Osteoporosis is a disabling skeletal disorder affecting 200 million people worldwide¹. Presently, the main challenge is to develop new more effective drugs with reduced long-term side effects. Monitoring the interactions of diseased bone tissues with drugs is fundamental for osteoporosis drug development and screening. In vitro cytotoxicity assays represent the most commonly used approach for screening and validation of drug candidates in the early stage of drug discovery². Most of these assays rely on two dimensional (2D) cell culture systems which do not allow for complex three dimensional (3D) spatial interactions (i.e. cell-cell and cell-extracellular matrix (ECM) interactions) typical in living bone and crucial for accurate results in toxicological testing³. Moreover, conventional 2D cytotoxicity screenings do not provide temporal information of the cellular response to drugs. Thus, there is a need for new and complementary 3D in vitro systems that can better mimic the in vivo microenvironment of bone architecture and enable in situ monitoring of bone cell response to osteoporosis drugs in real time.

Conductive 3D substrates and scaffolds (e.g. based on metal, carbon, conductive polymers, etc.) offer the possibility for in-situ electrical sensing while providing a more in-vivo like microenvironment for real-time monitoring of cell population processes⁴. In the fabrication of 3D electrodes, limitations for high throughput, reproducibility, large-scale production and costs still remain a critical issue. Carbonaceous materials⁵, such as graphene, graphene foam, CNTs, diamond-like carbon, carbon composites and pyrolysed carbon are emerging for development of 2D and 3D electrodes. The carbon MEMS (C-MEMS) technique is a very simple and cost-effective method for electrode fabrication⁶, where a patterned polymer template is heated to temperatures above 900 °C in inert atmosphere to produce pyrolysed carbon electrodes. This process enables easy and reproducible fabrication of carbon electrodes with possibility for tailoring ad-hoc designs and sensitivities for specific applications⁷. Moreover, pyrolysed carbon electrodes exhibit a wide electrochemical potential window, chemical inertness towards a range of solvents and electrolytes, good biocompatibility, and the possibility to tune the electrical and mechanical properties of the electrodes.

In order to monitor bone cell response to drugs in real time, 2D interdigitated pyrolysed carbon electrodes (IDE) have been designed and fabricated with the C-MEMS technique, using SU-8 as photoresist to define the polymer template on a Si-based substrate (Figure 1A). The IDEs are composed of two combs, one used as working (WE) and one used as counter electrode (CE).  Three different designs have been evaluated by varying finger width and spacing, i.e. width/spacing of 5/8.4, 12/15, 30/30 µm. Electrochemical characterization has been performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Figure 1B shows cyclic voltammograms related to the 30/30 µm design performed in a solution of 1 mM ruthenium (II/III) hexamine chloride in PBS at a scan rate of 100 mV/s. For the same chips impedance spectra were recorded in PBS in the frequency range of 0.1 – 100000 Hz by applying 1 mV sinusoidal potential (Figure 1C). Analogue behaviors have been observed for the other geometrical designs.

In order to validate the electrodes for impedance-based cell assays, adhesion and proliferation of a human osteoblastic model cell line (Saos-2) was assessed in real time. First, the effect of carbon-coated surfaces (i.e. laminin, collagen, fibronectin) on Saos-2 cells was evaluated and compared with the traditionally used polystyrene (data not shown). Fibronectin coating was shown to better promote cell adhesion and proliferation (Figure 1D inset). Saos-2 cells were seeded in a concentration of 140000 cells/cm² on a fibronectin coated (20 µg/mL) carbon chip maintained at 37° C in a humidified atmosphere having 5% CO₂ and impedance spectra were acquired every hour for 48 hours. Figure 1D shows the normalized impedance (Cell Index) profile over time, providing indication on the temporal evolution of cell adhesion and proliferation until reaching confluence. These results indicate that carbon electrodes are suitable for EIS-based cellular assays using osteoblastic cells and pave the way for the development of 3D in vitro cytotoxicity models of bone tissue for osteoporosis drug screenings.

References:

1. Rachner, T. D. et. al., Lancet 2011, 377, 1276–87.

2. Yasothan, U. et al., Nat. Rev. Drug Discov. 2008, 7, 725–726.

3. Elliott, N. T. & Yuan, F. J. Pharm. Sci. 2011, 100, 59–74.

4. Spegel, C. et al., Electroanalysis, 2008, 20, 680-702.

5. McCreery, R. L. et al., Chemical reviews 2008, 108, 2646–2687.

6. Madou, M. et al., Electrichemical Society Proceedings 1997, 97, 61–69.

7. Amato, L. et al., Advanced Functional Materials, 2014, 24, 7042–7052.