The in vitro effect of pH on osteoclasts and bone resorption in the cat: implications for the pathogenesis of FORL

Abstract

Dental disease due to osteoclast over-activity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline osteoclastic resorptive lesions (FORL) involve extensive resorption of the tooth leaving it liable to root fracture and subsequent tooth loss. The aetio-pathogenesis of FORL is not known. Recent work has shown that systemic acidosis causes increased osteoclast activation and that loci of infection or inflammation in cat mouth are likely to be acidotic. To investigate this, we generated osteoclasts from cat blood and found that they formed in large numbers (approximately 400) in cultures on bovine cortical bone slices. Acidosis caused an increase in the size of cells-in cultures maintained up to 14 days at basal pH 7.25, mean osteoclast area was 0.01 +/- 0.003 mm(2), whereas an 8.6-fold increase was observed in cells cultured between 11 and 14 days at pH 7.15 (0.086 +/- 0.004 mm(2)). Acidosis caused a modest increase in the number of osteoclasts. Exposure to pH 6.92 exhibited a 5-fold increase in the area of bone slices covered by resorption lacunae ( approximately 70% bone slice resorbed). In line with this finding, significant increases were observed in the expression of cathepsin K and proton pump enzymes (both approximately 3-fold) that are key enzymes reflective of resorptive activity in osteoclasts. These results demonstrate that acidosis is a major regulator of osteoclast formation and functional activation in the cat, and suggest that local pH changes may play a significant role in the pathogenesis of FORL.

Figure 1

Gigantic osteoclasts formed in vitro under acidic culture conditions. A: Low power transmitted light micrograph of TRAP‐stained osteoclasts on a bone slice formed during culture from day 7 to 14 under acid conditions (pH 6.8) showing extreme size (approximately 2 mm diameter) (numerous nuclei, >200, are present but obscured by the intense TRAP staining) (mag. 5×). B: Higher power reflected light micrograph of combined TRAP‐ and toluidine blue‐stained osteoclasts; detail of the area marked in (A) showing numerous resorption lacunae under and associated with two osteoclasts (examples are identified by arrows) (mag. 10×). C,D: Immunostaining of osteoclasts for cathepsin K formed under control (pH 7.4) (C) and acidic (pH 6.8) conditions (D). F‐actin rings are indicated by arrows in (D). Scale bar (in C) = 20 µm. These cells are representative examples used for the analyses summarized in Table 1.

Figure 2

Numbers of osteoclasts formed in cultures grown under modified pH conditions. The number of TRAP‐positive osteoclasts per bone slice (per 20 mm² area) in cultures exposed to altered pH from days 7 to 14 (A) and days 11–14 (B). *P = 0.004, **P = 0.002, ***P = 0.0001.

Figure 3

Area of osteoclasts formed in cultures grown under modified pH conditions. Mean area (mm²) of single osteoclasts in cultures exposed to altered pH from day 7 to 14 (A) and 11–14 (B). *P = 0.003, **P = 0.002, ***P = 0.001, ****P = 0.0001.

Figure 4

Percentage area of bone slice resorbed by osteoclasts formed in cultures grown under modified pH conditions. Percentage area of bone slice resorbed in cultures exposed to altered pH from day 7 to 14 (A) and 11–14 (B). *P = 0.03, **P = 0.007, ***P = 0.001, ****P = 0.0001.