Phosphate-assisted hydration of tricalcium silicate promotes hydroxyapatite formation and enhances osteogenic...

Tricalcium silicate (TCS) is a clinically established calcium silicate-based material. However, its highly alkaline hydration environment limits cellular compatibility. This study investigated whether direct incorporation of phosphate solution during TCS hydration promotes calcium-deficient hydroxyapatite (CDHAp) formation, modulates solution chemistry, and enhances osteogenic biological responses. TCS was mixed with KH₂PO₄ solutions at 0, 5, 10, 15, and 20 wt% (TP0-TP20) at a fixed liquid-to-powder ratio. Crystalline phase evolution was characterised by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) after simulated body fluid (SBF) immersion for up to 28 days. Surface morphology was examined by scanning electron microscopy (SEM). Solution pH, solubility, and ion release (Ca²⁺, Si⁴⁺, PO₄^3-) were quantified by ICP-OES. Osteogenic gene expression (RUNX2, OSX, COL1A1, OPN) was evaluated in MC3T3-E1 pre-osteoblasts by RT-qPCR. Cell viability and migration under physiological perfusion were assessed using a custom microfluidic Lab-on-a-Chip system. XRD and XPS confirmed concentration-dependent CDHAp formation in TP15 and TP20 from day 1, with peak intensity increasing progressively through day 28. TP0 showed CDHAp formation was insufficient to be clearly detected by XRD. Higher phosphate content progressively reduced solution pH and suppressed free Ca²⁺ accumulation, maintaining levels within 2-6 mM at early time points while elevating Si⁴⁺ release. TP15 and TP20 significantly upregulated RUNX2, OSX, and COL1A1 compared with TP0 (p < 0.05). Under microfluidic system, TP0 supported no viable cells, whereas TP20 demonstrated directional cell migration toward the specimen surface. Incorporating phosphate solution during TCS hydration accelerates CDHAp crystallisation, moderates excessive alkalinity, and enhances early osteogenic differentiation and cell migration under flow. These findings establish phosphate-modified TCS as a strategy to enhance HAp formation with enhanced chemical and biological properties.