In vitro osteoblastic differentiation and identification of rat bone marrow mesenchymal stem cells by whole bone marrow adherent culture / 中国组织工程研究

ABSTRACT

BACKGROUND: Many operations for isolating, purifying and identifying bone marrow mesenchymal stem calls (BMSCs) are complicated and cost much. Also they have great effect on cell activity. Whether whole bone marrow adherent culture can avoid above-mentioned disadvantages remains unclear. At present, many studies huve been done to confirm an effective and low cost method for isolating, purifying and identifying such cells.OBJECTIVE: This study is to in vitro induce and differentiate rat BMSCs by whole bone marrow adherent culture,and to identify the cells.DESIGN: A controlled observational experiment.SETTING: Qingdao University Medical College.MATERIALS This study was carried out in the Laboratory of Oral Cavity and Laboratory of Molecular Biology (provincial level) Qingdao University Medical College between November 2005 and March 2007. Twenty Wistar rats of either gender, aged 3 to 4 weeks, of SPF grade, weighing 120-150 g, were provided by the Qingdao Laboratory Center. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Fetal bovine serum (FBS, Hangzhou Sijiqing Bioengineering Material Research Institute), alkaline phosphatase (ALP) kit (Nanjing Jiancheng Bioengineering Research Institute), reverse transcription kit (American Promega Corporation) and primer (Shanghai Bioengineering Co.,Ltd.) were used in this study.METHODS: Adult rat BMSCs were isolated and cultured by whole bone marrow adherent culture. They were digested with 2.5 g/L trypse and inoculated at a density of 5 ×107 L-1 in 6-well culture plate. Then, the cells were divided into experimental group and control group. Inducing culture medium was added to experimental group, and the same amount of basic culture medium was added to control group. ① Cell differentiation and calcium tuberculation were observed under the inverted microscope. ② Biological characteristics of induced cells were detected by calcium tubercle Von Kossa and alizarin Bordeaux. ③ALP activity was detected by diazo salt staining. ④Human core binding factor alpha subunit-1 (Cbf α-1), osteocalcin (OCN) and osteoblast-specific Osterix (OSX) mRNA expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: ① Induction and differentiation results of cells. ② Biological characteristics of cells induced by rat BMSCs. ③ ALP activity. ④ Cbf α-1, OCN and OSX expressions.RESULTS: ①Inducing culture medium was added in the serial subcultivation. About 9 days later, cell clones were connected to each other. On about 21 to 28 days, some pykno-round mineralized tubercles appeared. Meanwhile,control cells were connected to each other, but they did not form the tubercle. ② In the experimental group, when MSCs were induced for 21 to 28 days, obvious round or oval calcified tubercles were seen by naked eyes. The results of Von Kossa staining exhibited black sediments, and those of alizarin Bordeaux staining exhibited salmon tubercles. Calcium tubercles were not found in the control group. ③The ALP activity after 2 weeks of induction was obviously increased in the experimental group, but was relatively weak in the control group. ④In the experimental group,Cbf α-1, OCN and OSX expressions were significantly increased after induction.CONCLUSION: After being in vitro induced and differentiated by whole bone marrow adherent culture, rat BMSCs exhibited morphological and biological characteristics similar to typical osteoblasts.