MSM improves chondrogenesis. MSM addition did not affect the cells number (A); it upregulated the chondrogenic transcription factor SOX9 (B) and COL2A1 expression (C) during chondrogenic differentiation. MSM enhanced SOX9 expression in chondrocytes and restored the negative effects of IL-1β (D). MSM supplement increased the production of aggrecan by chondrocytes, even in the presence of IL-1β (E) (a: control; c: MSM treated; c: IL-1β + MSM treated). MSM upregulated the expression of chondrogenic genes in zebrafish larvae after 7 (9 dpf) (F) and 14 (16 dpf) (G) days of treatment. *p<0.05; **p<0.01

MSM increases chondrogenic gene expression in middle-aged zebrafish. Alcian blue staining was enhanced in MSM-treated zebrafish (A) and the expression of chondrogenic genes was increased in fin (B) and in scale (C) after 14 days of treatment. Aggrecan protein levels increased in MSM-treated zebrafish fin (D) while no difference was observed in scale (D) (a: control fin; b: treated fin; c: control; d: treated scale). *p<0.05; **p<0.01

Effects of MSM in the early phase of osteogenic differentiation. After 3 and 7 days of osteogenic differentiation a reduced number of differentiating cells was observed (A). In MSM-treated cells SP7 gene expression was upregulated after 3 and 7 days of differentiation whereas RUNX2 was downregulated after 7 days of differentiation (B). RUNX2 protein levels were similar after 3 days of osteogenic differentiation (C) and significantly lower after 7 days of differentiation (D) in nuclei of MSM treated cells. *p<0.05; **p<0.01

Effects of MSM in the osteogenic maturation. RUNX2 gene expression was reduced while the expression of SPARC and SPP1 (A), as well the number of osteocalcin positive cells (B) increased in MSM-treated cells after 14 days of osteogenic differentiation. After 21 days of differentiation calcium deposition, evaluated by alizarin red staining (ARS), increased in cells treated with MSM (C). *p<0.05; **p<0.01

MSM modulates gene expression in zebrafish larvae. Increased expression of sp7 gene and reduced tnfrsf1b gene expression in larvae at 9 dpf (A). The fluorescence density produced by calcein staining was higher in larvae after 7 days of MSM treatment compared to untreated larvae (B) (a: control; b: MSM treated). Increased runx2b, sp7, col1a1, and reduced tnfrsf1b expression observed after 14 days of MSM treatment compared to control (C). Bone mineralization evaluated by calcein staining was higher in MSM-treated larvae compared to untreated larvae after 14 days of treatment as well (5D) (a: control; b: MSM treated). *p<0.05; **p<0.01

MSM increases mineral deposition in middle-aged zebrafish. Alizarin red staining (ARS) in adult zebrafish untreated (a) and treated (b) with MSM for 14 days. A Magnification 4×. Increased percentage of the positive ARS staining area in MSM-treated zebrafish (b) compared to untreated zebrafish (a). C Magnification 20×. *p<0.05;

MSM modulates gene expression in middle-aged zebrafish. Runx2a gene expression was downregulated in fin of MSM treated adult zebrafish (A); runx2b expression increased while tnfrsf1b expression decreased in scales of MSM-treated zebrafish compared to controls (B). Stronger Alcian blue as well as alizarin red staining areas were observed in fin of MSM-treated zebrafish (b) compared to control (a) (C); Magnification 4×. The Alcian blue staining was weak in scales of both control (a) and treated (b) zebrafish (D) whereas a stronger alizarin red staining area was observed in scales of MSM-treated zebrafish (E); Magnification 40×. *p<0.05

MSM affects miR146b expression and ERK signaling. MiR 146b expression was reduced in both fin and scale treated with MSM (A). Increased ERK levels and reduced pERK levels and pERK/ERK ratio were observed in both fin and scale of MSM-treated zebrafish; a: control fin; b: treated fin; c: control scale; d: treated scale (B). A schematic graphic showing as MSM promotes chondroblast progenitors and pre-osteoblast maturation by reducing ERK phosphorylation (C).