*2.11. Collagen Synthesis Rate*

Collagen content was quantified by a Sirius Red-based colorimetric assay as described by Park et al. [40]. The MC3T3-E1 cells were cultured in an osteogenic medium containing 10 mM β-glycerophosphate, 5 nM dexamethasone, 50 g/mL ascorbic acid, and *G. littoralis* (0 μg/mL and 20 μg/mL). After seven days, the cells were cultured in a medium containing bovine serum albumin (BSA) (3%) for two days. The cultured cells were gently washed twice with PBS, followed by fixation with Bouin's fluid (8.3% formaldehyde and 4.8% acetic acid in saturated aqueous picric acid, (O2N)3C6H2OH) for 1 h. After fixation, the fixative fluids were removed, and the cultured cells were washed with tap water for 10 min. The cultured cells were air-dried and stained with Sirius red dye (0.1% saturated picric acid) for 1 h on a shaker. The stained cells were washed with PBS and observed under a microscope. For quantitative analysis, the stained cell layer was washed extensively with 0.01 N HCl to remove the non-bound dye. The stained cells were dissolved in 0.2 mL of 0.1 N and shaken for 30 min. Then, the absorbance was measured at 540 nm.
