An Experimental Study On Percutaneous Autologous Cultured Bone Marrow Mesenchymal Stem Cell Grafting Exposed In Electromagnetic Field For Tibia And Fibula Fracture In Rat

Objective1. To investigate the effects of electromagnetic field (EMF) on the apoptosis, proliferation and expression of FGF-2 and FGFR-2 mRNA in rat bone marrow mesenchymal stem cells(BMSCs) in vitro.2. To study the therapeutic effect of Autologous Cultured Bone Marrow Mesenchymal Stem Cell Grafting exposed in electromagnetic field for tibia and fibula fracture in rat.Method1. The rat bone marrow mesenchymal stem cells were isolated and cultured in vitro. The morphology of the cells were observed with a inverted phase contrast microscope. The BMSCs of passage 3 were divided into different groups to be stimulated in the magnetic field exposure system. The intensity of generated peak magnetic field between coils was 1.0 mT. But the frequency of the EMF and the exposed time were different between groups. The apoptosis test was taken with Annexin V-FITC /PI method. And the proliferation of the cells was detected with MTT assay. Meanwhile, the growth curve was drawed. In addition, the expression of FGF-2 and FGFR-2 mRNA in the cells of each group was detected by reverse transcriptase -polymerase chain reaction(RT-PCR).2. Thirty rats were divided into BMSCs+EMF group,BMSCs group and control group randomly. The BMSCs were harvested from every rat in each group and amplified in vitro. Cells of BMSCs+EMF group were irradiated by pulsed electromagnetic fields of 15Hz,1.0mT. And cells of BMSCs group were adopted sham irradiation. After modeling artificial tibia and fibula fracture, the cells of BMSCs+EMF group and BMSCs group were injected into the fracture site percataneously. The rats of Control group were treated with 0.9% physiological saline in the same way instead of BMSCs. Radiographs were taken immediately postoperatively and every 1 week thereafter until sacrifice after week 4.The blood was drawn to measure the activity of Alkaline Phosphatase and the concentration of Bone Gamma-carboxyglutamic-acid-containing Proteins in serum immediately postoperatively and every 1 week respectively. Histological sections were taken to examine the change of contents in bony callus.Result1. After isolation and culture, BMSCs arranged spindle or polygonal. And the morphous of cells was same even irradiated by EMF. Cells were S-shaped growth curve. Cells exposed in the EMF (15Hz, 1mT, 2h/d ) grew most prosperitily, and the expression of both FGF-2 and FGFR-2 mRNA reached the peak among all groups.2. The activity of ALP(P<0.05) and the concentration of BGP(P<0.05)in serum enhanced in BMSCs+EMF group and BMSCs group, compared with control group. The BMSCs+EMF group achived complete bone healing, which showed the largest quantity of bony callus measured by X-ray and histology analysis in all groups.ConclusionModerate EMF stimulation (15Hz, 1mT, 2h/d ) can significantly promote the proliferation, and increase the expressions of FGF-2 and FGFR-2 mRNA in rat bone marrow mesenchymal stem cells in vitro.BMSCs can play a stimulative role during the process of fracture healing. What is more, EMF stimulation (15Hz, 1mT) can increase the bone-forming ability of BMSCs.