3 (OPN) expression; (a) COL10 and (e) OPN co-localise with (b,f) MG-H1 in human NP tissue; (i-l) negative control. Arrows indicate positive staining and DAPI nuclei staining.

Fig. 4

Mineralised deposits and expression of COL10 and OPN after 4 d AGE exposure indicate a direct relationship of AGEs on endochondral ossification in bovine cell culture. (a) Mineralised deposits were visible after 4 d AGE exposure in NP (top) and CEP (bottom). Inserts show higher magnification of representative regions. Arrows mark ectopic calcifications. (b) Immunocytochemistry with RAGE (top), COL10 (middle) and OPN (bottom) after 4 d culture. White arrows mark positive stained cells. (c) Quantification of RAGE, COL10 and OPN positive cells after 4 d culture; * p < 0.05; n = 4, COL10 and OPN; n = 4, RAGE.

Fig. 5

Inhibition of RAGE reduces effect of AGEs on NP cell differentiation, indicating potential pathway of endochondral ossification via the AGE/RAGE axis. (a) Representative images of protein expression determined by western blot of (left) COL10 (58 kDa) and (right) OPN (full length: 66 kDa & MMP cleaved: 32 kDa); (b) quantitative analysis of protein expression normalised to BASAL control (n = 3).