Table 4.

Brassicaceae-derived OSCs: effects on in vitro models of osteoclastogenesis and osteoblastogenesis.

Molecule (organosulfur compouds)	Experimental in vitro model	Concentration	Main effect	Specific outcomes	Authors	Ref
Sulforaphane *	MLO-Y4, an osteocyte – cell line	3-10-15-30-100 μM	Inhibits cells proliferation; induces apoptosis; and inhibits osteoclastogenesis	• ↓ viability and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-like assay (EZ4U) • ↑ in the activities of Caspase 3/7 and 8 (assay kit) • ↑ Fas mRNA expression (RT-PCR) • ↓ RANKL mRNA expression (RT-PCR)	Thaler et al.	(97)
Glucoraphanin *	In vitro culture of human mesenchymal stromal cells from tibial plateau	3.3-10-33-100 μM	Induction of osteogenesis	• ↑ mineralization (alizarin red staining) • ↑ BSP, CBS, SMAD-1 mRNA (RT-PCR) • ↓ ALP, WISP-1 mRNA (RT-PCR)	Gambari et al.	(98)
Brassica rapa L. root ethanol extract	MG-63 cells line	1-5-10-25-50 μg/ml	Increased osteogenesis	• ↑ viability (Wst-8 assay) • ↑ ALP activity (pNPP measurements) • ↑ collagen (Sirius Red) • ↑ mineralization (alizarin red staining)	Jeong et al.	(54)
Sulforaphane *	MC3T3-E1	3-10-15-20-30-100 μM SFN	Promotion osteoblast differentiation and induction of apoptosis	• ↓ cells proliferation (3-(EZ4U) • ↑ in the activities of Caspase 3/7 and 8 (assay kit) • ↑ Fas mRNA expression (RT-PCR) • ↑ mineralization (alizarin red staining) • ↑ RUNX-2 mRNA expression (RT-PCR)	Thaler et al.	(97)
Sulforaphane *	BMMSCs from long bones of 6-week-old C57BL/6 mice	3 μM	Promotes osteoblast differentiation	• ↑ mineralization (alizarin red staining) • ↑ RUNX-2 mRNA expression (RT-PCR)	Thaler et al.	(97)
Hot water extract of Brassica oleracea	RAW 264.7 cell line	200 g/mL	Inhibition of osteoclast formation	↓ osteoclasts in femur, when in combination with P. ginseng extract (TRAP staining)	Kang et al.	(99)
Sulforaphane *	RAW 264.7 cell line	3-10-15-30-100 μM	Reduces proliferation and induces apoptosis	• ↓ viability and metabolic activity (EZ4U) • No alteration in Acp5, Clcr, and CTSK mRNA expression (RT-PCR) • ↑ Tet1 and Fas-Caspase 8-Caspase 3/7 pathway (western blot, assay kit)	Thaler et al.	(97)
Sulforaphane *	RAW 264.7 cell line	1-2-5-10 μM	Inhibition of osteoclastogenesis	• ↓osteoclasts (TRAP staining) • ↑ NRF2 protein accumulation (western blot); ↑ HO1, NQO1, GCLC and GCLM mRNA (RT-PCR) • ↓ ROS (2′,7′-Dichlorofluorescin diacetate) • ↓ NFATc1, C-FOS, TNFα, TRAP, CTSK, MMP-9, DC-STAMP mRNA (RT-PCR)	Xue et al.	(100)
Sulforaphane *	RAW 264.7 cell line	0.01-0.1-0.5-1 μM	1. Inhibits osteoclastogenesis 2. Inhibits osteoclasts cells-fusion	• induced cytotoxicity at > 5 μM (CCK-8 assay) • ↓ osteoclasts (TRAP assay) • ↓NFATc1, TRAP, CTSK mRNA (RT-PCR) • ↓ OSCAR, DC-STAMP, OC-STAMP mRNA (RT-PCR) • ↑ phosphorylation of STAT1 (Tyr701) (western blot)	Takagi et al.	(101)
Sulforaphane *	RAW 264.7 cell line	0.01-0.1-1-10 μM	Inhibition of osteoclastogenesis	• ↓ osteoclasts • ↓NF-kappaB activation	Kim et al.	(102)
Sulforaphane *	RAW 264.7 cell line	0.5, 1, 2.5, 5, 10, 20 μM	Decreased viability and osteoclastogenesis	• Marked cytotoxicity at concentration > 5 μM, low cytotoxicity 1-2.5 μM (CCK-8 assay) • ↓osteoclasts (TRAP staining) • ↓ CTSK, MMP-9 mRNA and protein (RT-PCR) • ↓ in autophagosomes and LC3-II, Beclin1, and Atg5–Atg12 mRNA and protein; ↓ of JNK phosphorylation (RT-PCR, western blot) • ↓size of F-actin rings	Luo et al.	(103)
Sulforaphane *	Primary mouse osteoclasts from tibial and femoral bone marrow of 8-week-old C57BL/6 mice	3 μM	Inhibition of osteoclasts resorption	↓ resorption activity	Thaler et al.	(97)
Sulforaphane *	Primary osteoclast precursors isolated from BM of tibias and femurs of 8–12 weeks old male C57BL/6 mice	1-5 μM	Inhibition of osteoclastogenesis	↓ osteoclasts (TRAP staining)	Xue et al.	(100)
Sulforaphane *	BM cells obtained from the femur and tibia of 7–10-week-old ddY male mice	0.01-0.1-0.5-1 μM	Inhibition of osteoclastogenesis	• induced cytotoxicity at > 5 μM (CCK-8 assay) • ↓ osteoclasts (TRAP staining) • ↓ NFATc1, TRAP, CTSK mRNA expression (RT-PCR)	Takagi et al.	(101)
Sulforaphane *	BM cells isolated from femora and tibiae of 4- 6-week-old C57BL/6 mice	0.01-0.1-1-10 μM	Inhibition of osteoclastogenesis	• ↓ osteoclasts • Early inhibition of osteoclastogenesis • No effects on osteoclasts resorption • No effects on RANK or c-fms mRNA	Kim et al.	(102)
Sulforaphane *	BMMs from 5-week-old C57BL/6 female mice	1, 2.5, 5 μM	Decreased viability and inhibition of osteoclastogenesis	• Moderate cytotoxicity at concentration >2.5 μM (CCK-8 assay) • ↓ osteoclasts (TRAP staining)	Luo et al.	(103)
Sulforaphane *	Human monocytes isolated from peripheral blood of healthy volunteers	0.2-1-5 μM	Inhibition of osteoclastogenesis	• ↓ osteoclasts (TRAP staining) • ↑NRF2 accumulation (immunocytochemistry) • ↑ NQO1 and PRDX1 mRNA expression (RT-PCR)	Gambari et al.	(104)

