| BACKGROUND:Electromagnetic fields are composed of interdependent electric and magnetic fields,which are ubiquitous in nature and in the biological world and have a wide range of effects on biological processes.In the medical field,pulsed electromagnetic fields have been used to treat delayed fracture healing and nonunion for more than 40 years.Low-frequency pulsed electromagnetic field(PEMF)can be used to treat nonunion,osteoporosis and bone and joint diseases,promoting fracture healing,and is increasingly concerned and valued by its advantages of non-invasive,non-infectious,convenient treatment and small side effects.It can effectively increase bone mineral density by treating patients with osteoporosis.These findings have led to the widespread acceptance of pulsed electromagnetic fields to promote osteogenesis.Osteogenesis is a biological process with complex regulatory mechanisms,including the differentiation of precursor cells such as bone marrow mesenchymal stem cells(BMSCs)into osteoblasts and the process by which osteoblasts secrete bone matrix and mineralize to form bone tissue.Osteoblasts are direct functional cells of osteogenesis.Studies have shown that pulsed electromagnetic fields have a positive stimulating effect on the differentiation of bone marrow mesenchymal stem cells into osteoblasts,bone matrix synthesis,synthesis and secretion of growth factors.However.Adipose-derived stem cells(ASCs)possess some attractive characters for clinical application compared to BMSCs,such as abundant stem cells from lipoaspirates,faster growth,less discomfort and morbidity during surgery.ASCs can become adipocytes,osteoblasts,chondrocytes,myocytes,neurocytes,and other cell types.Thus,ASCs are a good alternative in clinical work im treatment of PEMF.However,there is still a lack of research on the role of pulsed electromagnetic fields in the osteogenesis of ASCs.OBJECTIVE:In this study,adipose-derived stem cells were used as an in vitro study model to study osteoblasts,trying to clarify the following questions:Firstly,whether PEMF can promote the proliferation of ASCs adipose-derived stem cells;Secondly,whether PEMF has an effect on the differentiation of ASCs adipose-derived stem cells into osteogenic differentiation;Thirdly,exploring what is the effect of PEMF on osteogenic differentiation of ASCs adipose-derived stem cells.MATERIAL AND METHODS:Human ASCs were divided into the control group(no PEMF exposure)and the experimental group(PEMF exposure at 2h/d).The pulse field parameters were as follows:intensity 1 mT,frequency 50 Hz.In this experimental study,we examined the effects of PEMF on promoting cell proliferation and osteogenic differentiation from several aspects:we demonstrated the proliferation and apoptosis of ASCs on the exposure of PEMF through CCK-8 cell proliferation assay.In order to prove the effect of PEMF on the osteogenic differentiation of ASCs,we set different PEMF time on stimulation for the expernmental group for 1 week,2 weeks,and 3 weeks,Detection of special markers of osteogenic differentiation(alkaline phosphatase)expression by ALP staining experiments.RNA expression and qRT-PCR were used to detect the expression of osteogenic special genes OPN(osteopontin),OCN(osteocalcin),Runx-2(one of the transcription factor Runxx family members),and Western Western blot and immunofluorescence staining assays were performed to detect the expression of their corresponding osteogenic proteins.RESULTS:Firstly,at the specific intensity(1 mT)and frequency(50 Hz)of PEMF,CCK-8 cell proliferation assay showed that PEMF can promote cell proliferation of human ASCs at an early stage.Secondly,we demonstrated that PEMF can promote the differentiation of adipose-derived stem cells into osteoblasts.In the ALP staining experiment,the expression of alkaline phosphatase in ASCs irradiated by PEMF was significantly increased in the experimental group.Bone-related gene expression increased after 2 weeks of PEMF exposure according to the results of RNA extraction and qRT-PCR detection experiments,the protein expression of OPN,OCN,and RUNX-2 also increased after a longer period(3 weeks)of PEMF treatment as determined by Western blotting and immunofluorescence staining.CONCLUSIONS:We found for the first time that PMEF has a role in stimulating cell proliferation of hASCs at an early period,subsequently promoting bone-related gene expression and inducing the expression of related proteins to stimulate osteogenic differentiation. |
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