Table 1.
Primers Used for Real-Time Polymerase Chain Reaction.
Figure 1.
Zirconium ion promotes HOB proliferation.
(A) The MTT assay was used to determine the HOB viability after HOBs were treated with zirconium solutions with different concentrations for 1, 3 and 7 days. Compared to the control and NaNO3-treated control, higher concentrations of zirconium significantly increased HOB viability. ZrO(NO3)2 at 5µM increased cell proliferation at D1 and D3 but not D7, whereas ZrCl4 at 5µM had no effects on HOB proliferation. However, higher concentrations of both of zirconium solutions significantly increased cell proliferation at D1, D3 and D7. Between the time points, viable cell number in each treatment group are significantly increased (p<0.01). (B) The graph shows the quantification of Ki67 positive cells to total cells. Compared to the control, the proportion of Ki67-positive cells is significantly increased in all treatment groups. There were no significant differences observed between the treatment groups at the different concentrations. (C-J) Representative images from each treatment group following KI67 immunostaining. Arrows indicate the positive staining of cells with the Ki67 antibody (C). Scale bar: 100 µm. Data are Mean±SD of three separate experiments. * p<0.05, **p<0.01 versus control.
Figure 2.
Effects of zirconium ion on mineralization of HOBs at D21 and D28.
(A, B) In the controls, patchy weak Alizarin Red S staining (asterisks, A) was observed in HOBs at Day21 and D28. (C-H) ZrO(NO3)2 and (K-P) ZrCl4 dramatically promoted the formation of mineralized matrix (arrows, C, E), compared to the control (A, B) and NaNO3-treated control (I and J). Scale bar: 100 µm.
Figure 3.
Zirconium ions up-regulate osteoblast genes in HOBs.
Real-time PCR was used to analyze the expression of osteoblast genes in zirconium-treated HOBs. (A) At D7, OPN gene expression was significantly up-regulated in HOBs treated with ZrO(NO3)2 at concentrations of 5, 50 and 500 µM, compared to the controls. At D3, only ZrO(NO3)2 at 500 µM increased OPN expression. ZrCl4 at 50 and 500 µM increased OPN expression at D3 and D7, but not 5 µM. (B) Compared to the controls, ZrO(NO3)2 at 50 and 500 µM up-regulated BSP expression at D7, but not at D3; whereas ZrCl4 at 50 µM and 500 µM increased BSP expression at D3 and D7. (C) At D3, ZrO(NO3)2 and ZrCl4 at 5 and 50 µM increased Runx2 expression. At D7, at all three different concentrations, ZrO(NO3)2 increased Runx2 expression; However, ZrCl4 increased Runx2 expression only at 50 and 500 µM. (D) ZrO(NO3)2 and ZrCl4 at 500 µM increased OC expression at D7. At D3, OC expression was up-regulated by ZrCl4 at 50 µM; However it was suppressed by ZrO(NO3)2 at 5 and 500 µM and ZrCl4 at 5 µM. (E) ALP expression was up-regulated by ZrCl4 at 50 µM at D3 but no consistent evidence of regulation of ALP gene expression was seen. OPN, osteopontin; BSP, bone sialoprotein; OC, osteocalcin; ALP, alkaline phosphatase. *p<0.05, **p<0.01 vs control.
Figure 4.
Zirconium ions up-regulate gene expression of BMP2 and BMPR in HOBs.
HOBs were treated with ZrO(NO3)2 and ZrCl4 solutions at the concentrations of 5, 50 and 500 µM for 3 and 7 days. Real-time PCR was employed to analyze the gene expression of BMP2 (A), BMP receptors BMPR1a (B), BMPR1b (C) and BMPR2 (D). (A) At D3 and D7, BMP2 gene expression was significantly up-regulated in HOBs treated with ZrO(NO3)2 at 500 µM and ZrCl4 at 50 µM and 500 µM, compared to the control. (B) At D3, BMPR1a expression was up-regulated in HOBs by ZrO(NO3)2 and ZrCl4 only at 500 µM, compared to the control. ZrO(NO3)2 and ZrCl4 at 50 and 500 µM significantly increased BMPR1a expression in HOBs treated for 7 days. BMPR1a gene expression was inhibited by ZrCl4 at 5 µM at D3. (C) BMPR1b expression was significantly increased in HOBs treated with ZrO(NO3)2 and ZrCl4 at 50 and 500 µM at D3 and D7. Increased BMPR1b expression was exhibited by ZrO(NO3)2 at 5 µM only at D7 whereas ZrCl4 at 5 µM only at D3, respectively, compared with the control. (D) Zirconium solutions had no effect on the BMPR2 expression in HOBs.*p<0.05, **p<0.01 vs control.
