The effect of pulsed electromagnetic field exposure on osteoinduction of human mesenchymal stem cells cultured on nano-TiO2 surfaces

Abstract

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with TiO2 nanostructured surface might be a great option in bone tissue engineering applications.

Fig 1. PEMF exposure of hBM-MSCs grown on TiO₂ variously interferes in the expression of voltage-gated calcium channels (VGCCs).

A) Current traces were elicited applying voltage pulses 100 ms long from –40 to +70 mV. The amplitude of the current recorded in Ba 108.8 mM (left traces) decreased after the application of nifedipine, (right traces), a specific blocker of L-type VGCC. B) Current-voltage relationships of the traces in A. C) Number of MSCs growing on TiO₂ and expressing VGCCs during osteogenesis without/with PEMF exposure. Data were normalized to the number of cells grown on glass and subjected to the same treatment. D) Normalized VGCC I_max value for the same population as in C. E) Number of cells grown on TiO₂ and exposed to PEMF normalized to the unstimulated sample and plotted in relation to the growing culture medium. F) Maximal current values of cell grown on TiO₂ and exposed to PEMF normalized to the unstimulated sample and plotted in relation to the growing culture medium. G) The expression of VGCC proteins carrying the currents as the one reported in panel A was confirmed by immunolocalization in MSCs grown on TiO₂ during osteogenesis (OM) and under PEMF stimulation (OM/PEMF+). No fluorescence was observed in hBM-MSCs grown in PM s a control (data not shown).

Fig 2. Gene expression of the indicated bone-specific markers as determined by qRT-PCR.

hBM-MSCs were seeded and cultured in osteogenic medium on TiO₂ nanostructured surface with/without PEMF stimulation for 7 (A) and 28 (B) days, respectively. Statistical significance values are indicated as *** p < 0.001 and # p > 0.05.

Fig 3. CLSM images of bone proteins.

Immunolocalization of COL-I (A, B), OSC (C, D) and OPN (E, F) on cells cultured on TiO₂ surface in OM conditions for 28 days, unstimulated (A, C and E) or PEMF-stimulated (B, D and F). Arrows indicate the distribution of the immuno-stained proteins. Magnification 40X; the scale bar represents 50 μm. Nuclei (blue) were counterstained with Hoechst 33342.

Fig 4

ALP activity (A) and immunolocalization (B) of hBM-MSCs cultured for 28 days onto TiO₂ with/without PEMF stimulation. A) ALP activity determined colorimetrically, corrected for the protein content measured with the BCA Protein Assay Kit and expressed as millimoles of p-nitrophenol produced per min per mg of protein. Bars express the mean values ± SEM of results from three measurements in two separated experiments (* p < 0.05). C). Immunolocalization of ALP following incubation with rabbit anti-human ALP primary antibody and detected with goat anti-rabbit secondary antibody (Alexa flour 488). Nuclei (in blue) were counterstained with Hoechst 33342. Magnification 40X; the scale bar represents 50 μm.

Fig 5. Representative CLSM images of calcium deposits during the osteogenic differentiation after calcein staining.

Mineralized matrix regions were stained green with calcein and nuclei were stained blue with Hoechst 33342. Magnification 20X; the scale bar represents 50 μm.

Fig 6. Dot Blot of the indicated bone-specific proteins released in the osteogenic medium.

The culture medium of hBM-MSCs on TiO₂ with/without PEMF exposure was collected at day 28 and analysed as reported in the Material and Methods Section. Statistical significance values are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001.