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. 2015 Aug 24;230(12):3049–3056. doi: 10.1002/jcp.25041

Figure 1.

Figure 1

The effect of pH on mineralisation by primary rat osteoblasts and ecto‐nucleotidase mRNA expression. Osteoblasts were cultured at pH 6.9 or 7.4 for up to 14 days. A: Low power scans demonstrate a lack of alizarin red staining at pH 6.9 compared to pH 7.4. Phase contrast micrographs show the presence of collagenous matrix but the absence of alizarin red staining at pH 6.9 indicates that mineralisation has failed to occur. Scale bars: tissue culture wells = 5 mm, phase contrast micrographs = 500 μm. B: Culture at pH 6.9 results in the complete abolition of mineralised nodule formation. Values are means ± SEM (n = 6 replicate wells), ***P < 0.001. C, D: Ecto‐nucleotidase mRNA expression was investigated in differentiating (day 7) and mature, bone forming (day 14) osteoblasts at physiological pH7.4 using qRT‐PCR. Expression levels are given as relative to TNAP (Alpl) expression. The rank order of mRNA expression in differentiating osteoblasts was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > Alpl > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. In mature, bone forming osteoblasts the rank order of expression was NTPdase 6 > Enpp1 > NTPdase 4 > Alpl > NTPdase 5 > Enpp3 > NTPdase 3 > Enpp2 > NTPdase 1 > NTPdase 2. E: RT‐PCR analysis of mRNA at day 7, 10 and 14 showed that compared to pH 7.4 culture at pH 6.9 upregulated the expression of Enpp1 mRNA in osteoblasts, at all stages of differentiation. In contrast, Alpl, Enpp2, Enpp3 and NTPdase 2 mRNA expression was decreased in acid. mRNA expression of NTPdase1, 3‐6 and Ank was unchanged. F: qRT‐PCR analysis of osteoblasts at day 10 (the stage when the largest effects were seen with RT‐PCR) showed that culture under acid conditions resulted in a 2.8‐fold increase in Enpp1 mRNA expression; expression of Alpl and Enpp3 was decreased 3.2‐ and 2.5‐fold, respectively. The mRNA expression of the other ecto‐nucleotidases was not significantly altered. All qPCR reactions were carried out in triplicate using RNAs derived from four different osteoblast cultures. Values are means ± SEM, *P < 0.05.