Combinatorial effect of Si4 +, Ca2 +, and Mg2 + released from bioactive glasses on osteoblast osteocalcin expression and biomineralization

Abstract

Osteocalcin (OCN) expression is an essential osteogenic marker of successful bone regeneration therapies. This study hypothesizes that Si4+ and Ca2+ combinatorial released by bioactive glass enhance osteoblast biomineralization through up-regulation of OCN expression; and Mg2+ 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], Si4++Ca2+) after 20days, and by 45S5 GCM and 6P53-b GCM (Si4++Ca2++Mg2+) after 30days. 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 30days while control treatments exhibited no mineral content. The formation of well-defined mineralized nodules (distinct PO43− [960cm−1] and CO32− [1072cm−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 Si4+, Ca2+, and Mg2+, it was found Mg2+ 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 Mg2+ release down-regulated OCN expression and delayed osteoblast biomineralization. These results indicate that Si4+, Ca2+, and Mg2+ combinatorially regulate osteoblast OCN expression and biomineralization.