Calcium is the most abundant stored mineral in the human body and is especially vital for bone health; thus, calcium deficiency can cause bone-related diseases, such as osteopenia and osteoporosis. However, a high concentration of serum calcium, which is commonly known as hypercalcemia, can also lead to weakened bones and, in severe cases, osteosarcoma. Therefore, it is necessary to maintain the concentration of calcium that is appropriate for bone biology. In the present study, we aimed to elucidate the effects of high concentration of calcium, approximately 2 folds the normal calcium level, on osteoblast differentiation. The CaCl2 treatment showed dose-dependent suppression of the alkaline phosphatase activity and mineralized nodule formation. Calcium showed cytotoxicity at an extremely high concentration, but a moderately high concentration of calcium that results in inhibitory effects to osteoblast differentiation showed no signs of cytotoxicity. We also confirmed that the CaCl2 treatment repressed the mRNA expression and protein abundance of various osteogenic genes and transcriptional factors. Considered together, these results indicate that a high concentration of calcium negatively regulates the osteoblast differentiation of C2C12 cells.

Introduction
Materials and Methods
    1. 세포 배양 및 조골세포 분화 유도
    2. Alkaline phosphatase 염색과 Alizarin red S (ARS) 염색
    3. 세포 독성 분석
    4. RNA 추출 및 real-time polymerase chain reaction(RT-PCR)
    5. 단백질 추출, 전기영동 및 Western blot
    6. 통계 분석
Results
    1. CaCl2의 농도에 따른 세포 독성 평가
    2. CaCl2의 ALP 염색 결과
    3. 고농도의 CaCl2가 골 형성 유전자의 발현에 미치는 영향
    4. 고농도의 CaCl2가 조골세포 분화관련 유전자의 단백질 수준에 미치는 영향
Discussion
Acknowledgements
Conflicts of Interest
References

• Ye Jin Lee(Department of Oriental Pharmacy, College of Pharmacology, Wonkwang University)
• Younho Han(Department of Oral Pharmacology, College of Dentistry, Wonkwang University) Correspondence to