Pulsed electromagnetic fields increase osteogenetic commitment of MSCs via the mTOR pathway in TNF-α mediated inflammatory conditions: an in-vitro study

Abstract

Pulsed electromagnetic fields (PEMFs) have been considered a potential treatment modality for fracture healing, however, the mechanism of their action remains unclear. Mammalian target of rapamycin (mTOR) signaling may affect osteoblast proliferation and differentiation. This study aimed to assess the osteogenic differentiation of mesenchymal stem cells (MSCs) under PEMF stimulation and the potential involvement of mTOR signaling pathway in this process. PEMFs were generated by a novel miniaturized electromagnetic device. Potential changes in the expression of mTOR pathway components, including receptors, ligands and nuclear target genes, and their correlation with osteogenic markers and transcription factors were analyzed. Involvement of the mTOR pathway in osteogenesis was also studied in the presence of proinflammatory mediators. PEMF exposure increased cell proliferation and adhesion and the osteogenic commitment of MSCs even in inflammatory conditions. Osteogenic-related genes were over-expressed following PEMF treatment. Our results confirm that PEMFs contribute to activation of the mTOR pathway via upregulation of the proteins AKT, MAPP kinase, and RRAGA, suggesting that activation of the mTOR pathway is required for PEMF-stimulated osteogenic differentiation. Our findings provide insights into how PEMFs influence osteogenic differentiation in normal and inflammatory environments.

Conflict of interest statement

O.D.- payed consultant of the Magdent ltd. Company. S.B.- Co-founder of Magdent ltd.

Figures

Figure 1

MSCs subjected to PEMF irradiation in the presence of proinflammatory cytokines for 30 days. (A) MTT proliferation assay. Results are expressed as mean ± SD of at least 3 independent experiments, *p < 0.05. (B) DNA content quantification. Results are expressed as mean ± SD of at least 3 independent experiments, *p < 0.05.

Figure 2

Morphologic analyses of MSCs. Phalloidin-labeled F-actin (red), DAPI nuclear staining (blue) and overlaid fluorescent image of immunostained cellular components (merged) for the MSCs of the control and PEMF-treated groups. After 7 days of culture, the cells were well-colonized throughout the implant surface, demonstrating a star-like shape associated with osteoblastic features. The cells were also able to spread after 7 days. PEMF irradiation resulted in a greater number of cells that were attached to the surfaces.

Figure 3

Analyses of cell adhesion properties in normal conditions (A) and in the presence of inflammation (B) were conducted by searching for the expression of molecules involved in hyaluronian synthesis (HAS1), i.e., extracellular receptor for hyaluronic acid (CD44), integrin (ITGA1, 2, 3, 4), and cadherin family cell adhesion molecules (NCAM; VCAM; PCAM). The results are reported as an increase in the gene expression value in samples of cells cultured on implants with MED device compared to the same gene expression obtained in normal conditions.

Figure 4

Real-time PCR for principal osteogenic markers, such as Runx, osteopontin, osteonectin, osteocalcin, collagen type I, wnt, foxO, ALP, BMP2, and BMP7 was performed in order to evaluate the commitment of stem cells onto an osteoblastic phenotype. The cells were cultured in the (A) presence and (B) absence of inflammatory conditions, and the variations obtained in normal implants versus implants + MED were compared.

Figure 5

Quantification of intracellular ALP activity (expressed as U/mL) in MSC exposed to PEMFs and in non-exposed MSC in the presence and absence of an inflammatory environment at 15 and 30 days. Results are expressed as mean ± SD of at least 3 independent experiments, *p 

Figure 6

The osteogenic properties of MSCs seeded in the osteogenic medium have been evaluated as their ability to produce a mineralized extracellular matrix by means the ARS test. staining on implant (A); on the medium (B) and the quantification of ARS staining (C). Results are expressed as mean ± SD of at least 3 independent experiments, **p = 0.01.

Figure 7

Gene expression of mTOR activity: (A) positive regulator, (B) negative regulator. (C) downstream effector: positive regulation, and (D) downstream effector: negative regulation.

Figure 8

Real-time PCR analysis of mTOR pathway markers. Gene expression levels of the selected markers are reported as ration of MSC coltured on active implants in presence of osteogenic medium and Rapamicin implants with passive implants in presence of osteogenic medium and Rapamicin. Results are expressed as mean ± SD of at least 3 independent experiments, **p = 0.01.

Figure 9

MSC were treated with inflammatory cytokines in the presence and absence of PEMFs. The results of the effect on inflammatory/anti-inflammatory activities of the active implants on MSC indicate a significant increase of in vitro expression of IL-10 (that exerts anti-inflammatory activity) in the presence of PEMFs generated by the MED device. Conversely, there is a reduction of expression of inflammatory cytokines, such as IL-1, in the presence of PEMFs. No significant difference in the expression of the other tested cytokines is evident.