In the present study, the combined effects of physical treatment (PEMF) and biochemical stimuli (osteo-induction medium) on the osteogenic differentiation of AECs were investigated. The major findings of this study are: (1) Combined application of PEMF and osteo-induction medium to AECs has stronger effects on osteogenic differentiation, than either treatment alone; (2) Activation of signaling pathways, such as BMP-2 and Wnt/β-catenin, might be important in mediating the PEMF and/or osteo-induction medium-induced osteogenic differentiation of AECs; (3) Nrf2/Keap1, master regulators of ROS, might be implicated in the PEMF and/or osteo-induction medium-induced osteogenic differentiation of AECs; (4) The expression of integrinβ1 mRNA was up-regulated in the process of osteogenic differentiation of AECs induced by PEMF and/or osteo-induction medium. Our data indicate that PEMF could play an important role in the modulation of osteogenic differentiation of AECs. These observations also establish possible links of integrinβ1 and Nrf2/Keap1 in mediating PEMF and/or osteo-induction medium-induced osteogenic differentiation of AECs.
PEMF could interfere with cellular growth, proliferation and differentiation, as recently demonstrated in osteoblasts . PEMF is also capable of regulating Ca2+ homeostasis and promoting fracture healing . There have been several studies to demonstrate the inductive effect of PEMF in the osteoblast differentiation of MSCs [11, 12, 14, 22, 23]. Tsai et al. reported that PEMF could induce early onset of osteogenic differentiation of MSCs on the basis of ALP activity and stimulate the gene expression of ALP and Runx-2 at day 7 but lower at day 10 in the process of osteogenic induction . The similar results were reported by Sun et al. who found that exposure to PEMF significantly increased ALP gene expression during the early stages of osteogenesis and enhanced mineralization near the midpoint of osteogenesis . Additionally, Song et al. revealed that PEMF could up-regulate the gene expression of Runx-2, bone sialoprotein (BSP) and osteopontin (OPN); enhance the alkaline phosphatase activity and calcium deposition in a time-dependent manner. Furthermore, MEK/ERK signaling pathway might be mediated in this process of osteogenic differences of MSCs .
The present study showed that PEMF stimulation alone could induce the expression of osteoblast markers ALP and OC at both gene and protein levels at specific time point(day 7). The osteo-induction medium was also able to induce the osteogenesis of AECs as reported in the previous literature . Moreover, combined application of PEMF and osteo-induction medium led to significant up-regulation of ALP and OC expression and promoted the obvious extracellular matrix calcification. Our findings demonstrated the synergistic effects of physical (PEMF) and biochemical stimuli (OM) on the osteogenic induction of AECs. The effect of PEMF on the osteoblast differentiation of AECs may depend on the specific parameters of PEMF, such as waveform, duration, frequency and magnetic flux, as well as different cell types [22, 24]. This may be the reason why the effects of PEMF alone on the stimulation of osteogenic differentiation of AECs were found most obvious at day 7. Therefore, further studies are necessary to determine the optical parameters of PEMF in the osteogenic differentiation of AECs.
Among the intracellular signals involved in PEMF actions, the activation of bone morphogenetic proteins (BMPs) plays important roles. Runx2, as a downstream regulator of BMP-2 signaling, is necessary for osteoblast differentiation . Wnt/β-catenin signaling is also of crucial importance for MSCs osteogensis . Our results showed that the gene expression of BMP-2, Runx2 and β-catenin were all up-regulated in the osteogenic differentiation of AECs induced by PEMF alone at the specific time point (day 7). Osteo-induction medium alone or combined with PEMF exposure could induce BMP-2, Runx2 and β-catenin gene expression especially at the early stage of osteogenic induction (day 3). Additionally, similar gene expression profiles of BMP2, Runx2 and β-catenin were observed. These results may be due to the fact that both BMP-2/Runx2 and Wnt/β-catenin signaling could play important role in activating the osteogenic induction of AECs at the early-stage, while down-regulation of these signals are required for the late-stage of osteogenesis and matrix mineralization [26, 27]. Therefore, the present observations showed that PEMF and/or osteo-induction medium-induced the osteogenic differentiation of AECs may be via activation of both BMP-2 and Wnt/β-catenin signaling.
Another pathway implicated in the PEMF action is the generation of reactive oxygen species (ROS) [28, 29]. Recent study showed that Nrf2/Keap1, master regulator of ROS generation, would be required for intestinal stem cells (ISCs) proliferation . In our study, the induction expression of Nrf2 and Keap1 were observed in the treatment of PEMF and/or osteo-induction medium, and the gene expression of Nrf2 and Keap1 exhibited similar profiles during the osteogenesis of AECs. As key regulators of ROS generation, Nrf2/Keap1 might be of potential importance during osteogenic differentiation of AECs induced by PEMF and/or the osteo-induction medium.
The mechanism involving how the cells sense and transduce physical stimulation such as PEMF into biochemical signals has remained elusive. Integrins function as one of the mechanoreceptors, which are capable of switching mechanical strain to biochemical signals [20, 30]. This process is comprised of binding to the extracellular matrix (ECM) ligands and activating the specific signaling pathways which would be involved in the mechanical-induced differentiation of cells . Kasten et al. reported that certain biological functions of MSCs would be performed under the circumstance of integrin-mediated mechanical forces . Franceschi et al. found that the application of mechanical force to osteoblasts could activate the MAPK signaling through integrin α2 and β1 . However, little is known about the role of integrins in the differentiation of AECs. The current study showed that, integrinβ1 gene expression was up-regulated in the PEMF and/or osteo-induction medium-induced osteogenic differentiation of AECs. We propose that integrinβ1 might act as a receptor, which can be inducible in response to the physical stimulation, especially to the PEMF.
Our preliminary study demonstrates the role of BMP-2, Wnt/β-catenin, Nrf2/Keap1 and integrinβ1 in the osteogenic differentiation of AECs, only from the perspective of gene expression. Even though these molecules/pathways may be critical in mediating PEMF/osteo-induction medium enabled osteogenic differentiation, it is difficult to reveal the exact mechanisms without further testing, such as pathway-specific approaches. Therefore, additional experiments are needed to explore the mechanisms on how these signalings, ROS and integrinβ1 play role in regulating the PEMF-induced osteogenic differentiation of AECs.