Combinatorial effect of Si^{4 +}, Ca^{2 +}, and Mg ^{2 +} released from bioactive glasses on osteoblast osteocalcin expression and biomineralization

Osteocalcin (OCN) expression is an essential osteogenic marker of successful bone regeneration therapies. This study hypothesizes that Si ^{4 +} and Ca^{2 +} combinatorial released by bioactive glass enhance osteoblast biomineralization through up-regulation of OCN expression; and Mg^{2 +} release delays such enhancement. Osteoblasts (MC3T3-E1) were treated with ionic products of bioactive glass dissolution (6P53-b experimental bioactive glass and 45S5 commercial Bioglass™). Results showed that gene expressions, including OCN and its up-stream transcription factors (Runx2, ATF4, MSX1, SP7/OSX), growth factors and signaling proteins (BMP2, BMP6, SMAD3), were enhanced in both 45S5 and 6P53-b glass conditioned mediums (GCMs). This up-regulation led to enhanced mineral formation by 45S5 glass conditioned mediums ([GCM], Si^{4 +} + Ca^{2 +}) after 20 days, and by 45S5 GCM and 6P53-b GCM (Si^{4 +} + Ca^{2 +} + Mg^{2 +}) after 30 days. In examining the extracellular matrix generated by cells when exposed to each GCM, it was found that 45S5 GCM had slightly elevated levels of mineral content within ECM as compared to 6P53-b GCM after 30 days while control treatments exhibited no mineral content. The formation of well-defined mineralized nodules (distinct PO₄^{3 -} [960 cm^{- 1}] and CO₃^{2 -} [1072 cm^{- 1}] peaks from Raman Spectra) was observed for each GCM as the soluble glass content increased. In examining the individual and combined ion effects between Si ^{4 +}, Ca^{2 +}, and Mg^{2 +}, it was found Mg ^{2 +} down-regulates OCN expression. Thus, ions released from both 45S5 and 6P53-b bioactive glasses up-regulate OCN expression and biomineralization while 6P53-b GCM Mg^{2 +} release down-regulated OCN expression and delayed osteoblast biomineralization. These results indicate that Si ^{4 +}, Ca^{2 +}, and Mg^{2 +} combinatorially regulate osteoblast OCN expression and biomineralization.