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. 2015 Nov 11;7(11):9265–9284. doi: 10.3390/nu7115465

Figure 2.

Figure 2

Osteoblast viability, differentiation and transcription in culture, after exposure to serum harvested from mice raised either under control conditions or treated with pomegranate peel extract. * p < 0.05 PGPE versus Control condition. (A) Cell proliferation test performed on MC3T3-E1 cells cultured in minimal medium (2% Fetal Bovine Serum, FBS), supplemented with serum from mice given either physiological serum (1.25% FBS + 0.75 Control mice serum) or pomegranate peel extract, PGPE (1.25% FBS + 0.75 PGPE mice serum), after 2 and 7 days of treatment. Results are expressed as mean ± standard error of the mean (SEM) (n = 6). (B) Alkaline phosphatase, ALP activity of MC3T3-E1 cells cultured in optimized medium with 10% FBS or with 7.5% FBS + 2.5% serum from mice fed either physiological serum (Control) or pomegranate peel extract (PGPE), after 0, 2, 7 and 14 days of treatment. Results are expressed as mean ± SEM (n = 8). (C,D) Mineralized nodules stained with alizarin red S on Day 21 in MC3T3-E1 cells cultured in optimized (C+) medium with 10% FBS or with 7.5% FBS + 2.5% serum from mice exposed to either physiological serum (Control) or pomegranate peel extract (PGPE). (C) Representative image of alizarin red staining for each condition. (D) Relative quantification of alizarin red staining with picture analyser software ImageJ. Results are presented as mean ± standard deviation, SD (n = 6). (E) Transcriptomic analysis of the main osteoblast markers in MC3T3-E1 cells cultured in optimized medium after 7 days of treatment with serum from mice exposed to either physiological serum (Control) or pomegranate peel extract (PGPE). Transcriptomic analysis of MC3T3-E1 mRNA levels determined by Taqman Low-Density Arrays are presented as fold change compared to the Control condition (fold change = 1) as mean ± SD (n = 6). Genes: ALP: alkaline phosphatase; βCat: β-catenin；Coll1a1: collagen 1a1; Coll2a1: collagen 2a1; DDR2: discoidin domain receptor 2; BSP: bone sialoprotein; Lrp5; OCN: osteocalcin; OPG: osteoprotegerin; osteri RunX2 and Wnt.