A Novel In Vitro System for Comparative Analyses of Bone Cells and Bacteria under Electrical Stimulation

Abstract

Electrical stimulation is a promising approach to enhance bone regeneration while having potential to inhibit bacterial growth. To investigate effects of alternating electric field stimulation on both human osteoblasts and bacteria, a novel in vitro system was designed. Electric field distribution was simulated numerically and proved by experimental validation. Cells were stimulated on Ti6Al4V electrodes and in short distance to electrodes. Bacterial growth was enumerated in supernatant and on the electrode surface and biofilm formation was quantified. Electrical stimulation modulated gene expression of osteoblastic differentiation markers in a voltage-dependent manner, resulting in significantly enhanced osteocalcin mRNA synthesis rate on electrodes after stimulation with 1.4V_RMS. While collagen type I synthesis increased when stimulated with 0.2V_RMS, it decreased after stimulation with 1.4V_RMS. Only slight and infrequent influence on bacterial growth was observed following stimulations with 0.2V_RMS and 1.4V_RMS after 48 and 72 h, respectively. In summary this novel test system is applicable for extended in vitro studies concerning definition of appropriate stimulation parameters for bone cell growth and differentiation, bacterial growth suppression, and investigation of general effects of electrical stimulation.

Figure 1

(a) Confocal 3D laser scanning image of the surface roughness measurement of corundum-blasted Ti6Al4V electrodes with 20x magnification. Dashed horizontal line shows the measured path with respective roughness values above. Red bar represents 100 µm. (b) 40x magnified image of the surface of a corundum-blasted Ti6Al4V electrode. Red bar represents 50 µm.

Figure 2

(a) Three-dimensional technical drawing of stimulation system including coordinate grid and round coverslip on bottom. (b) Top view technical drawing of stimulation system with coordinates. Red dots show coordinates where measurements for validation were done. (c) Composition of the electrical stimulation chamber. (d) Triangular electrodes with contact rods separated by insulator.

Figure 3

Representative image of the set-up for validation of electric potential distribution obtained from numerical simulations within the stimulation system. Electric potentials were measured with a pick-up electrode (white frame) connected to an oscilloscope.

Figure 4

(a) Numerical simulation of electric potential in V. (b) Numerical simulation of electric displacement field norm in V/m at the bottom of stimulation system. Red dots show coordinates where measurements for validation were done.

Figure 5

(a) Live/dead staining of human osteoblasts on coverslips and titanium electrode after three days of electrical stimulation with 1.4V _RMS and under control conditions. Live cells display green fluorescence. Dead cells appear red. White bars represent 100 µm. (b) Metabolic activity of human osteoblasts on titanium electrode and coverslips after three days of electrical stimulation with 0.2V _RMS or 1.4V _RMS compared to controls. Values are presented as median and single values. n ≥ 3.

Figure 6

Relative gene expression of human osteoblasts on titanium electrode (a) and coverslips (b) after three days of electrical stimulation with 0.2V _RMS or 1.4V _RMS compared to controls. Values are presented as boxplots, while whiskers denote minimum and maximum. n ≥ 6. ^∗ p < 0.05 (Kruskal-Wallis Test followed by Dunn's Multiple Comparison Test). Col I: collagen type I, ALP: alkaline phosphatase, OC: osteocalcin.

Figure 7

Procollagen type I in supernatant of human osteoblasts cultures after three days of electrical stimulation with 0.2V _RMS or 1.4V _RMS compared to controls. Values are presented as boxplots, while whiskers denote minimum and maximum. n ≥ 5, ^∗∗ p < 0.01 (Kruskal-Wallis Test followed by Dunn's Multiple Comparison Test).

Figure 8

CFU/ml of planktonic S. epidermidis from the supernatant of controls and with 0.2V _RMS and 1.4V _RMS treated samples over 72 h. Values are presented as boxplots, while whiskers denote minimum and maximum. n ≥ 4. ^∗ p < 0.05 (Kruskal-Wallis Test followed by Dunn's Multiple Comparison Test).

Figure 9

CFU/ml of adherent S. epidermidis from the electrode surface of controls and with 0.2V _RMS and 1.4V _RMS treated samples over 72 h. Values are presented as boxplots, while whiskers denote minimum and maximum. n ≥ 4.

Figure 10

Biofilm mass quantification via crystal violet staining of S. epidermidis formed biofilm in relation to the surface area of the coverslip of controls and with 0.2V _RMS and 1.4V _RMS treated samples over 72 h. Values are presented as boxplots, while whiskers denote minimum and maximum. n ≥ 4.

Figure 11

Determined minimal inhibitory concentrations (MIC) of gentamicin (a) and levofloxacin (b) using E-Test stripes with samples of the supernatants of controls and with 0.2V _RMS and 1.4V _RMS treated samples over 72 h. Values are presented as median and single values. n ≥ 3.