Figure 5. Adherent LPS inhibits osteoblast differentiation and mineralization in vitro.

DNA measurements of MC3T3-E1 pre-osteoblast cells at one or eight days after plating on rigorously cleaned discs (open circles) or discs with adherent LPS (closed circles) (A). Fluorescent microscopic images of cells stained with Texas Red X phalloidin and DAPI on rigorously cleaned discs or discs with adherent LPS at one day in culture (B). Images are representative based on microscopic analysis of discs. Expression of runx2 (C), osterix (D) and alkaline phosphatase (E) normalized to GAPDH on rigorously cleaned discs (open circles) or discs with 2.7 × 10⁴ EU/m² of adherent LPS (closed circles). Alkaline phosphatase activity at eight days in culture on rigorously cleaned discs (open circle) or discs with adherent LPS (closed circles) (F). The percentage of surface area mineralized as assessed by xylenol orange staining at thirty days in culture on rigorously cleaned discs (open circles) or discs with adherent LPS (closed circles) (G). Representative rigorously cleaned discs and discs with adherent LPS, which are closest to the mean in G (H). Statistical analysis was by Two-Way ANOVA, * denotes p<0.05 compared to rigorously cleaned. Each data point represents the mean ± SEM of three to four independent experiments containing three to four discs per group, each assayed in triplicate (A, C–F), viewed using fluorescence microscopy (B), or analyzed for the percent of surface area mineralized (G).