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 Si ⁴⁺ (from Na ₂SiO ₃) and Ca ²⁺ (from CaCl ₂:H ₂O) ion treatments both individually (0.4 mM each + control treatment) and combinatorially (0.4 mM Si ⁴⁺+ 0.4 mM Ca ²⁺+ control treatment) and compared to control treated (α-minimum essential medium, 10% fetal bovine serum, and 1% penicillinstreptomycin) 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 Si ⁴⁺ or Ca ²⁺ ion treatments enhanced Col(I)α1, Col(I)α2, Runx2, and OCN expression, while increased Si ⁴⁺+Ca ²⁺ ion treatments enhanced OCN expression. Moreover, it was found that a Si ⁴⁺/Ca ²⁺ ratio of unity was optimal for maximal expression of OCN. Collagen fiber bundles were dense, elongated, and thick within extracellular matrices (ECM) exposed to Si ⁴⁺ and Si ⁴⁺+ Ca ²⁺ treatments, while collagen fiber bundles were sparse, short, and thin within Ca ²⁺ 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 Si ⁴⁺ enhanced osteoblast gene expression and ECM formation at higher levels than Ca ²⁺. These results support the larger concept that ions (possibly released from bioactive glasses) could control bone formation by targeting osteoblast marker expression.