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Einfluss von elektrischen Feldern auf nanotubulären TiO2-Schichten auf die osteogene Differenzierung von mesenchymalen Stammzellen

Fachliche Zuordnung Biomaterialien
Beschichtungs- und Oberflächentechnik
Zellbiologie
Förderung Förderung von 2014 bis 2021
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 257236827
 
Erstellungsjahr 2021

Zusammenfassung der Projektergebnisse

In regenerative medicine, in spite of the long history over 50 years and beneficial effects of electric fields on bone regeneration, the electric stimuli-sensing mechanism in bone is poorly understood. For bone regeneration, biocompatible TiO2 implants have been widely used in dental and orthopedic fields. Previously, we have developed vertically aligned TiO2 nanotube (NT) layers where individual tube diameters were precisely adjustable in the range of 15–150 nm using anodic self-ordering oxidation of titanium at different voltages. Further, we demonstrated that osteogenic differentiation of mesenchymal stem cells (MSC) is stimulated responding to the topographic cues from ordered TiO2 nanotube layers with distinct nanoscale geometry. Biocompatible TiO2 implant NT surfaces can be the potential candidate of substrate for electric field (EF)-guided bone regeneration. Thus, in our study funded by DFG, we proposed the combination of an EF with a nanoscale topographic cue of TiO2 nanotube surfaces to induce synergistic osteogenic stimulation of MSC. The goal of our project aimed to establish an osteo-inductive TiO2 nanotube implant system via liposomal delivery of target molecules under controlled EF stimulation. At first, we established a direction-controlled direct current (DC) EF setup and applied constant electric fields under physiologically relevant current flow level (400 mVnominal) to MSC on TiO2 nanotube surfaces. Notably, EF with a nanoscale topographic cue of TiO2 NT synergistically induced much higher osteogenic stimulation than in the absence of EF and/or NT surface topographic cue. Further, we identified, as an EF-biosensing mechanism, that EF immediately triggered long-standing intracellular calcium increase of MSC, resulting in osteogenic induction via downstream calcium signaling pathway. We further identified the EF-triggered target molecules (Ca2+, ATP and gap junction agonists) which mediate EF-triggered downstream calcium signaling and its propagation, and thus we further focused on 1) optimization of NT surface and 2) establishment of liposome-encapsulated EF-target molecule delivery in order to accelerate EF responses and minimize EF application time with help of stimulatory effects of target molecules. For the optimization of NT surface, we first developed controlled NT morphology with diameters of 15, 50, 100 and 150nm in different length scale, and evaluated the surface area and surface charge of the nanotubular surface. In addition, we improved the electrical conductivity of TiO2 nanotubular layers by an optimized thermal reducing in Ar/H2 environment. Through the optimization of NT surface, we found that spaced nanotube morphology with 150nm diameter size has remarkable advantages over classical close-packed nanotubes, in respect to available surface area and higher adsorption efficiency with target molecule distribution in depth. For establishment of liposome-encapsulated EF-target molecule delivery, we first established in vitro short-term alternating current (AC) EF system demonstrating the superior EF efficiency over DC EF on human MSC differentiation. Then, we optimized EF-guided modulation of the speed and duration in adsorption and release of liposome-encapsulating target molecules (Ca2+, ATP and fluorescent dyes) from TiO2 NT surfaces. Further, ex vivo application revealed that ATP- or ICG-encapsulating liposomes can efficiently move toward calvarial periosteal layer by EF guidance, leading to intracellular delivery of target molecules, confirmed by molecular imaging using multispectral optoacoustic tomography and by intracellular calcium imaging. Under in vitro and ex vivo established EF modality with spaced NTs, in near future, 3D spaced NT implants in preparation can be applied on in vivo large animal study. Based on our previous collaborative in vivo studies on titanium nanotube implants in minipigs with the Department of Oral and Maxillofacial Surgery (University Erlangen-Nürnberg), we have a potential to exploit large animal experiments using TiO2 spaced NT implants delivering target molecules via EF. The in vivo study by enhancement of osteo-induction via strategic advantages (nano-scale topography, EF stimulatory effect, and target molecule delivery), may give us an option to accelerate bone healing in compromised/aged patients with metabolic or chronic inflammatory diseases which hamper bone-implant osseo-integration.

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