Viability of osteoblasts under cell hypoxia condition

Abstract

Objective: To investigate the effects of hypoxia condition and hypoxia-reoxygenation condition on the cell viability, apoptosis rate and gene expression of osteoblasts cultured in vitro. Methods: The cranium osteoblasts from newborn Sprague Dawley rats within 48 hours were cultured and purified through tissue block method.The morphological changes of cells were evaluated by the Alizarin Red S staining and Alkaline phosphatase staining.The third-generation osteoblasts were cultured in normal condition for 36 hours (group A), in hypoxic condition for 24hours (group B), in hypoxic condition for 24hours thereafter reoxygenated for 12 hours (group D), in hypoxic condition for 36 hours (group C). The cell viability of osteoblasts was tested via MTT assay.The apoptosis rate of osteoblasts was tested by FCM (flow cytometry). Quantitative PCR and Western blot methods were used to determine Collagen type Ⅰ, Bone morphogenetic protein 2 (BMP-2), Runt-related transcription factor 2 (RUNX-2), Transforming growth factor-β1(TGF-β1) expression levels. Results: The cell viability of osteoblasts decreased, group A(99.1%±8.3%), group B(90.9%±9.4%), group C(79.9%±8.7%), group D(73.0%±8.2%), group D <group C <group B <group A (F=37.886, P=0.000); the apoptosis rate of osteoblasts increased, group A(1.9%±1.3%), group B(16.3%±2.5%), group C(28.2%±4.2%), group D(33.5%±3.6%), group D >group C >group B >group A(F=26.198, P=0.000); the mRNA expressions of Col Ⅰ, BMP-2, RUNX-2, TGF-β1 decreased under hypoxia and hypoxia-reoxygenation condition, and group D< group C< group B< group A (F=13.082, P=0.006; F=7.088, P=0.017; F=6.857, P=0.038; F=51.368, P=0.000); the protein expressions of Col Ⅰ, BMP-2, RUNX-2, TGF-β1 decreased under hypoxia and hypoxia-reoxygenation condition, and group D< group C< group B< group A (F=8.114, P=0.013; F=28.935, P=0.000; F=9.857, P=0.007; F=46.541, P=0.000). Conclusion: Hypoxia condition and hypoxia-reoxygenation condition decreased the cell viability, increase the apoptosis rate and suppress the expressions of associated genes.Hypoxia-reoxygenation condition aggravate the injure of osteoblasts preconditioning under hypoxia condition.