The Effect of Dyes on the Calcification of Hypertrophic Rachitic Cartilage in Vitro

The calcification of rat hypertrophic cartilage slices in vitro is markedly inhibited by preliminary exposure to metachromatic and other basic dyes. The dyes are effective at 10(-3) to 10(-4)M in the absence of calcium and phosphate. This inhibition does not occur at the same low dye concentration if calcium and phosphate are present. Neither ion alone is effective in preventing the inhibition. The inhibitory action can be removed by placing slices which have been treated with basic dye in a solution which contains calcium and phosphate ions, plus an acid dye, Orange G. Most acid dyes do not inhibit calcification, except at very high concentrations. Alizarin and quinalizarin are exceptional, and produce marked inhibition at 10(-3)M, an effect which is perhaps attributable to the tendency of these dyes to form lakes with calcium. Rachitic cartilage slices which no longer calcify in inorganic phosphate as a result of treatment with basic dyes show normal calcification in the presence of organic phosphate esters. These results are discussed in terms of the possibility that chondroitin sulfate ester participates in normal calcification.