Electrical impulse effects on degenerative human annulus fibrosus model to reduce disc pain using micro-electrical impulse-on-a-chip

Abstract

Electrical stimulation of cells and tissues for therapeutic benefit is a well-established method. Although animal studies can emulate the complexity of an organism's physiology, lab-on-a-chip platforms provide a suitable primary model for follow-up animal studies. Thus, inexpensive and easy-to-use platforms for in vitro human cell studies are required. In the present study, we designed a micro-electrical impulse (micro-EI)-on-a-chip (micro-EI-chip), which can precisely control electron density and adjust the frequency based on a micro-EI. The micro-EI-chip can stimulate cells at various micro-EI densities (0-500 mV/mm) and frequencies (0-300 Hz), which enables multiple co-culture of different cell types with or without electrical stimulation. As a proof-of-concept study, a model involving degenerative inflamed human annulus fibrosus (hAF) cells was established in vitro and the effects of micro-EI on inflamed hAF cells were evaluated using the micro-EI-chip. Stimulation of the cells (150 mV/mm at 200 Hz) inhibited the secretion of inflammatory cytokines and downregulated the activities of extracellular matrix-modifying enzymes and matrix metalloproteinase-1. These results show that micro-EI stimulation could affect degenerative diseases based on inflammation, implicating the micro-EI-chip as being useful for basic research of electroceuticals.

Figure 1

Summary of establishment of the degenerative inflamed hAF model by treatment with pro-inflammatory soluble factors and assessment of the effects of micro-EI on inflamed hAF in a micro-EI-chip. (a) Scheme of the experimental design. (b) (1) 3D hydrogel formation between the intervertebral disc cell and macrophage channel. (2) Human THP-1 cells (non-activated macrophages) treated with PMA to stimulate differentiation of activated-macrophages in the upper channel. After 48 h, the disc cells were seeded and cultured in the IVD cells channel. (3) Disc cells were stimulated by soluble factors derived from activated macrophages, which can induce inflammatory responses on disc cells. (4) Micro-EI was applied to the inflamed disc cells for 48 h. (c) Micro-EI-chip platform designed for culturing and stimulating hAF cells. (d) Whole experimental process. Abbreviations are; Micro-EI, micro-electrical impulse; hAFs, human annulus fibrosus cells; and PMA, phorbol myristate acetate.

Figure 2

Differentiation of monocyte THP-1 cells and cytokines secretion by activated macrophage-like cells. (a) Pro-inflammatory cytokines collection process. (b) Representative images of monocyte THP-1 cells differentiated into activated macrophage-like THP-1 cells. Human monocyte THP-1 cells treated with PMA for differentiation for 48 h. (c) Production of inflammatory cytokines secreted by activated macrophage-like THP-1 cells for 96 h. (d) Characterization of the diffusion profile of applied macrophage derived-soluble factors over time. Values are mean ± SEM of five or six independent experiments. Blue corresponds to a normalized concentration of a soluble factor = 0, whereas red corresponds to 1.0. ns = no significant difference. The scale bar = 100 μm.

Figure 3

Production of inflammatory mediators and ECM enzymes on hAF co-cultured with THP-1 cells (with or without PMA). Production of (a) IL-1β, (b) TNF-α, (c) IL-8, and (d) IL-6 on hAFs induced by macrophage-like THP-1 cells. Production of ECM-modifying enzymes (e) MMP-1 and (f) TIMP-2. Values are Relative Expression (±SEM) of five or six independent experiments. Abbreviations are: ECM, extracellular matrix; hAFs, human annulus fibrosus cells; TNF-α, tumour necrosis factor-alpha, IL-1β, interleukin-1 beta; IL-6, interleukin 6; IL-8, interleukin 8; MMP-1, matrix metalloproteinase-1; and TIMP-2, tissue inhibitor of metalloproteinase-2.

Figure 4

Production of inflammatory mediators on inflamed hAFs stimulated by micro-EI, mediated via the expression and translocation NF-κB p50. (a) Expression of inflammatory mediators on hAFs stimulated with micro-EI. (b) Fluorescence image of NF-κB p50 protein levels in hAF stimulated by micro-EI at 200 Hz with 150 mV/mm. (c) Quantification of the fluorescence intensity and preferential distribution of NF-κB p50 protein levels in hAFs. Values are Relative Expression (±SEM) of five or six independent experiments. Abbreviations are: TNF-α, tumour necrosis factor-alpha, IL-1β, interleukin-1 beta; IL-6, interleukin-6; IL-8, interleukin-8; micro-EI, micro-electrical impulse; hAFs, human annulus fibrosus cells; and NF-κB, nuclear factor-kappa B.

Figure 5

Production of ECM-modifying enzymes and kinetic characteristics of inflamed hAFs stimulated by micro-EI. (a) MMP-1 activity analysed by collagen zymography. (b) Relative optical density of MMP-1 activity from zymography. (c, d) Production of MMP-1 (c) and TIMP-2 (d). (e) Production ratio of MMP-1/TIMP-2. (f) Rate of cell migration. (g) Rate of cell growth. (h) LDH cytotoxicity of micro-EI stimulation on hAF. Values are Relative Expression (±SEM) of five or six independent experiments. Abbreviations are: ECM, extracellular matrix; micro-EI, electrical impulse; hAFs, human annulus fibrosus cells; MMP-1, matrix metalloproteinase-1; TIMP-2, tissue inhibitor of metalloproteinase-2; and LDH, lactate dehydrogenase.