Measurement of oxygen consumption rate of osteoblasts from Sprague-Dawley rat calvaria in different in vitro cultures

摘要

The oxygen consumption rates of Sprague-Dawley (SD) rat’s osteoblasts cultured in different?in vitro?media were on-line measured by using precision dissolved oxygen and respirometry instrumentation. The osteoblasts, isolated from the calvaria of neonatal SD rat by an enzymatic digestive process of 0.25% trypsin solution and 0.1% type II collagenase, were diluted to 1 × 106?cells/ml in fresh media and passaged in T-25 flasks in an incubator. After two (2) passages, the cells were cultured directly in T-flasks and encapsulated medium by calcium alginate microcapsules, respectively. The cells were evaluated through live/dead assay, hematoxylin-eosin (HE) and alkaline phosphatase (ALP) staining. Moreover, Von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation. Following this, the oxygen consumption rates of osteoblasts in the earlier mentioned different cultures were measured on-line. The results showed that the osteoblasts performed well in its major functions after being examined by cellular morphology and viability, growth curve in growth kinetics, HE, ALP, Von-Kossa and Alizarin Red stains, which were capable of being used for further investigation as seed cells in bone tissue engineering. The osteoblasts cultured in static T-flask and encapsulated medium?in vitro?both proliferated well, and the oxygen consumption rates were 5.56 × 10-6?and 1.25 × 10-7?μmol/(min·cell), respectively. By measuring the oxygen consumption rates of osteoblasts in different cultures?in vitro, it would provide significant instruction and model function for further fundamental investigation and clinical application in bone tissue engineering.