Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model

Abstract

Introduction: The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA).

Methods: Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving four TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for eight weeks. At baseline, Week 4 and Week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (Week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS5)) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE(2) and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an α-threshold of 0.05.

Results: A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at Week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE(2) (P = 0.02), nitrites/nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone.

Conclusion: Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.

Figure 1

Schematic representation of the study design.

Figure 2

Kinetic gait analysis. Peak vertical force (mean (standard deviation)) recorded before (baseline) and four and eight weeks after anterior cruciate ligament transection in dogs. Line plots representation includes respective values for A) placebo-control and B) tiludronate treated dogs. There was a significant time effect (P < 0.01), a group effect (P = 0.05) and a time per group interaction (P < 0.01) with PVF value reaching 35% higher in tiludronate than in placebo-control at Week 8 (P = 0.04).

Figure 3

Immunohistochemistry. (A) Expression of matrix metalloproteinase 13 (MMP-13), ADAMTS5 and cathepsin K in representative sections of cartilage (MMP-13 and ADAMTS5) and subchondral bone (cathepsin K) from placebo-treated dogs with osteoarthritis (OA) and tiludronic acid (TA: 2 mg/kg/2 weeks)-treated dogs with OA. Positive cells are shown by dark brown staining (original magnification ×100). (B) Levels of MMP-13, ADAMTS5 and cathepsin K as determined by immunostaining. The values are expressed as the mean ± SEM. P-values were calculated by using a generalized linear model under logistic polytomous distribution function using the proportional odds assumption.

Figure 4

Histomorphometry. Representative histological sections of calcified cartilage and subchondral bone in osteoarthritic dogs that received either placebo (n = 8) or treatment with tiludronate (n = 8) 2 mg/kg/2 weeks. Specimens were selected from lesional areas of the tibial plateaus (Fast green/Safranin O staining, original magnification ×63).