Si⁴⁺, Ca²⁺ and Mg²⁺ Combinatorially Regulate Osteoblast Osteocalcin Expression

Method: Osteoblasts (MC3T3-E1) were treated with ionic products of bioactive glass dissolution (6P53-b experimental bioactive glass and 45S5 commercial Bioglass^TM) each in αMEM.  Ion extracts were supplemented with fetal bovine serum (FBS, 10%), penicillin streptomycin (pen-strep, 1%), ascorbic acid (AA, 50 ppm), and glycerol-2-phosphate (βGP, 10 mM) to make 45S5 and 6P53-b glass conditioned medium (GCM).  Each GCM was measured for ion constituency using inductively coupled plasma mass spectrometry (ICP-MS).  Control treatments were made from αMEM with the same supplements.  Gene expression was assayed using quantitative RT-PCR, protein expression was assayed using ELISA, and biomineralization was stained using Alizarin red.  For all assays, comparison of gene expression and protein expression was made between GCM and control treated cells (P<0.05 for statistical significance).

Result: ICP-MS results showed that both glasses released Si⁴⁺ and Ca²⁺, while 6P53-b released additional Mg²⁺.  Endogenous OCN expression was enhanced in 45S5 GCM treated cells on days 2 and 6 (12- and 46-fold, respectively) and in 6P53-b GCM treated cells on day 2 and 20 (4-fold).  Both GCM treatments enhanced extracellular OCN expression on day 30 (3-fold [6P53-b GCM], 5-fold [45S5 GCM]).  Alizarin staining showed early signs of biomineralization on day 20 for 45S5 GCM treated cells whereas this occured on day 30 for 6P53-b GCM treatments.

Conclusion: Bioactive glasses that release Si⁴⁺ and Ca²⁺ alone up-regulated OCN expression and biomineralization while the combined release of Si⁴⁺, Ca²⁺, and Mg²⁺ dampened OCN up-expression and delayed  biomineralizatin.