Protective effect of zoledronic acid on articular cartilage and subchondral bone of rabbits with experimental knee osteoarthritis

Abstract

Subchondral bone reabsorption and remodeling are responsible for the initiation and progression of osteoarthritis (OA). Zoledronic acid (ZOL), a third-generation bisphosphonate (BIS), is an inhibitor of bone reabsorption. However, the intervention effect of ZOL on OA has not been fully characterized and remains to be directly demonstrated in animal experiments. The present study examined the microscopic and macroscopic changes in the anterior cruciate ligament transection (ACLT) model of OA in rabbits and evaluated the effects of ZOL on cartilage degeneration and subchondral bone loss. A total of 32 New Zealand white rabbits were randomly divided into four groups: High-, medium- and low-dose ZOL groups, which received an intravenous injection of 250, 50 and 10 µg/kg ZOL, respectively, after modeling, as well as an untreated group. The bone mineral density (BMD) of the knee joint was evaluated by dual-energy X-ray absorptiometry scanning immediately after modeling and at 4 and 8 weeks. At week 8, quantitative measurement of cartilage was performed by a specialized magnetic resonance imaging (MRI) technique, including three-dimensional fat-suppressed spoil gradient-recalled sequence and T2 mapping. The rabbits were sacrificed by air embolism after anesthesia and both knee joints were harvested and evaluated by general and histological observation. Toluidine blue and hematoxylin and eosin staining were used to assess histological changes in the articular cartilage. Quantitative analysis of cartilage histopathology was performed according to the Mankin scoring system. The BMD of ACLT joints dropped after modeling, which was effectively suppressed by ZOL at the high and medium dose but not the low dose. MRI scans demonstrated that in the untreated group, articular cartilages on ACLT knees were thinner than those on normal knees. The high dose of ZOL preserved the cartilage tissue thickness more efficiently than the medium and low doses. Observation of specimens and pathological slices revealed that the articular cartilage degeneration in the high-dose ZOL group was lightest, while that in the medium- and low-dose ZOL group was moderate, and the untreated group exhibited the most severe defect. The untreated group had the highest Mankin score, whereas the high-dose ZOL group had the lowest score. In conclusion, ZOL increased the subchondral bone density, improved the microstructure and reduced the degeneration of articular cartilage in OA according to morphological as well as quantitative observation. ZOL exerted significant chondroprotective effects in a dose-dependent manner. A favorable chondroprotective effect was induced at the dose of 250 µg/kg. ZOL may represent a novel promising drug to complement the treatment of OA.

Keywords: articular cartilage; bone mineral density; osteoarthritis; subchondral bone; zoledronic acid.

Figures

Figure 1.

DXA scanning in five compartments of (A) the right knee and (B) the left knee. In total, the bone mineral density of five compartments [femoral medial condyle (left 1, right 5), femoral lateral condyle (left 2, right 6), tibial medial condyle (left 3, right 7), tibial lateral condyle (left 4, right 8), and whole knee joint of the right (10) and left (9) control was evaluated by DXA]. DXA, dual-energy X-ray absorptiometry.

Figure 2.

T2 mapping in saggital position of (A) the normal knee joint and (B) the OA joint. (Aa) In the original T2-mapped image, the normal articular cartilage displayed homogeneous hyperintensity and (Ab) in the T2-mapped pseudo-color image, the normal articular cartilage was uniform light green and subchondral bone was yellow-green. (Ba) In the T2-mapped image of the OA joint, OA cartilage had a slightly inhomogeneous hyperintensity and (Bb) in the T2-mapped pseudo-color image, OA cartilage was less uniformly green and subchondral bone was orange (B2). OA, osteoarthritis.

Figure 3.

Representative images of the macroscopic appearance of osteoarthritic articular surface of the femur (left) and tibia (right) of the knees subjected to anterior cruciate ligament transection. (A) High-dose ZOL (250 µg/kg); (B) medium-dose ZOL (50 µg/kg); (C) low-dose ZOL (10 µg/kg); (D) untreated group and (E) sham-operated knee. The articular surface of the normal knee exhibited no detectable changes. Cartilage erosion as well as strip and osteophyte formation in the margin were observed in D. The surface in A had reduced damage with slight roughness and degeneration. In B and C, the defect was more severe than that in A. ZOL, zoledronic acid.

Figure 4.

Cartilage cells of femoral condyle and tibial plateau from normal knees and knees subjected to anterior cruciate ligament transection (hematoxylin and eosin staining; magnification, ×100). (A) High-dose ZOL (250 µg/kg); (B) medium-dose ZOL (50 µg/kg); (C) low-dose ZOL (10 µg/kg); (D) untreated group and (E) sham-operated knee. In D without ZOL intervention, the arrangement of chondrocytes was more irregular and their number was lower compared with those in the joints in A and B. Furthermore, the articular cartilage layer was damaged and clear boundaries between cartilage layers were lost. ZOL, zoledronic acid.

Figure 5.

Cartilage cells of femoral condyle and tibial plateau from normal knees and knees subjected to anterior cruciate ligament transection (toluidine blue staining; magnification, ×100). (A) High-dose ZOL (250 µg/kg); (B) medium-dose ZOL (50 µg/kg); (C) low-dose ZOL (10 µg/kg); (D) untreated group and (E) sham-operated knee. In D, the arrangement of chondrocytes was irregular with clusters. The contour of the subchondral bone was destroyed. The matrix was severely hypochromic. In comparison, in A and B, the articular surface was smoother and the cartilage cells were regularly arranged without any obvious loss of cells. ZOL, zoledronic acid.

Figure 6.

Mankin scores of the (A) high-dose ZOL (250 µg/kg), (B) medium-dose ZOL (50 µg/kg), (C) low-dose ZOL (10 µg/kg) and (D) untreated groups. ZOL, zoledronic acid.

Figure 7.

Microscopic observation of subchondral bone of femur (hematoxylin and eosin staining; magnification, ×40). (A) High-dose ZOL (250 µg/kg); (B) medium-dose ZOL (50 µg/kg); (C) low-dose ZOL (10 µg/kg); (D) untreated group and (E) sham-operated knee. In D, the subchondral bone cells were disordered and the matrix was dyed unevenly. The bone trabecula was sparse with obvious breakdown. The lesion was reduced in A, B and C. Particularly in A, cells were more regularly arranged compared with those in D. The bone trabecula was thick and densely distributed. No cracks were observed and the matrix was dyed evenly. ZOL, zoledronic acid.