Apoptosis of rat osteoblasts in process of calcification in vitro.

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 phenotype 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.