Computational simulations of asymmetric fluxes of large molecules through gap junction channel pores.

Gap junction channels are formed out of connexin isoforms, which enable molecule and ion selective diffusion amongst neighboring cells. HeLa cells expressing distinct connexins (Cx) allow the formation of heterotypic channels, where we observed a molecular charge-independent preferential flux of large fluorescent molecules in the Cx45 to Cx43 direction. We hypothesize that the pore's shape is a significant factor along-side charge and transjunctional voltages for this asymmetric flux. To test this hypothesis, we developed a 3D computational model simulating Brownian diffusion of large molecules in a gap junction channel pore. The basic pore contour was derived from x-ray crystallographic structures of Cx43 and Cx26 and approximated using basic geometric shapes. Lucifer yellow dye molecules and cesium counter-ions were modeled as spheres using their respective Stokes radii. Our simulation results from simple diffusion and constant concentration gradient experiments showed that only charged particles yield asymmetric fluxes in heterotypic pores. While increasing the inner mouth size resulted in a near-quadratic rise in flux, the rise was asymptotic for outer mouth radii increase. Probability maps and average force per particle per pore section explain the asymmetric flux with variation in pore shape. Furthermore, the simulation results are in agreement with our in vitro experimental results with HeLa cells in Cx43-Cx45 heterotypic configurations. The presence of asymmetric fluxes can help us to understand effects of the molecular structure of the pore and predict potential differences in vivo.

Modulating OPG and TGF-ß1 mRNA expression via bioelectrical stimulation.

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 ß1 (TGF-ß1) 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-ß1 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-ß1 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-ß1 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-ß1 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-ß1 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.