Most in vitro studies were conducted using purified OSCs (6 studies, 15 in vitro models; sulforaphane, glucoraphanin); while only a few used water or ethanol extracts from Brassicaceae edible plants (2 studies, 2 in vitro models; Brassica rapa, Brassica oleracea). Most studies showed increased osteogenesis and decreased osteoclastogenesis. Notably, only the effects of purified OSCs (labeled with * in the table) can be attributable to OSCs. The concentrations tested ranged from 0.01 to 100 μg/ml. Murine in vitro models of osteoclastogenesis: osteoclasts derived from bone marrow of femora and tibiae of mice, RAW 264.7 cell line. Human in vitro models of osteoclastogenesis: human monocytes isolated from peripheral blood of healthy volunteers. Murine in vitro models of osteoblastogenesis: MC3T3-E1 (Mouse C57BL/6 calvaria cells line); murine bone marrow (BM) cells; bone marrow-derived mesenchymal stem cells (BMMSCs), bone marrow macrophages (BMMs). Human in vitro models of osteoblastogenesis: MC3T3-E1, MSCs isolated from human tibial plateau. Osteocyte – cell line: MLO-Y4. Functional assays for osteoclastogenesis: tartrate-resistant acid phosphatase positive (TRAP staining); pit assay. Functional assays for osteoblastogenesis: Alizarin red staining (marker of mineralization), Sirius red assay (marker of collagen I), p-nitrophenyl phosphate (pNPP) quantification. Proliferation/viability assays: cell counting kit-8 (CCK-8) cell viability assay, water-soluble tetrazolium-8 (WST-8) assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-like assay (EZ4U). Markers of osteoclasts: nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), cathepsin K (CTSK), receptor activator of NF-KB (RANK), osteoclast stimulatory transmembrane protein (OC-STAMP), tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor-κB ligand (RANKL), dendritic cell specific transmembrane protein (DC-STAMP), reactive oxygen species (ROS), c-fos, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), matrix metallopeptidase 9 (MMP-9), osteoclasts-specific activating receptor (OSCAR), acid phosphatase 5, tartrate resistant (ACP5), calcitonin receptor-like receptor (Clcr), colony-stimulating factor-1 receptor (c-fsm), c-fos. Markers of osteoblastogenesis: cystathionine-β-synthase (CBS), bone sialoprotein (BSP), SMAD family member 1 (SMAD-1), alkaline phosphatase (ALP), WNT1-inducible-signaling pathway protein 1 (WISP-1), osteocalcin (OCN), runt-related transcription factor 2 (RUNX-2). Markers of cell viability – apoptosis: Fas, Caspase 3/7 and 8, nuclear factor erythroid-derived 2-related factor 2 (NRF2), heme oxygenase-1 (HO1), NAD(P)H: quinone oxidoreductase 1 (NQO1), peroxiredoxin-1 (PRDX-1), glutamate cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), peroxiredoxin 1 (PRDX-1), microtubule-associated protein 1A/1B-light chain 3 (LC3-II), beclin1, autophagy related 5 (ATG5), Jun N-terminal kinases (JNK), autophagy related 12 (Atg12). ↑ means up-regulation; ↓ means down-regulation