Casein phosphopeptides induces intracellular calcium changes and cell function modulation in primary human osteoblasts

Caseinphosphopeptides (CPPs) are a family of peptides originating from in vivo and in vitro hydrolysis of casein. These nutritional compounds display the ability to bind and solubilize minerals such as calcium. The importance to enhance the mineral uptake by the intestine relies on two findings: i) the inverse correlation found between the colon tumor incidence and the absorption of calcium from diet as put in evidence by recent epidemiologic studies; ii) minerals, especially calcium and zinc, are fundamental for improving and maintaining a correct bone mass. On this basis, CPPs were hypothesized to increase the calcium absorption and retention in vivo, with potential effects on bone mineralization (1). Notwithstanding, there are controversial reports on CPP action. The methodological approach used by different laboratories to study calcium absorption and bone mineralization resulted unable to out light whether the peptides have a specific effect on bone metabolism besides the enhancement of calcium availability. We have therefore designed the following study to evaluate a possible direct role of CPPs in bone cell metabolism. Primary human osteoblasts were established in culture using trabecular bone samples obtained from waste materials during orthopedic surgery of patients without metabolic or malignant bone disease. Cytosolic calcium changes were measured by video-microscopy using the fura-2 method on single cells. A mixture of CPPs of commercial origin as well as pure synthetic CPPs were used. The administration of CPPs to human osteoblasts caused an immediate but transient intracellular calcium change in a dose dependent manner. This CPP-induced effect is not cytotoxic and is triggered by an influx of the extracellular ions through the cell plasma membrane. The osteoblast pre-treatment with the active form of vitamin D, known to differentiate human osteoblast, does not affect the cell responsiveness to CPP administration. 4 hours of cell incubation with CPPs induced an increase of the expression of alkaline phosphatase, while 24 hours of cell incubation induced the increase of activity of alkaline phosphatase and of Runx2 expression, both markers of osteoblast differentiation. The same CPP treatment caused a small but significative reduction in cell rate proliferation and a slight increase in apoptosis activity. These results open the possibility to use CPPs, a nutrient component of the western diet, as tools for improving osteoblast mineralization and differentiation. Further studies are in progress to develop the way to use CPPs in bone tissue repairing and associated pathology.