Histological analysis of calcification in explanted tibiae.

<p>(A) Midsagittal sections of tibiae were stained with Alizarin red after explantation or cultured seven days under hypoxic or normoxic conditions. (B) Image analysis was used to determine the length of the intensely calcified tissue, which was taken as the broken line indicated in ‘A’. (C) The area of calcification was used to determine relative calcification of the samples. (D) Higher magnification microphotographs were used to investigate the calcification of the hypertrophic cartilage that resides on top of the intensely stained bone. The dashed line represents the osteochondral interface. (N = 15). * = P<0.05 compared to freshly isolated tibiae. # = P<0.05 compared to normoxic condition of the same time point.</p

Explanted tibiae cultured 21 days under hypoxic or normoxic conditions.

<p>(A) Microphotographs of representative tibiae at different points in time. (B) Using image analysis the average tibiae lengths were calculated. (N = 18). * = P<0.05. ** = P<0.01.</p

Effect of hypoxic and normoxic culture conditions on Frzb and Dkk1 protein levels.

<p>(A) Frzb and Dkk1 levels were quantified in the conditioned medium of tibiae, which were cultured for 7 days without receiving new medium in either hypoxia or normoxia (N = 5). (B) The effect of oxygen levels on Frzb and Dkk1 on protein activity over time was studied by exposing culture medium containing 10% fetal bovine serum to either hypoxia or normoxia for 7 days in 37°C. (N = 4). Frzb and Dkk1 protein levels were analyzed using ELISA. * = P<0.05 compared to normoxic condition of the same time point.</p