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    • Bone remodeling is a highly integrated process

    2018-10-20

    Bone remodeling is a highly integrated process of resorption by osteoclasts and formation of bone tissue by osteoblasts, which results in precisely balanced skeletal mass with renewal of the mineralized matrix (Ashton et al., 1980). Hartig et al. reported that a 16Hz EMF enhances osteoblast activity while reducing osteoclast formation, shifting the balance towards osteogenesis (Hartig et al., 2000). Sun et al. investigated the effect of a 15Hz, 1.8mT pulsed EMF (PEMF) on cell proliferation, alkaline phosphatase (ALP) activity, gene expression, and mineralization of the ECM in hBMSCs. Their osteogenic differentiation resulted in a significantly altered temporal how to find the molarity of a solution of osteogenic-related genes, including a 2.7-fold increase in expression of the key osteogenic regulatory gene RUNX2/CBFA1, compared to untreated controls (Sun et al., 2010). In addition, cell exposure to PEMF significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. Increased cell numbers were observed at late stages of osteogenic culture with this same PEMF exposure. The production of ALP, an early marker of osteogenesis, was significantly enhanced at day 7 when exposed to PEMF treatment in both basal and osteogenic cultures as compared to untreated controls. Furthermore, the expression of a key osteogenic regulatory gene RUNX2/CBFA1 and ALP, was also partially modulated by PEMF exposure, indicating that osteogenesis in hBMSCs was associated with the specific PEMF stimulation (Tsai et al., 2009). Tsai et al. reported similar results when they isolated hBMSCs from adult patients and cultured them in osteogenic medium for up to 28days. Using a PEMF stimulation of 7.5Hz, greater cell numbers were observed compared with controls (Tsai et al., 2007). The production of ALP was significantly enhanced at day 7 on both basal and osteogenic cultures as compared to untreated controls. Also the expression of early osteogenic genes RUNX2/CBFA1 and ALP was indicative of PEMF stimulation. ALP accumulation produced by the hBMSCs, along with Ca deposits reached their highest levels at day 28. EMF alone, and in combination with nanomagnetic particles (MPs), has also been used to promote the differentiation potential of hBMSCs. Kim et al. investigated the effect of both EMF and MPs on hBMSCs by treating them with 50μg/ml of Fe3O4 MPs and/or an exposure of 45Hz, 1mT intensity EMF (Kim et al., 2015). Cells were exposed to EMF twice every 8h/day for 7days. Treatment with MP, and/or then exposure to EMF did not cause cytotoxic effects. Strong expression of osteogenic markers OSTEOCALCIN, OSTEOPONTIN, and OSTEONECTIN was observed in the cells treated with MPs, EMF alone, MP alone, or a combination of MP and EMF, as compared with controls. Quantitative RT-PCR revealed that mRNA expression levels of OSTEOCALCIN, OSTEOPONTIN, OSTEONECTIN, COLLAGEN I (COL1A1), COLLAGEN III (COL3A1), BONE MORPHOGENETIC PROTEIN 2 (BMP2), BONE SIALOPROTEIN (IBSP), and RUNX2 were significantly increased in cells treated with MPs, than those exposed to EMF. Furthermore, the mRNA expression of calcium channels, CACNA1C, CACNA1E, CACNA1G and CACNA1l, was activated during osteogenic differentiation. BONE SIALOPROTEIN, BMP2, OSTEOPONTIN and OSTEONECTIN, as well as the phosphorylated extracellular signal-regulated kinase, p-ERK, were all increased in the cells treated with MPs alone, EMF alone, and MP+EMF, compared with the control group. Florescence-activated cell sorting (FACS) analysis of CD73, CD90, and CD105 showed a decrease in these hBMSC cell surface markers in the cells treated with MPs, compared with those exposed to EMF. This was also seen in the cells treated with MPs, then exposed to EMF, as compared with control. Cell mitochondrial activity among the four groups was similar, showing an increase in ALP activity. Frequencies used thus far for stimulating and enhancing osteogenesis have varied from 7.5 to 75Hz (De Mattei et al., 1999; Lohmann et al., 2000; Schwartz et al., 2008; Sun et al., 2009; Trock DH et al., 1993; Tsai et al., 2007; Tsai et al., 2009), and have revealed that not only frequency, but also the direction of the EMF makes a difference in the results. For example, hBMSCs exposed to positive (30/45Hz, 1mT) and negative (7.5Hz, 1mT) EMF for osteogenic differentiation reported increases in ALP mRNA expression. These data indicate that the effect of EMF on osteogenic differentiation is significantly dependent on the direction of the EMF exposure. It is important to point out that the effects occurring at 7.5, 15, 45, and 75Hz are harmonic waves and these pulsed patterns going from lower to higher order harmonics cause a decrease in relative energy states (Poon et al., 1995).