Apoptosis of rat osteoblasts in process of calcification in vitro

KEY WORDS cultured cells; skull; osteoblasts; physiologic calcification; alkaline phosphatase; apoptosis AIM: To establish a cell model of osteoblasts retaining their differentiated phenotype in culture and observe the apoptosis of osteoblasts in the process of calcification using a novel acetylcholinesterase (AChE) staining method. METHODS: Osteoblasts were isolated enzymatically from skull of newborn SD rats; alkaline phosphatase (AKP) activity was determined by reformed cobalt method and azo dye method; mineral deposition was assessed with Von Kossa staining and Fluo-3 staining; a novel AChE staining method was used to assay cellular apoptosis based on the higher expression of AChE in apoptotic cells. RESULTS: During the 44 d of cells cultured, primary rat skull-derived osteoblasts progressively developed into a bone-like tissue of multi-layered nodules of cells with mineralized extra-cellular matrix and the apoptotic cells increased while the matrix calcificated. CONCLUSION: The pheno-type of developmental sequence of rat skull-derived osteoblasts can reflect the maturation of osteoblasts in vitro. It is a convenient model for the research of osteoblasts biology