Nutr Res Pract.  2010 Oct;4(5):356-361.

Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells

• Seo HJ
• Cho YE
• Kim T
• Shin HI
• Kwun IS

Abstract

Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 1, 3, 15, 25 uM) along with a normal osteogenic medium (OSM) as control for 1, 5, 10 days. As measured by MTT assay for mitochondrial metabolic activity, cell proliferation was stimulated even at low zinc treatment (1-3 micrometer) compared to OSM, and it was stimulated in a zinc concentration-dependent manner during 5 and 10 days, with the most pronounced effect at 15 and 25 uM Zn. Cellular (synthesized) alkaline phosphatase (ALP) activity was increased in a zinc concentration-dependent manner, so did medium (secreted) ALP activity. Cellular collagen concentration was increased by zinc as time went by, therefore with the maximum zinc stimulatory effect in 10 days, and medium collagen concentration showed the same pattern even on 1 and 5 day. This zinc stimulatory effect of collagen synthesis was observed in cell matrix collagen staining. The study results imply that zinc can increase osteogenic effect by stimulating cell proliferation, ALP activity and collagen synthesis in osteoblastic cells.

Keyword

Zinc; MC3T3-E1 cells; proliferation; ALP activity; collagen synthesis

MeSH Terms

Alkaline Phosphatase
Cell Proliferation
Collagen
Durapatite
Osteoblasts
Osteogenesis
Zinc
Alkaline Phosphatase
Collagen
Durapatite
Zinc

Figure

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  Fig. 1 Proliferation of osteoblastic MC3T3-E1 cells by zinc treatment for 1, 5 and 10 days. Cell proliferation of MC3T3-E1 cells by zinc treatment were measured by MTT assay. The values are presented as %OSM as normal osteogenic medium. Different superscripts mean significant difference by zinc treatments at P < 0.05 by Tukey, ANOVA. Data presented as mean ± SEM (n = 10).

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  Fig. 2 ALP activity in osteoblastic MC3T3-E1 cells by zinc. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells (synthesized ALP, A) and the media (secreted ALP, B) treated with zinc. Different superscripts mean significant different among zinc treatments at P < 0.05 by Tukey, one-way ANOVA. Data presented as mean ± SEM (n = 3). ALP one unit of ρ-nitrophenol as products being converted from p-nitrophenyl phosphate as substrate for 30 minute reaction time. (cellular ALP 1 unit = a nmole ρ-nitrophenol/mg of protein/30 min, medium ALP 1 unit = 1 nmole p-nitrophenol/ml/30 min)

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  Fig. 3 Collagen synthesis in osteoblastic MC3T3-E1 cells by zinc. Cellular and medium collagen concentration was measured as described in Methods. Different superscripts mean significant difference among zinc treatments at P < 0.05 by Tukey, one way ANOVA. Data presented as mean ± SEM (n = 3).

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  Fig. 4 Collagen staining by zinc treatment in MC3T3-E1 cells for 1, 5 and 10 days. Cell matrix collagen was stained using Van Gieson's staining method as described on Method. Magnification rate, ×100 (1days) or ×40 (5 and 10 days). Representative image of 8 replicates in 24-well plate.

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  Fig. 5 Alizarin Red S staining for Ca deposits (bone nodule) by zinc treatment in MC3T3-E1 cells for 1, 5 and 10 days. Cells were cultured at 24-well plates for 1, 5, and 10 days with the 0-25 mM zinc treatment. Extracellular matrix Ca deposits for matrix mineralization was measured using Alizarin red S dye which binds with Ca. Magnification rate, ×100. Representative image of 8 replicates in 24-well plate.

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