Bone cells control initial steps of mineralization by forming matrix vesicles (MVs) in which calcium and phosphate ions are accumulated. Under physiological conditions, MVs are released to the extracellular matrix (ECM) and Ca2+ and Pi form hydroxyapatite (HA). Growing minerals break the MVs membrane and are deposited in ECM. Under pathological conditions the mineralization process becomes deregulated, which leads to the formation of calcium phosphate deposits. It is suggested that members of the annexins family and tissue-nonspecific alkaline phosphatase (TNAP) (activators of ossification) as well as fetuin-A (a potent inhibitor of calcification) play an important role in regulation of mineralization both in healthy and pathological conditions.
We used two human cell lines: osteoblastic hFOB 1.19 and osteosarcoma Saos-2. These cells were stimulated for mineralization for 7 days in the presence of ascorbic acid and β-glycerophosphate. Electron microscopy data revealed that MVs in osteoblastic cells are small and single-walled, whereas in osteosarcoma they are big and multi-compartmental (called then multivesicular bodies). The results from the X-ray microanalysis showed significant differences between calcium to phosphate ratio in minerals formed by both cell lines. In osteoblastic cells the atomic ratio of calcium to phosphate was close to HA (e.g. 1.67), but osteosarcoma cells formed abnormal mineral with the ratio calcium to phosphate shifted towards the phosphate. In addition enzymatic assay indicated of divergences in TNAP activity in osteosarcoma cells in comparison to osteoblasts, while western blot analysis revealed changes in distribution of AnxA2, AnxA6 and fetuin-A in both cell lines.
Concluding, not only differences between the amount and size of MVs in pathological vs physiological conditions, but also in the activity and distribution of vesicular proteins engaged in the process of mineralization were observed.
17 May 2014 - 20 May 2014