Clodronate exerts an anabolic effect on articular chondrocytes mediated through the purinergic receptor pathway

Abstract

Objective: Bisphosphonates are commonly used anti-osteoporotic drugs which have controversial effects on joint diseases including osteoarthritis. Certain bisphosphonates have been shown to have anabolic effects on cartilage which could have important ramifications for their proposed effects in vivo; however, the underlying mechanisms are poorly understood. Thus, the purpose of this study was to characterize the effects of clodronate on primary articular chondrocyte metabolism and to determine the underlying signaling pathways responsible.

Design: The effects of clodronate and pamidronate on extracellular matrix (ECM) biosynthesis, accumulation and MMP-13 activity were observed in high density, 3D cultures of bovine articular chondrocytes for up to 4 weeks were evaluated. Mechanisms were delineated by measuring intracellular Ca(2+) signaling and the effects of pharmacologic inhibition of the purinergic receptor pathway.

Results: Clodronate (100 μM) induced an anabolic effect (increased biosynthesis by 13-14%) which resulted in an 89-90% increase in ECM accumulation after 4 weeks of culture and without an associated effect on matrix turn-over. Stimulation by clodronate resulted in a 3.3-fold increase in Ca(2+) signaling and pharmacological inhibitor experiments suggested that the anabolic effects exerted by clodronate are transduced through the purinergic receptor pathway.

Conclusions: These findings support the previous notion that certain bisphosphonates may be useful as adjunctive therapies to potentially ameliorate progression of cartilage degeneration and improve arthritis management.

Keywords: Anabolism; Bisphosphonates; Calcium signaling; Chondrocytes; Clodronate; Purinergic signaling.

Conflict of interest statement

Conflict of interest

None.

Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Figures

Fig. 1

The effect of short-term clodronate and pamidronate exposure on collagen and proteoglycan synthesis by articular chondrocytes. The effect of clodronate and pamidronate on collagen (A) and proteoglycan (B) synthesis by chondrocytes was determined 24 h after exposure to the bisphosphonate. Data was normalized to control (as described in the Methods) and expressed as the mean with errors bars representing the 95% confidence interval (N = 3 donors; total n = 6 samples/group) and open circles representing individual data points. *denotes a significance difference between all other groups (panel A: P = 0.018; panel B: P = 0.036).

Fig. 2

The effect of long-term exposure to clodronate and pamidronate on chondrocyte ECM. Histological and immunohistochemical assessment of cartilaginous tissue constructs after 4 weeks of exposure to clodronate (100 μM) or pamidronate (100 μM). Tissue sections were stained with hematoxylin and eosin (H&E) as well as collagen II and collagen I assessed by immunohistochemical methods (positive protein staining appears in brown). Original magnification of 40 × and the scale bar represents 100 μm.

Fig. 3

The effect of clodronate on calcium transients in chondrocytes. Overall cell fluorescent intensity (arbitrary units) of individual chondrocytes pre-incubated with Fluo-4 using confocal microscopy is shown here for cultures supplemented with (top trace) or without (bottom trace) clodronate (100 μM). Fluorescent intensity was determined from images captured every 5 s over a 300 s imaging period.

Fig. 4

The effect of purinergic receptor pathway inhibitors on calcium transients induced by clodronate. (A) Fold changes in intracellular Ca2+ signaling of Fluo-4 labeled chondrocytes stimulated by clodronate (100 μM) after pre-incubation with pharmacological inhibitors to block discrete elements of the purinergic receptor pathway (Table I). Confocal images (640 × 640 pixels) were captured every 5 s over a 300 s imaging period and the number of cells experiencing multiple transients were counted. For each inhibitor, the number of cells experiencing multiple transients was normalized to vehicle-only control (shown in parentheses) and expressed as the mean with errors representing the 95% confidence interval (N = 3 donors; total n = 8–9 samples/group) and open circles representing individual data points. (B) Fold changes in intracellular Ca2+ signaling of Fluo-4 labeled chondrocytes pre-incubated with FFA or dansylcadaverine (Table I) and stimulated by clodronate (100 μM) with or without additional ATP stimulation (100 μM). The number of cells experiencing multiple transients was normalized to vehicle-only control and expressed as the mean with errors representing the 95% confidence interval (N = 3 donors; total n = 6–8 samples/group) and open circles representing individual data points. A,Bdenotes a significant effect of clodronate (P < 0.001 and P = 0.045, respectively), a,b,cdenotes a significant effect of the inhibitor (P < 0.001, P = 0.030 and P = 0.006, respectively), and ddenotes a trend of the inhibitor (P = 0.093). *denotes a significant effect of ATP (P < 0.001).

Fig. 5

Schema of proposed clodronate signaling pathway. Proposed clodronate signaling pathway. Pharmacological inhibitors used to block discrete elements of the signaling pathway (irrespective of their effect) are shown.