Figure 5.
Zirconium ions elevate protein levels in the BMP/SMAD signaling pathway.
(A) Western blotting was used to determine the expression levels of BMP2, SMAD1 and phospho-SMAD1/5 (p-SMAD1/5) in primary HOBs treated with ZrO(NO3)2 and ZrCl4 for 7 days. β-actin was used as the loading control. (B) Quantification of Bmp2 protein bands indicated ZrO(NO3)2 at 500 µM increased Bmp2 protein level compared to the untreated and NaNO3 controls. Compared to the controls, ZrCl4 treatment increased BMP2 at the concentrations of 5 and 50 µM, but not at 500 µM. (C) Treatment of HOBs with ZrO(NO3)2 at 50 and 500 µM increased SMAD1 protein expression, but SMAD1 levels were reduced at 5 µM, compared to the controls. ZrCl4 treatment increased SMAD1 protein level at 5 and 50 µM but not 500 µM. (D) Both ZrO(NO3)2 and ZrCl4 treatment significantly and dose dependently increased the p-SMAD1/5 levels in HOBs. *p<0.05, **p<0.01 vs control.
Figure 6.
Localization of BMP2 in HOBs treated with zirconium solutions.
In untreated control (A, A’) and NaNO3-reated control (E,E’)HOBs, BMP2 localized strongly in the cell nuclei (arrows) but weakly detected in the cytoplasm. In ZrO(NO3)2-treated HOBs (B-D), BMP2 is strongly localized in nuclei. Localization of BMP2 protein is also strongly observed in the cytoplasm of HOBs treated with ZrO(NO3)2 at 50 and 500 µM (arrows, C,C’,D,D’). Localization of BMP2 in ZrCl4-treated HOBs (F-H) is similar to that in ZrO(NO3)2-treated HOBs. Scale bar: 50 µm.
Figure 7.
Localization of phospho-SMAD1/5 in HOBs treated with zirconium solutions.
Strong p-SMAD1/5 nucleus localization is detected in the control (arrows, A,A’) and NaNO3-reated control (E,E’) HOBs. In HOBs treated with ZrO(NO3)2 at 5 µM (B,B’), p-SMAD1/5 has similar localization in the nucleus as in controls. (C-D) In the HOBs treated with ZrO(NO3)2 at 50 and 500 µM, p-SMAD1/5 localization is strongly detected the nuclei and in cytoplasm (arrows, C,C’,D’D’). Treated with ZrCl4 at different concentrations of 5, 50 and 500 µM, HOBs show the similar p–SMAD1/5 localization exhibited in the nucleus and/or cytoplasm, as seen in HOBs treated with ZrO(NO3)2. Scale bar: 50 µm.
Figure 8.
Noggin suppressed osteogenic gene expression in HOBs treated with ZrCl4.
Combined culture of HOBs with ZrCl4 and noggin (500 ng/ml) suppressed: (A) OPN gene expression at D3 (ZrCl4 at 50 and 500 µM) and D7 (ZrCl4 at 500 µM); (B) BSP gene expression at D7 (ZrCl4 at 500 µM); (C) Runx2 gene expression at D3 (ZrCl4 at 50 and 500 µM) and at D7 D7 (ZrCl4 at 500 µM); and (D) OC gene expression at D7 (ZrCl4 at 500 µM). *p<0.05, **p<0.01 vs the same concentration of ZrCl4.