| Obiective (1) Isolation and in vitro culture of Sparague-Dawley (SD) rat bone marrow derived mesenchymal stem cells (BMSCs), identify the immunophenotype characteristic of the cultured cells and evaluate the differentiation potential for osteogenesis, chondrogenesis and adipogenesis.(2) To study the cell proliferation characteristic of BMSCs by MTT assay, and study the osteogenic effects of extremely low frequency electromagnetic fields (ELF-EMF) on BMSCs by detecting the gene expression level of osteogenic indices and the formation of calcium nodules.(3) To study the osteogenic effect of ELF-EMF on co-culture of rat BMSCs with osteoblasts and explore the potential mechanism of in vivo osteogenesis promoted by ELF-EMF by decteing the gene and protein expression level of osteogenic markers in both BMSCs and osteoblasts and the formation of calcium nodules.(4) To study the effect of ELF-EMF on the chondrogenesis of BMSCs by pellet culture under controlled condition by detecting the GAG/DNA content and expression of chondrogenic markers in pellet cultures.Methods Percoll density gradient centrifugation combined with plating culture method was used to isolate and purify rat bone marrow derived mesenchymal stem cells;. Cells at the third to sixth passage, which showed better morphology and cell proliferation ability, were chosen for the following experiments.(1) Cell immunophenotype identification was carried out by cell immunohistochemisty and flow cytometry technology. Muti-lineage differentiation potential of the cells, which contains osteogenesis, chonderogenesis and adipogenesis, was also evaluated by multi-differentiation inductive culture. Thus, we could affirm that the cultured cells were BMSCs which have muti-lineage differentiation potential.(2) In order to study the osteogenic effect of ELF-EMF on rat bone marrow mesenchymal stem cells, in vitro cultured BMSCs were randomly divided into three groups:control group, ELF-EMF exposure group and osteogenic induction combined ELF-EMF exposure group. Cell proliferation ability was assessed by MTT assay. On day14, cells in each group were randomly chosen for total RNA extraction, Real-Time PCR reaction were carried out to detect the gene expression level of osteogenic indices which contain ALP, Colla2, Runx2and Bglap(3) In order to study the effect of ELF-EMF on simulated in vivo situation, rat osteoblasts were isolated and cultured in vitro, Transwell cell-culture-chamber polyester inserts were used to establish co-culture system between BMSCs and osteoblasts. Co-cultures were randomly divided into ELF-EMF exposure group and nonexposure group, single cultured osteoblasts and BMSCs were set as single culture group. On day14, cells in each group were randomly chosen for total RNA extraction and protein extraction, Real-Time PCR reaction was carried out for detecting the gene expression level of Runx2, Sp7, ALP, Bmp2, Coll a2and Bglap, and Western blotting assay was carried out to dectet the protein expression level of Run2, Sp7, Bmp2, Bglap. Cells in each group were also randomly chosen for alizarin red staining to evaluate the formation of calcium nodules.(4) In order to study the chondrogenic effect of ELF-EMF on rat bone marrow derived mesenchymal stem cells, cells at passage five were centrifuged into cell pellets for pellet culture under controlled condition. Pellet cultures were randomly divided into three groups: non-drug-treated ELF-EMF exposure group, drug-treated nonexposure group and drug-treated ELF-EMF exposure group. The ELF-EMF exposure groups were treated with1.0mT,50Hz electromagnetic fields for4hper day. The culture medium of drug-treated groups contains FGF-2and TGF-β3. After three weeks, cell pellets in each group were randomly chosen for total RNA extraction, Real-Time PCR was carried out for detecting the gene expression of chondrogenie markers containing Aggrecan, Col2α1,Col10α1and Sox9. Glycosaminoglycan (GAG) content of pellet cultures were determined using the dimethylmethylene blue (DMMB) dye-binding assay. Alcian blue staining was also carried out for evaluating the chondro genesis.Result (1) The obstained cells were observed under inverted microscope, primary bone marrow derived mesenchymal stem cells were spindle-shaped and adherea on the bottom of the falsk after48hours’ culture. The cells grew rapidly and formed tightly packed whorls after4days. Cell cultures could reached100%confluency after9-12days. After osteogenic inductive culture for14days, a lot of calcium nodules formed; after adipogenic inductive culture for14days, the large fat droplets formed; after chondrogenic inductive culture for21days, a lot of cartilage matrix secreted and cartilage excavation formed. The flow cytometry analysis result show that the expression rate of CD29+, CD44+, CD90+are over95%, while the expression rate of CD34+is less than5%, which indicate that less hematopoietic cell lines were contained and the uniformity of cells. Thus, the obstained cell were affirmed to be mesenchymal stem cells which have the ability of multi-lineage differentiatioa(2) Extremly low frequency electromagnetic fields could significantly promote the proliferation of rat bone marrow derived mesenchymal stem cells, but the proliferation rate of cells in osteogenic inductive culture was not significant different from the control. Real-Time PCR result showed that the gene expression of Runx2, ALP, Collα2and Bglap in osteogenic induced groups increased significantly, the gene expression of these indices in normal ELF-EMF exposed groups did not show significant differences with the control groups. All these showed that ELF-EMF as a single factor could not promote osteogenic differentiation of BMSCs.(3) Real-Time PCR results showed that the the gene expression level of Runx2, Sp7, ALP, Colla2and Bglap were relatively low in single cultured rat bone marrow derived mesenchymal stem cells. The gene expression of these indices in co-cultured BMSCs and osteoblasts up-regulated in varying degrees. The gene expression level of ALP, Bmp2and Coll α2in co-cultured osteob lasts under ELF-EMF exposure was even higher. It was worth notice that, the gene expression of Bmp2in BMSCs did not show significant difference between single culture groups with co-cultured groups, while it sharply up-regulated in EMF-ELF exposed co-cultured groups. Western blotting assay showed the protein expression level of Runx2, Sp7, Bmp2and Bglap was consistent with the PCR results. No calcium nodule formed in single cultured BMSCs, a lot of calcium nodules formed in co-cultured BMSCs and ELF-EMF treated co-cultured BMSCs. Compared to single cultured osteob lasts, osteob lasts in co-cultured groups and ELF-EMF treated co-culture groups could form more calcium nodules, especially in the ELF-EMF treated co-cultured osteob last, and it showed a sharp contrast with the single cultured osteob lasts.(4) Glycosaminoglycan (GAG)/DNA assay showed no glycosaminoglycan content existed in non-drug-treated pellet cultures, the GAG content in drug-treated and ELF-EMF exposed groups were higer than the nonexposed pellet cultures. Alcian blue stain showed increased cartilage matrix secretion in drug-treated groups, while no cartilage matrix secretion in non-drug-treated groups. ELF-EMF could enhance the secretion of cartilage matrix in drug-treated cell pellets. Real-Time PCR results showed that TGF-β3could significantly promote the gene expression of Aggrecan, Col2α1, Col10α1and Sox9in rat BMSCs. Compared to drug-treated groups, the gene expression level of Aggrecan, Col2α1, Col10α1were much higher in drug-treated groups which combined with ELF-EMF exposure.Conclusions (1) Through the method of Percoll density gradient centrifugation combined with plating culture, we can obtain high purity rat bone marrow derived mesenchymal stem cells.(2) Electromagnetic fields can promote the proliferation rate of rat bone marrow derived mesenchymal stem cells. But as a single factor, ELF-EMF can not promote the osteo genic differentiation of bone marrow mesenchymal stem cells.(3) In simulated in vivo circumstance, ELF-EMF can significantly promote osteogenic differentiation of rat bone marrow derived mesenchymal stem cells, Bmp2-mediated cellular interaction might play an important role in the osteogenic differentiation induced by ELF-EMF exposure.(4) As a single factor, ELF-EMF can not promote chondrogenic differentiation of rat bone marrow derived mesenchymal stem cells. FGF-2and TGF-β3can significantly induced chondrogenic differentiation of BMSCs, this effect can be increased when exposed to ELF-EMF. |
PDF Full Text Request