FO increases mitochondrial activity in preosteoblast MC3T3-E1 cells and decreases total cell number. MC3T3-E1 cells were treated with FO (0–200 µg/mL) or 100 nM DEX for 7 days. (A) Mitochondrial activity was measured on day 1 (D1), day 3 (D3), day 5 (D5), and day 7 (D7), and (B) cell images were collected by Ezscope i900PH phase contrast microscope (×10). Scale bar shows 100 μm. (C) Under the same experimental conditions, cell viability and total cell count were measured using flow cytometric analysis. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas. DEX; dexamethasone, and UT; untreated control.

FO upregulates the specific marker gene expression responsible for osteoblast differentiation in MC3T3-E1 cells. (A) MC3T3-E1 cells were treated with 100 µg/mL of FO and 100 nM DEX for 7 days. The expression of mRUNX2, mALP, mCol1a1, mOCN, mOSX, mBMP2, and mBMP4 was measured on day 1 (D1), day 3 (D3), day 5 (D5), and day 7 (D7). (B) The cells were also treated with 50 µg/mL and 100 µg/mL of FO or 100 nM DEX for 5 days. The expression of mRUNX2, mALP, mCol1a1, mOCN, mOSX, mBMP2, and mBMP4, was observed. mGAPDH was used as a house keeping gene. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and DEX; dexamethasone.

FO upregulates ALP expression and activity in preosteoblast MC3T3-E1 cells. (A) MC3T3-E1 cells were treated with FO (50 µg/mL and 100 µg/mL) or DEX (100 nM) for 7 days. Representative images from three biological replicates were collected using the Toup View software after staining the cell for ALP activity (as described in the Materials and Methods section) on day 3 (D3), day 5 (D5), and day 7 (D7). (B) The relative ALP staining density was represented using mean integrated pixel density. (C) ALP activity was measured at 520 nm using a tartrate-resistant acid phosphatase (TRACP) & alkaline phosphatase (ALP) assay kit. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001 and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and DEX; dexamethasone.

FO activates osteoblast-specific protein expression and mineralization/calcification in osteosarcoma MG-63 human osteoblast-like cells. (A) MG-63 cells (3 × 10³ cells/mL) were seeded overnight and then treated with FO (50 µg/mL and 100 µg/mL) or DEX (100 nM) for 7 days. Protein was extracted and western blotting analysis was performed using each specific antibody. β-Actin was used as the internal control of protein expression. All protein expression was normalized by the density of β-actin. The nuclear localization of runt-related transcription factor 2 RUNX2 (B) and osterix OSX (C) was measured by immunostaining. (D) ALP activity (mineralization) was measured using a TRACP & ALP assay kit. (E) In vitro calcification was detected by alizarin red staining. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and DEX; dexamethasone. FO; fermented extract of C. gigas and DEX; dexamethasone.

FO promotes vertebrae formation in zebrafish larvae. (A) Zebrafish larvae (3 dpf) were treated with FO (50 µg/mL and 100 µg/mL) and GP (4 mM) for 6 days (i.e., until 9 dpf) and then stained with calcein to visualize vertebrae formation. Relative intensity of calcein (bottom, left) and relative bone area (bottom, middle) were calculated using the Image J software and normalized against the GP-treated group. The number of vertebrate in each fish was manually counted. All of them are shown in the yellow box. Total fish length is 3.4 ± 0.4 mm. (B) Under the same experimental conditions, total mRNA from the 9 dpf zebrafish larvae was subjected to RT-PCR in order to determine zALP, zRUNX2a, zRUNX2b, zCol1a1, zOCN, zBMP2, and zBMP4 expression. zβ-Actin was used as a house keeping gene. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and GP; β-glycerophosphate.

FO promotes caudal fin regeneration in adult zebrafish. Adult zebrafish (n = 15) were immersed in FO for 12 days post amputation (dpa). Next, fish caudal fins were subjected to double fluorochrome labeling with calcein and alizarin red at 6 and 12 dpa, respectively. Bone regions labeled with calcein (green) and alizarin red (red) indicate new bone regeneration at 6 and 12 dpa. Relative intensity of each bone vein was calculated using the Image J software. Amputation axis is also indicated (dashed line). Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and GP; β-glycerophosphate.

FO enhances osteogenesis via crosstalk with the Wnt/β-catenin pathway. MC3T3-E1 cells were treated with 100 µg/mL of FO and 100 nM DEX for 7 days. (A) The cells were harvested at day 1 (D1), day 3 (D3), day 5 (D5), and day 7 (D7). Next, western blot analysis was performed to quantify β-catenin expression. (B) The cells were treated with 50 µg/mL and 100 µg/mL of FO or 100 nM DEX for 3 days. Total protein was extracted to quantify β-catenin expression in the cytosol and nucleus. (C) To evaluate the effect of β-catenin/TCF signaling, TOPFlash activity was determined. (D) Zebrafish larvae (3 dpf; n = 20) were pretreated with FH535 for 24 h and then treated with 100 µg/mL of FO or 100 nM DEX. The larvae were stained with calcein to observe vertebrae formation and mineralization 10 dpf. Relative calcein fluorescence intensity and total bone area were quantified using the Image J software and the number of vertebrae was manually counted. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas, DEX; dexamethasone, GP; β-glycerophosphate, dpf; days post fertilization, and UT; untreated group.

FO increases the expression of β-catenin, which translocates to the nucleus to transactivate osteogenesis-related genes. Under normal condition, β-catenin is phosphorylated by GSK-3β and consequently degraded through proteasome. FO inhibits degradation of β-catenin and increases the movement of β-catenin to nucleus. FO; fermented extract of C. gigas and GSK-3β; glycogen synthase kinsase.