Repetitive short-span application of extracellular calcium is osteopromotive to osteoprogenitor cells

Extracellular calcium ([Ca²⁺ ]_E ) concentration has been suggested to stimulate osteoblastic activity; thus, calcium can be used to enhance fracture healing. However, systemic administration of calcium at high dose levels may cause physiological problems such as hypercalcaemia. Short-span application of single or repetitive [Ca²⁺ ]_E stimulus may be suggested as a novel regimen to reduce such side effects. However, osteopromotive effect of such short-term [Ca²⁺ ]_E stimulus on osteoprogenitor cells has not yet been evaluated yet. This study investigated the effects of [Ca²⁺ ]_E dose (6 and 18 mM) and regimen (single, repetitive, and continuous) on viability, proliferation, osteogenic gene expression, and mineral formation by osteoprogenitor cells. BMP-2 treatment was set as the positive control group. It was observed that repetitive and continuous calcium stimulation resulted in significant enhancement of osteoblastic activity. A 6 mM [Ca²⁺ ]_E significantly increased cell viability and proliferation in all three regimens, and the expression of osteogenic transcription factors was significantly upregulated by continuous application of 6 mM [Ca²⁺ ]_E . It was observed that application of [Ca²⁺ ]_E repetitively at 18 mM had an osteopromotive effect to an extent that was as pronounced as BMP-2. Continuous application of 18 mM [Ca²⁺ ]_E provided the greatest degree of osteogenic activity among all groups. This study demonstrated that repetitive [Ca²⁺ ]_E exposure from the basal aspect of cells resulted in upregulation of osteogenic transcription factor and bone formation. The knowledge gained from the dose and treatment regimen of calcium therapy is important in setting the guidelines for developing approaches to treat fractures.