The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture

Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca²⁺ and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca²⁺ concentration were examined in two- and three-dimensional cultures. The present results indicate that 2-4 mM Ca²⁺ is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6-8 mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10mM) are cytotoxic. Purely from the perspective of calcium deposition, higher concentrations lead to increased accumulation of Ca²⁺. Culturing cells in phosphate-containing gel in media with Ca²⁺ also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca²⁺ concentrations around 5 mM is useful for both HAp deposition and osteoblast behavior.