Effect Of PEMF On Proliferation, Differentiation And Gene Expresstion Of Osterix In Osteoblasts

ObjectiveTo investigate the influence of Pulsed Electromagnetic Field (PEMF) on cell proliferation, differentiation and gene (mRNA) expression of osteogenesis specific transcription factor-Osterix (OSX) of calvaria-derived osteoblasts in SD rat, and to explore the direct effects of PEMF on osteoblasts and the possible mechanism in its treatment usage for osteoporosis and related bone metabolism abnormality.Methods1. Cell isolation, culture and identification of oseteoblastsCell isolation and culture of oseteoblasts:Calvarias were taken out of the new-born Sprague-Dawley rats younger than 24h under the aseptic conditions. Osteoblasts were isolated via the trypsinization and collagenase digestion technique and improved tissue-culture method. Cells were cultured in a humidified atmosphere of 5% CO2 at 37℃. Osteoblasts were purified and subcultured by way of differential adhesion. The 4th generation purified osteoblasts were chosen for the experiment.Identification for osteoblasts:(1) Cell morphological characteristics and cell viability were monitored and photographed under inverted phase contrast microscope.(2) Alkaline phosphatase (ALP) staining was used for osteoblasts identification.2. To investigate the effects of PEMF on the proliferation, differentiation and mRNA expression of OSX in osteoblasts:Purified osteoblasts at passage 4 were counted, and then seeded in 96-well plates,24-well plates and 25 cm2 culture flasks, respectively. After 24 hours of incubation, osteoblasts became adherent. Subsequently, they were transferred into serum-free culture medium and incubated for another 24h for cell synchronization. After that, osteoblasts were divided into two groups:control group and PEMF group. Cells in the control group were cultured in normal medium. Osteoblasts in the PEMF group were interfered by PEMF of 12Hz and 11mT for 60min a day, totally for 3 days in all. The following measurements were performed at the next day after intervention.(1) To detect the proliferation ability of osteoblasts via MTT analysis.(2) To assay ALP activities in cell lysate and supernatant fluid of osteoblasts through disodium phenyl phosphate colorimetric determination, so as to reveal the differentiation capacity of osteoblasts.(3) To analyze the mRNA expression of OSX by fluorescence quantitative PCR.Results1. Cell isolation, culture and identification of osteoblastsCells were obtained with trypsin-collagenase digestion and improved tissue-culture method, and cell morphological characteristics and growth consistent with the feature of osteoblasts. ALP staining proved that the cultured cells were osteoblasts. Highly purified osteoblasts were used for experiment.2. The effectiveness of PEMF on osteoblastsThe proliferation of osteoblast in PEMF group (0.448±0.074) was significantly higher than that in the control group (0.239±0.116) (P<0.01). However, when compared with that of the control group (2.552±0.493,0.745±0.372), the ALP activities decreased markedly in both osteoblast lysate (0.853±0.276) and the supernate (0.620±0.175) of osteoblast, especially those in lysate (P<0.01, P<0.05, respectively). The OSX mRNA expression in PEMF group (0.357±0.007) was significantly regulated down, when compared with that in the control group (1.000±0.072)(P<0.01). ConclusionPEMF of 12Hz, 11mT and 60min every day for 3 days can promote the proliferation of osteoblasts in its early stage, but inhibit cellular differentiation and down-regulate the mRNA expression of OSX.