Proliferation and Cluster Analysis of Neurons and Glial Cell Organization on Nanocolumnar TiN Substrates

Abstract

Biomaterials employed for neural stimulation, as well as brain/machine interfaces, offer great perspectives to combat neurodegenerative diseases, while application of lab-on-a-chip devices such as multielectrode arrays is a promising alternative to assess neural function in vitro. For bioelectronic monitoring, nanostructured microelectrodes are required, which exhibit an increased surface area where the detection sensitivity is not reduced by the self-impedance of the electrode. In our study, we investigated the interaction of neurons (SH-SY5Y) and glial cells (U-87 MG) with nanocolumnar titanium nitride (TiN) electrode materials in comparison to TiN with larger surface grains, gold, and indium tin oxide (ITO) substrates. Glial cells showed an enhanced proliferation on TiN materials; however, these cells spread evenly distributed over all the substrate surfaces. By contrast, neurons proliferated fastest on nanocolumnar TiN and formed large cell agglomerations. We implemented a radial autocorrelation function of cellular positions combined with various clustering algorithms. These combined analyses allowed us to quantify the largest cluster on nanocolumnar TiN; however, on ITO and gold, neurons spread more homogeneously across the substrates. As SH-SY5Y cells tend to grow in clusters under physiologic conditions, our study proves nanocolumnar TiN as a potential bioactive material candidate for the application of microelectrodes in contact with neurons. To this end, the employed K-means clustering algorithm together with radial autocorrelation analysis is a valuable tool to quantify cell-surface interaction and cell organization to evaluate biomaterials’ performance in vitro.

Adhesion of Neurons and Glial Cells with Nanocolumnar TiN Films for Brain-Machine Interfaces

“…Our results show the lowest adhesion forces on gold substrates for both neuronal and glial cells. Since bad cell adhesion can also reduce proliferation, our results go in line with our previous findings that neurons and glial cells show a reduced proliferation rate on gold compared to ITO, TiN, and nanocolumnar TiN [30]. However, reduced proliferation rates could also be partly attributed to the influence of differentiative processes on the neuron-like SH-SY5Y cells caused by the surface nanotopography of the substrate material as shown recently [43][44][45].…”

“…As we have shown previously, U-87 MG cells showed the poorest cell proliferation on gold substrates (the cell number increased on gold by a factor of 1.6 and nearly tripled on ITO, TiN, and nanocolumnar TiN with additional growth time) [30]. Thus, the available space for spreading was largest on gold.…”

“…Even though very large cells were seen on TiN nano, the number of very small cells was highest. Since the SH-SY5Y cells organized in clusters on the TiN and TiN nano surface in contrast to ITO and Au [30], cells in the center of the clusters could not be included in our evaluation because of the high density, which resulted in optical separation of adjacent cells becoming impossible. Thus, our evaluation only included cells in less dense areas, viz.…”

“…In contrast, U-87 MG cells proliferated much better on ITO, TiN, and nanocolumnar TiN substrates but still lacked a significant increase in cell size. The cells tended to form evenly distributed cell patterns instead of agglomerations and clusters on these materials [30] which would allow spreading in the empty intercellular spaces. However, we saw no significant changes in cell area, and thereby, cell adhesion for the glial cells.…”

“…A possible candidate is titanium nitride (TiN), which exhibits an increased surface area and allows the shrinking of microelectrode size without losing detection sensitivity [26][27][28][29]. By further increasing the surface area of TiN with a nanocolumnar pattern, we have previously shown that neurons and glial cells cultured on these surfaces exhibit a much better proliferation behavior, in contrast to conventional electrode materials such as gold and indium-tin-oxide (ITO) [30]. In addition to research on multielectrode arrays, the functional properties of primary cortical neurons and neuron-like cells have also been studied on promising organic [31][32][33][34], as well as inorganic [35], memristive brain-machine interfaces.…”