Table 1.
Primers used to amplify the human BMP2 gene.
Table 2.
The Primers Correlated with SNPs of BMP2 gene.
Figure 1.
Application of mechanical stress by uniaxial cyclic stretch.
The transfected C3H10T1/2 cells were cultured on a deformable silicon membrane coated with gelatin. After reaching confluence, the cells were subjected to uniaxial cyclic stretch. After cyclic stretch, gene expression analyses were performed. In the same conditions, the cells inoculated in the stretch apparatus and not subjected to mechanical stress were as control.
Figure 2.
The computerized tomography (CT) and sequencing results of two genotypes of OPLL patients.
(A: 55-year-old man; B: 50-year-old woman). (Aa) The CT showed patient had ossification of the posterior longitudinal ligament in the cervical spine (C2-C3, C4-C5, C6-C7, segmental type). (Ab) The direct sequencing result of the PCR products showed the TG heterozygosis mutation of the rs2273073 (T/G) polymorphism of the BMP2 gene. (Ba) The CT showed patient had ossification of the posterior longitudinal ligament in the cervical spine (C4, C4-C5, mixed type). (Bb) The direct sequencing result of the PCR products showed the AT heterozygosis mutation of the rs235768 (A/T) polymorphism of the BMP2 gene.
Table 3.
Genotypic and allelic distributions of two SNPs between OPLL cases and controls.
Figure 3.
Histological and immunohistochemical examination of ligament tissues from OPLL and non-OPLL patients.
(A) Histological examination of ligament tissues from non-OPLL patient, showing normal morphology and texture (Masson staining ×400); (B) Histological examination of ligament tissues from OPLL patient, showing endochondral ossification. Cartilage was stained blue, there were more red dye with the maturing of bone (Masson staining ×400); (C) Ligament tissues from non-OPLL patient with normal structures were un-positive for BMP2 (IHC staining ×400); (D) Ligament tissues from OPLL patient with endochondral ossification structures were positive for BMP2. Representative brown signals are indicated by the arrows (IHC staining ×400). Scale bar = 50 um in all.
Figure 4.
Western blotting analysis of BMP2 protein levels in different groups.
(WT: wide-type) (A) BMP2 protein can be detected in all the groups. The ligaments tissues from OPLL patients with different genotypes were showed higher expression. (B) Quantification of BMP2 protein expression in the OPLL patients with different genotypes were higher than the ligament tissues from non-OPLL patients and bone chips from trauma patients, but no statistical difference was seen between each group of themselves. ** P<0.01 versus control.
Figure 5.
Morphology changes of the transfected cells after mechanical stress.
(WT: wide-type) The stretched cells (b-g and i-n) aggregated in different degree along the direction of force and manifested to the osteoblastic differentiation when compared with the static cells (a and h). (a-g) were observed through white light vision and (h-n) were the same view through evaluation for GFP expression with fluorescence inversion microscope. Scale bar = 50 mm in all.
Figure 6.
Western blotting analysis of BMP2 protein levels induced by uniaxial cyclic stretch in different groups.
(WT: wide-type) (A) BMP2 protein can be detected in all the groups and the expression levels were up regulated after mechanical stress was applied. (B) Quantification of BMP2 protein expression in all the groups. The expression of BMP2 protein in the C3H10T1/2 cells transfected by pcDNA3.1-BMP2 (rs2273073 (T/G)) and pcDNA3.1-BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the other stretched groups (P<0.01) and the corresponding static groups (P<0.05). **P<0.01 versus un-transfected in the static condition; ##P<0.01 versus un-transfected in the stretched condition; &&P<0.01 versus the other stretched groups; $ P<0.05 versus the corresponding static groups.