## 3 Results and Discussion
### Biomineralization Analysis  
To quantitatively evaluate mineralization, Alizarin Red S (ARS) staining was performed after 21 days of MG-63 cell culture in osteogenic medium. Figure 17 presents the amount of ARS-bound dye normalized to membrane mass (mg/mg), which reflects the extent of extracellular calcium deposition on each scaffold surface, a hallmark of osteoblast differentiation.  
Pristine PLA membranes (sample 1) exhibited the lowest mineralization value (0.00092 mg/mg), confirming their limited osteoinductive capacity due to a lack of bioactive sites and ionic stimulation. This finding is consistent with prior studies highlightingPLA membranes modified with MgO (samples 2-4) demonstrated a concentration-dependent increase in mineral deposition, with sample 3 (0.25 g MgO) showing the highest value among them (0.00155 mg/mg). Magnesium ions are well-known to stimulate osteogenic activity, primarily by upregulating alkaline phosphatase (ALP) and modulating intracellular calcium pathways [33; 37]. Previous research has shown that MgO-containing materials enhance matrix mineralization and improve cell-matrix interactions [36; 38].  
Membranes modified with AuNPs alone (samples 5-7) exhibited relatively lower mineralization, though sample 7 (2 mL AuNPs) showed improved calcium deposition (0.00104 mg/mg) compared to PLA alone. This suggests that AuNPs exert a mild promotive effect on osteogenesis, likely through cytoskeletal modulation and protein adsorption rather than direct ionic bioactivity [40; 43]. Still, their effect appears to be less potent than that of MgO, particularly in stimulating mineral accumulation over a 21-day culture period.  
The most pronounced mineralization effects were observed in dual-functionalized membranes containing both MgO and AuNPs. Sample 9 (0.25 g MgO + 1 mL AuNPs) exhibited the highest ARS staining value (0.00195 mg/mg), suggesting a strong synergistic enhancement in calcium deposition. Sample 10 (0.5 g MgO + 2 mL AuNPs) also demonstrated elevated mineralization (0.00164 mg/mg). These results indicate that the dual incorporation of nanoparticles creates a bioactive interface that supports osteoblast maturation and mineral output. Similar synergistic effects have been described in previous studies on nanocomposite scaffolds, where ionic and structural cues together promote bone-like tissue formation [34; 44; 50].  
These findings are consistent with earlier trends observed in this study, namely, improvements in wettability, swelling, buffering capacity, and cell viability, all of which contribute to the scaffold's biofunctionality. Enhanced surface hydrophilicity promotes protein adsorption and cell adhesion, while Mg\({}^{2+}\) release and localized AuNP cues facilitate intracellular signaling required for differentiation [25; 29].  
Figure 17: Biomineralization study (Alizarin Red S).  
Taken together, the ARS staining results confirm the superior osteoinductive capacity of PLA/MgO/AuNP membranes, particularly at optimized concentrations of both nanoparticles. These hybrid nanocomposites represent a promising platform for guided bone regeneration, combining structural suitability with biochemical functionality supported by cumulative evidence across in vitro models [35, 36, 50].