IN the preceding paper [Robison and Soames, 1930] it was shown that, although bone slices from rachitic animals will calcify in solutions of inorganic salts supersaturated with respect to the bone phosphate, at lower concentrations of calcium and inorganic phosphate calcification will only occur in presence of phosphoric ester. At critical concentrations of the inorganic radical as little as 0*5 mg. per 100 cc. of phosphorus in the form of ester is sufficient to determine calcification, this amount being similar to that found in plasma. The solutions used for these experiments contained no protein and no other organic constituent but phosphoric ester. The concentration of calcium ions was, therefore, higher than in plasma even when the total calcium content was the same. The solutions further differed from plasma in containing much less bicarbonate. The experiments described in the present paper deal with the effect of bicarbonate, sodium, potassium and magnesium on calcification in protein-free solutions at different levels of calcium and inorganic phosphate, in absence and in presence of phosphoric ester. Experiments on the effect of antiseptics, of previous treatment of the bone slices with organic solvents or of desiccation in vacuo have also been carried out. The results of these have set in sharp distinction two mechanisms in calcifying hypertrophic cartilage, the phosphatase mechanism which can produce in the matrix a local supersaturation sufficiently great to cause deposition of the bone phosphate, and the second, still obscure, mechanism, which favours, and may determine, deposition of the bone phosphate from its supersaturated solution. Method of experiment. The experiments were carried out by comparing the calcification in vitro of slices cut under sterile conditions from the bones of rachitic rats (fed three to four weeks on McCollum diet 3143). The distal heads of the femora and the proximal heads of the tibiae and humeri were cut longitudinally by hand, 12-14 slices, about 1 mm. thick, being in this way obtained from each rat.
EXPERIMENTS on the deposition of barium, strontium and magnesium salts in bone slices in vitro have been previously reported [Robison and Rosenheim, 1934] and have shown that the calcifying mechanism is not entirely specific for the calcium salt of bone, although the properties of this salt are very specially appropriate to the function which it fulfils. Deposits of the phosphates (or carbonatophosphates) of these allied metals were produced in the matrix ofthe hypertrophic cartilage by agency of the phosphatase mechanism. The behaviour of strontium showed further resemblance to that of calcium in that its salts could also be deposited in the cartilage from supersaturated inorganic solutions, in absence of added phosphoric ester, that is, by the agency of the second mechanism. This reaction of hypertrophic cartilage towards strontium salts gained interest as a result of the experiments of Sobel et al. [1934] who showed that the substitution of SrCO3 for CaCO3 in a rickets-producing diet caused the development in rats of a severe type of rickets which was not cured by vitamin D therapy. Bone slices from these rats also failed to become calcified in vitro in
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