| BackgroundThe key of bone tissue engineering was to improve osteogenic ability to repair bone defects, and its major point was enough seed cells. At present, the seeds cells of tissue-engineered bone were usually mesenchymal stem cells and embryonic stem cells. However, the proportion of MSCs in the bone marrow was very low and the proliferation rates of mesenchymal stem cells was low, it is hard to supply enough cells in short time. Also, MSCs were difficult to purify. Moreover, the cell biological function of MSCs was vulnerable by the factors of age. Embryonic stem cells (ESCs) were isolated from inner cell mass of mammalian blastocysts and primordial germ cells, which had two major characteristics, one is unlimited proliferation, another is the capcity of differentiation into all cell types. As the most potential seeds cells of bone tissue engineering, ESCs could be induced into osteoblasts, the number of osteoblasts differentiated and ESCs'functional status were one of the main factors that affected the osteogenesis effects in tissue engineering. At present, the method that differentiating ESCs to osteoblasts mainly was to add chemical compounds, the differentiation rate of osteoblast was too low to meet the enormous require of tissue engineering of seeds cells.As a safe, simple and effective factors, PEMFs had been widely used in clinical treatment of fracture nonunion, delayed union and false joints, and low-frequency, low field strength PEMFs would not lead to gene mutation and chromosomal aberrations. Therefore, that PEMFs was began to be applied to bone tissue engineering and it showed that PEMFs had great significance. Our recent study showed that a specific field strength and frequency PEMFs could improve the osteoblasts differentiation rates of bone marrow MSCs. Considering similarities between ESCs and MSCs in many respects, especially for the same reaction to the same osteogenic inducer, we speculated that pulsed electromagnetic fields also played a role on ESCs to differentiate into osteoblasts. ObjectiveTo observe the effect of pulsed electromagnetic fields on murine embryonic stem cells differentiating to osteogenic cells, and explore a new strategy of inducing ESCs to differentiate into osteoblasts.Methods1. ESCs culture and identification: Mouse embryonic stem cells from cell line C57BL/6X129 were expanded in feeder cells of mouse embryonic fibroblast (MEF) combinated with leukemia inhibitory factor (LIF). Non-differentaition were identified by Alkalescence phosphate (AKP) staining.2. Osteogenic induction: embryonic bodies of 4 days old derived from mES cells were inoculated into cell culture plates with a concentration of 5×104/mL, and adherent culture was used for embryonic single-cells in DMEM basic medium. From the 5+14days of EBs differentiation, pulsed electromagnetic fields, chemical inducer and the combined stimulation were used. And alizarin red staining, immunohistochemistry staining of osteocalcin, real-time PCR of osteogenic key factors Osterix and Cbfa-1/Runx-2 were used to observe the situation of osteogenic differentiation.Results1. ESCs culture and identification: ESCs were inoculated to feeder layer pre-treatment byγ-ray. After 24h, cells were attached to the MEF to clonal grow by the composition of a few to several dozen cells. 48h after, the cloning of ESCs increased several times, showing a colony of cells. Selecting better growth of ESCs to do alkaline phosphatase staining, cell clones were blue-violet, and the feeder layer cells are not colored.2. Osteogenic induction:(1) alizarin red staining: The size of calcium nodules in PEMFs group, induction medium group and combined culture group increased 123%,229%,262% respectively compared with control group, the differences were significant, P <0.01, among which combined culture group increased 62% and 10% compared with PEMFs group (P<0.01) and induction medium group (P<0.05).(2) Immunohistochemistry staining of osteocalcin: There were positive green fluorescent particles in PEMFs group, induction medium group and combined culture group; however, control group was negative. Among them, the osteocalcin secreted in combined culture group was the most.(3) Real-time PCR: The expression level of osterix in PEMFs group, induction medium group and combined culture group was130%(P<0.05),220%(P<0.01),410%(P<0.01) higher than that in control group respectively. Among them, that in combined culture group was the highest and had significant differences with that in PEMFs group and induction medium group, P <0.01. The expression level of Cbfa-1/Runx-2 in PEMFs group, induction medium group and combined culture group was 140%(P<0.05),210%(P<0.01),290%(P<0.01) higher than that in control group respectively. Among them, that in combined culture group was the highest and had significant differences with that in PEMFs group and induction medium group, P <0.01.ConclusionES cells can be expanded in vitro without differentiation under the condition of feed layer pre-treatment byγ-ray combinated with leukemia inhibitory factor (LIF). Pulsed electromagnetic fields could promote mouse embryonic stem cells to differentiate into the osteoblasts, and application combining pulsed electromagnetic fields with conventional chemical inducer showed a synergistic effect, whose induction effect was better than that of both individual uses. The mechanism of PEMFs promoting ESCs to differentiate into osteoblasts might be related to BMP-2 signal transduction pathway.
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