The effect of insulin and calcitonin on osteoblast proliferation and biomineralization
Alali, Ahmad Y A A
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OBJECTIVE: Insulin and calcitonin are involved in bone remodeling. Our aim was to determine the effect of insulin and calcitonin when added separately and in combination to mouse calvarial cultures on osteoblast proliferation and osteoblast mediated biomineralization in medium containing ascorbic acid and beta-glycerol phosphate. Our hypothesis is that both hormones in combination will cause a synergistic increase in osteoblast proliferation and biomineralization. METHODS: Osteoblasts were isolated from neonatal mouse calvaria and cultured under conditions that allow quantification of cellular proliferation and biomineralization. In our proliferation model, osteoblasts were grown in medium containing fetal calf serum stimulated by insulin, calcitonin, both, or culture medium alone that served as control. Proliferation was measured using a hemocytometer (Neubauer cell chamber). Osteoblast biomineralization was estimated by assessing cellular calcium uptake and secreted alkaline phosphatase activity. In the biomineralization model, osteoblasts were grown in the presence of ascorbic acid and betaglycerol phosphate and stimulated by factors used in the proliferation study. Calcium uptake was measured by an Arsenazo III microplate calcium assay and alkaline phosphatase activity was measured by enzyme release from the calvarial cellular layer. Histological and quantitative histomorphometric evaluations measured mineral deposition. RESULTS: Osteoblast proliferation was significantly increased by insulin or calcitonin alone but not by both together. All treatments, especially calcitonin, significantly increased calcium uptake. Only the combination of insulin and calcitonin significantly increased alkaline phosphatase activity. Discussion: Our findings suggest that insulin and calcitonin increase proliferation and biomineralization controlled by osteoblasts.