Si and Ca Individually and Combinatorially Target Enhanced MC3T3-E1 Subclone 4 Early Osteogenic Marker Expression

This study tests the hypothesis that silicon and calcium ions combinatorially target gene expression during osteoblast differentiation. MC3T3-E1 subclone 4 osteoblast progenitors (transformed mouse calvarial osteoblasts) were exposed to Si⁴⁺ (from Na₂SiO₃) and Ca²⁺ (from CaCl₂:H₂O) ion treatments both individually (0.4 mM each + control treatment) and combinatorially (0.4 mM Si⁴⁺ + 0.4 mM Ca²⁺ + control treatment) and compared to control treated (α-minimum essential medium, 10% fetal bovine serum, and 1% penicillin-streptomycin) cells. Cell proliferation studies showed no significant increase in cell density between treatments over 5 days of culture. Cellular differentiation studies involved addition of ascorbic acid (50 mg/L) for all treatments. Relative gene expression was determined for collagen type 1 (Col(I)α1/Col(I)α2), core-binding factor a (cbfa1/Runx2), and osteocalcin (OCN), which indicated osteoblast progenitor differentiation into a mineralizing phenotype. Increased Si⁴⁺ or Ca²⁺ ion treatments enhanced Col(I)α1, Col(I)α2, Runx2, and OCN expression, while increased Si⁴⁺ + Ca²⁺ ion treatments enhanced OCN expression. Moreover, it was found that a Si⁴⁺/Ca²⁺ ratio of unity was optimal for maximal expression of OCN. Collagen fiber bundles were dense, elongated, and thick within extracellular matrices (ECM) exposed to Si⁴⁺ and Si⁴⁺ + Ca²⁺ treatments, while collagen fiber bundles were sparse, short, and thin within Ca²⁺ and control treated ECM. These results indicated that individual ions enhance multiple osteogenic gene expression, while combined ion treatments enhance individual gene expression. In addition, these results indicated that Si⁴⁺ enhanced osteoblast gene expression and ECM formation at higher levels than Ca²⁺. These results support the larger concept that ions (possibly released from bioactive glasses) could control bone formation by targeting osteoblast marker expression.