Casein phosphopeptides: their effect on calcification of in vitro cultured embryonic rat bone

The effect of casein phosphopeptides (CPP) and enzymatically dephosphorylated, otherwise identical peptides (DPP) on Ca solubility and mineralization was studied in an in vitro model. The model consisted of explanted embryonic rat bone rudiments (femora, tibiae, and metatarsals), cultured in Bigger's medium at two different concentrations of total calcium (1.78 mM and 3.24 mM). The medium enabled growth of the explants, but minor or no mineralization took place during a culture period of 11 days. The medium was unstable with respect to Ca solubility, and measurements with a selective electrode showed that during most of the time the actual concentration of soluble, ionic Ca2+ was not higher than about 0.7 mM. CPP added at a concentration of 10 mg/100 ml of the medium vastly improved Ca-solubility under culture conditions. The mean concentration of soluble, ionic Ca2+ in the presence of the peptides was 1.4 mM at total calcium = 1.78 mM and 2.3 mM at the higher total calcium level (3.24 mM). Rudiments cultured in the presence of CPP showed impressive increases in calcification of their diaphyseal area. This progress in mineralization was quantitatively assayed by measuring the projected dark diaphyseal area at the beginning and at the end of the culture period. It was also verified by histological sections of the rudiments. DDP did not enhance Ca solubility of the culture medium, nor did explants cultured in the presence of 10 mg/100 ml of DPP show any significant progress in mineralization. This indicated that the activity of CPP was dependent on the presence of serine-linked phosphate groups.