Measurement of oxygen consumption rate of osteoblasts from Sprague-Dawley rat calvaria in different in vitro cultures
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 Tflasks 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.
Key words: Bone tissue engineering, osteoblast, respiration rate, in vitro culture, encapsulation.