Growth, attachment, proliferation and apoptosis in standard density calvaria cell cultures of BSP-/- and BSP+/+ mice.

<p>(A) Time-course of <i>in vitro</i> cell growth in BSP+/+ and BSP-/- MCC cultures N = 12 dishes/time point. (B) Number of adherent BSP-/- and BSP+/+ MCC 30 min, 1 hr and 2 hrs after plating. N = 4 dishes/group. (C and D) <i>In vitro</i> proliferation in BSP+/+ and BSP-/- MCC cultures assayed by BrdU incorporation (C, N = 12 dishes/group) and KI67 labelling (D, N = 6 coverslips/genotype/time-point, 10 fields analysed/coverslip, results expressed as number of labelled cells/1000). (E) <i>In vitro</i> apoptosis in BSP+/+ and BSP-/- MCC cultures assayed by TUNEL at D6. **: p< 0.01, ***: p< 0.001 vs BSP+/+; Mann-Whitney U Test and (D) contingency table with χ₂ test.</p

Expression of OPN protein in standard density MCC cultures.

<p>(A) OPN amounts were assayed in the cell layer and culture medium of BSP+/+ and BSP-/- MCC cultures at D3, 6, 14 and 17. Results are normalized on total cell layer proteins. N = 6 dishes/time point. *: p<0.05, **: p<0.01 vs time-matched BSP+/+, Mann-Whitney U Test. (B) BSP+/+ and BSP-/- MCC cultures were arrested at D10 and immunolabeled for OPN. Nuclei were labeled with DAPI and the same fields were imaged in phase contrast microscopy. White bar = 40μm.</p

Time-course expression of PHEX (A) and cathepsin B (CatB, B) in BSP+/+ and-/- MCC cultures plated at standard and high density.

<p>N = 3 dishes/group. * p< 0.05 vs time-matched BSP+/+. (C) Quantification and (D) micrography of mineralized colonies at D17 in standard BSP-/- and BSP+/+ MCC cultures treated from D2 to D17 with 1μg/ml protease inhibitor mix (Leu.+Pep. = leupeptin + pepstatin A). N = 6 dishes/group. **: p< 0.01 vs genotype-matched control; ##: p<0.01 vs BSP+/+. Mann-Whitney U Test.</p

Effects of BSP deletion on the time-course of osteoblast marker and SIBLING expression in standard density MCC cultures.

<p>(A-D) Time-course of expression of osteoblast-associated gene: osteocalcin (OCN), type 1 collagen (Col1α1), alkaline phosphatase (ALP), osterix (OSX) and Runx2 and SIBLING genes: osteopontin (OPN), bone sialoprotein (BSP), DMP1 and MEPE at successive time-points of BSP+/+ (A, C) and BSP-/- (B, D) MCC cultures. The arrow marks the appearance of bone nodules. N = 3 dishes/group. *: p< 0.05 vs time-matched BSP+/+; #: p<0.05 vs D3 to D14. Mann-Whitney U Test.</p

<i>In vitro</i> osteoblast differentiation in standard and high density BSP+/+ and BSP-/- MCC cultures.

<p>(A) Micrographs (Insets: higher magnification) and (B) quantification of total mineralized colonies formed at D14 in BSP+/+ and BSP-/- MCC cultures grown at standard (5000 cells/cm²) and high density (25000 cells/cm²). N = 3 dishes/group. Expression of osteoblast-associated (C and D) and SIBLING genes (E and F) at D14 in BSP+/+ (C, E) and BSP-/- (D, F) cultures. N = 3 dishes/group. Quantification of OPN in the cell layer (G) and the culture medium (H) of BSP+/+ and BSP-/- standard and high density cultures. Results are normalized on total cell layer proteins. N = 6 dishes/group. *: p< 0.05, **: p<0.01 vs BSP+/+; #: p< 0.05, ##: p<0.01 vs standard density. Mann-Whitney U Test.</p

<i>In vitro</i> osteoblast differentiation in mouse bone marrow stromal cell (A-C) and mouse calvaria cell (MCC, D-F) cultures.

<p>(A) micrographs and (B) quantification of unmineralized, ALP+ colonies (CFU-ALP) in bone marrow stromal cell cultures (D12) from BSP-/- and +/+ mice. N = 13 dishes/genotype. (C) Time-course of <i>in vitro</i> cell growth in BSP+/+ and BSP-/- bone marrow cultures. N = 12 wells/time point. (D) Quantification of total colony forming units-fibroblasts (CFU-F) and CFU-ALP in low density cultures (50 cells/cm²) of BSP+/+ and BSP-/- MCC at D15. N = 5 dishes/group. (E) Micrographs of ALP+ and Von Kossa (VK) stained standard density (5000 cells/cm²) MCC cultures at D20. (F) Quantification of mineralized colonies (CFU-OB) in standard density BSP-/- and BSP+/+ MCC cultures at D20. N = 3 dishes/group. *: p<0.05, **: p< 0.01 vs BSP+/+; Mann-Whitney U Test.</p

Newborn morphology and growth of BSP+/+ and BSP−/− mice.

<p>(A) general aspect of BSP+/+ (+/+) and BSP −/− (−/−) newborn mice. (B) Kinetics of weight gain during the growth of BSP+/+ and BSP−/− mice. M, males; F, females. Data are Mean±SEM of N = 4 to 31 mice. Note the log scale.</p

PCR primer sequences in 5′-3′ direction.

<p>The meaning of the abbreviations is explained in the text.</p

Growth plate and osteoblast marker protein expression and blood levels in newborn and adult BSP+/+ and BSP−/− mice.

<p>QRT-PCR was performed 6 days after birth on collected whole femurs and tibias including the growth plates, ground and extracted in Tri-reagent. Messenger RNA expression of growth plate regulators IHH, PTHrP and IGF-1 (A), markers of bone formation Runx2, Osx and Ocn (B) and SIBLING proteins DMP1, MEPE and Opn (C) were assessed in samples from BSP+/+ (N = 5) and BSP−/− (N = 9) mice. Expression levels are normalized on the housekeeping gene GAPDH, and on Runx2 levels for Opn (C). ELISA assay of Opn (D) in the serum of 6 days (N = 5 pools), 35 days and 12 month old (N = 5 male) BSP+/+ and BSP−/− mice. Data are Mean±SEM; *: p<0.05, **: p<0.01, ***: p<0.001 vs BSP+/+, Mann-Whitney U test.</p

Growth plate kinetics of newborn, 3 week old, 10 and 16 week old BSP+/+ and BSP−/− mice.

<p>Data are Mean±SEM of N = 7 to 8 newborn and 4 to 6 adult mice; *:p<0.05, **:p<0.001 vs BSP+/+ Mann-Whitney U Test. Micrographs of the growth plate in the distal femur of BSP+/+ and BSP−/− newborn and 3, 10 and 16 week old mice.</p