Modulating OPG and TGF-β1 mRNA expression via bioelectrical stimulation

Abstract

Background: Bone remodeling is a lifelong process that ranges from orthodontic tooth movement/alignment to bone damage/healing, to overall bone health. Osteoprotegerin (OPG) and transforming growth factor β1 (TGF-β1) are secreted by osteoblasts and participate in bone remodeling. OPG promotes bone remineralization and stabilization prominent in post-mechanical repositioning of the teeth in the dental alveolus. TGF-β1 participates in regulatory processes to promote osteoblast and osteoclast equilibrium. In the context of orthodontic tooth movement, post-treatment fixation requires additional, exogenous, stabilization support. Recent research showcases supplementary solutions, in conjunction to standard tooth fixation techniques, such as OPG injections into gum and periodontal tissues to accelerate tooth anchorage; however, injections are prone to post-procedure complications and discomfort. This study utilizes noninvasive bioelectric stimulation (BES) to modulate OPG and TGF-β1 as a novel solution to regulate bone remineralization specifically in the context of post-orthodontic tooth movement.

Purpose: The aim of this study was to investigate a spectrum of BES parameters that would modulate OPG and TGF-β1 expression in osteoblasts.

Methods: Osteoblasts were cultured and stimulated using frequencies from 25 Hz to 3 MHz. RT-qPCR was used to quantify changes in OPG and TGFb-1 mRNA expression.

Results: OPG mRNA expression was significantly increased at frequencies above 10,000 Hz with a maximum expression increase of 332 ± 8% at 100 kHz. Conversely, OPG mRNA expression was downregulated at frequencies lower than 1000 Hz. TGF-β1 mRNA expression increased throughout all stimulation frequencies with a peak of 332 ± 72% at 250 kHz. Alizarin Red tests for calcium, indicated that mineralization of stimulated osteoblasts in vitro increased 28% after 6 weeks in culture.

Discussion: Results support the working hypothesis that OPG and TGF-β1 mRNA expression can be modulated through BES. Noninvasive BES approaches have the potential to accelerate bone remineralization by providing a novel tool to supplement the anchorage process, reduce complications, and promote patient compliance and reduce post-treatment relapse. Noninvasive BES may be applicable to other clinical applications as a novel therapeutic tool to modulate bone remodeling.

Keywords: Bone remodeling/regeneration; Electrophysiology; Gene expression; Growth factor; Mineralization in vitro; Orthodontic tooth movement; Osteoblast.

Graphical abstract

Fig. 1

Bioelectric Stimulation System. Cells were plated in each dish and cultured to 80% - 100% confluency. Once confluent, cells were stimulated using an electrode array (shown at the top of panel A), which was inverted and introduced into the 6-well dish where cells were grown. Each well received uniform stimulation via a pair of carbon electrodes positioned at opposite sides (panel B).

Fig. 2

Cell Growth Curves. Cell growth comparison between control (squares) and bioelectrically stimulated (circles) osteoblasts. Cells were counted 3 and 5 days after stimulation. No significant changes were observed in cell growth rates.

Fig. 3

Cumulative Results of OPG and TGF-β1 mRNA Expression in Osteoblasts Due to Bioelectric Stimulation. Panel A (OPG): Frequency-specific responses to BES produced two apparent regions of statistically significant influence for OPG. Low frequencies (between 100 Hz and 1000 Hz) resulted in a reduction of expression from baseline. Higher frequencies (between 10,000 Hz and 500,000 Hz) resulted in upregulation. Panel B (TGF-β1): Frequency-specific responses to BES produced two apparent regions of statistically significant influence for TGF-β1. Low frequencies (between 75 Hz and 10,000 Hz) resulted in an increase of expression from baseline. Higher frequencies (between 100,000 Hz and 1000,000 Hz) also resulted in upregulation. Left-y-axis is Log fold change due to treatment and tick marks on right-y-axis denote fold change due to treatment. Circles represent predicted values using a generalized additive model. * Indicates significantly different from no change, p < 0.05. OPG: Osteoprotegerin; TGF-β1: transforming growth factor β1.

Fig. 4

BES increases the mineralization rate of osteoblasts in culture. A) micrography of osteoblasts treated with Alizarin Red after 1 week. B. Osteoblasts treated with Alizarin Red after 4 weeks C. Coverslips from Stimulated (column S) and Control (Column C) at wells S5 and C5, we added a coverslip with cells not treated with Alizarin Red, and covered with glycerin, for optimization. D) Plot of the aggregated measurements of Alizarin absorbance at 405 nm. Notice that there is a significant increase in the amount of mineralization due to stimulation.