Osteoblastic cell interactions with nanoporous titanium surfaces

Ariganello, Marianne B (Department of Mechanical Engineering, University of Ottawa)
Gerson, Eleanor (Department of Mechanical Engineering, University of Ottawa)
Nour, Elias (Department of Mechanical Engineering, University of Ottawa)
Ribeiro, Victor (Department of Mechanical Engineering, University of Ottawa)
Singla, Aarti (Department of Mechanical Engineering, University of Ottawa)
Variola, Fabio (Departments of Mechanical Engineering and Physics)

Introduction

It is understood that the surface of a biomaterial provides unique physicochemical cues to adhering cells. Controlling the surface properties of a material at the scale at which cells function, namely the nanoscale, may provide a way to tailor cellular responses without the use of pharmaceutical agents such as growth factors. As a result, surface properties at the level of the cell are gaining increasing attention over micro- and macro-level properties. In this work, we investigated the role of two chemically generated nano-porous titanium surfaces and assessed whether the resulting nanoscale physicochemical properties favored osteoblast attachment and growth. Improved osteoblast responses are expected to translate into enhanced healing and stronger in vivo bone-biomaterial adhesion. Although orthopedic devices are currently utilizing surface texturing to improve osseointegration, most devices still require "good quality bone" in sufficient quantity to ensure success. With an aging (and increasingly morbid) global population, there exists a need to develop materials that can better integrate with bone cells to expand the inclusion criteria for dental and orthopedic devices so that more patients can benefit from these technologies. A better understanding of cellular interactions with nanotextured surfaces may allow for a more sophisticated approach to improving designs that are already macroscopically optimized.

Materials and Methods

To generate two different nanoporous surfaces, commercially pure titanium (cpTi) disks were subjected to the following treatments: (i) anodization in 0.5% HF at 40V for 30 min and (ii) etching for 2 hrs with a 50:50 mixture of 30% H2O2 and concentrated H2SO4 (Piranha solution). A JEOL high resolution field emission Scanning Electron Microscope (FE-SEM) and a WI Tec Alpha 300R Atomic Force Microscope (AFM) were exploited to characterize the 2- and 3-dimensional (e.g. micro and nanoroughess) topography of nanoporous surfaces. Untreated, mechanically polished Ti disks were used as controls, thereby creating a total of three different conditions for in vitro cultures. Two osteoblastic cell lines (mouse, MC-3T3, and human, Saos-2) were cultured on the three Ti surfaces for 48 hrs. Samples were subsequently analyzed, using a combination of immunofluorescence and SEM, for differences in cell attachment (number of nuclei), spreading (cell area), proliferation (Ki-67 staining) and focal adhesions (FA, vinculin and actin staining). Experiments were repeated in triplicate with a minimum of three samples per condition to ensure statistical significance.

Results

While anodization in 0.5% HF is well know to generate surfaces characterized by arrays of titania nanotubes,1 the experimental parameters used in this study yielded a different nanotopography which differed from those previously reported for this treatment. Specifically, the resulting surface exhibited a three-dimensional sponge-like structure characterized by an interconnected network of nanosized pores about 100 nm in diameter. Piranha treatment similarly produced a nanoporous surface however this process generated smaller nanopores (20-25 nm in diameter),2 that were homogenously distributed across the surface. AFM analysis revealed that the nanoporous surfaces generated by anodization exhibited increased microscale surface roughness compared to the chemically etched surface (see Table 1 and Figure 1) Both treated surfaces showed higher roughness values than the cpTi surface. Our initial cell results demonstrated that MC-3T3 cell attachment on both the control and Piranha-treated surfaces were similar (there were no statistic differences between cell number and number of FAs per cell, Figure 2). In contrast, compared to the cpTi control, less MC-3T3 cells attached to the sponge-like anodized surface, however cells were more spread and contained fewer FAs per cell. The human Saos-2 cells were slightly more selective for the Piranha-treated Ti surface, resulting in a higher number of attached cells after 48 hrs of culture. Similar to MC-3T3s, Saos-2 cells were also more spread on the Piranha surface compared to the control, although Saos-2 cells on both surfaces contained equivalent numbers of FAs per cell.

Discussion and Conclusion

Surface modifications at the scale of the cell’s sensing apparatus have a great potential to influence cell behavior and activity. Our data demonstrate that osteoblast cells are detecting the physiochemical differences generated by nanopatterning the titanium surface as evidenced by differential attachment, spreading and/or points of adhesion. It is important to also recognize that the increase in surface roughness on these patterned surfaces may play a more significant role in vivo, where the nano-scale roughness can enable a greater initial physical interlocking and could lead to enhanced primary stability of some implants. Future work will need to investigate more functional markers of bone activity to confirm whether these differences in morphology and attachment translate to stronger bone adherence and/or more rapid bone formation.

Table 1: Root mean scale (RMS) micro- and nano-scale surface roughness of the studied Ti surfaces

Figure 1: Representative AFM topographies of Machine Polished, Acid Etched and Anodized CpTi surfaces

Figure 2: Representative SEM images of MC-3T3 cells cultured on machine polished, acid etched and anodized cpTi surfaces, scale bar = 200 μm. Insert: 1000x magnification of anodized cpTi surface, scale bar = 200 nm

Acknowledgements

This study was made possible by support from CFI and NSERC.

References

1. Brammer, K.S.; Frandsen, C.J.; Jin, S. TiO2 nanotubes for bone regeneration, Trends Biotechnol. 2012, 30, 315-22. 2. Yi, J.H.; Bernard, C.; Variola, F.; Zalzal, S.F.; Wuest, J.D.; Rosei, F.; Nanci, A. Characterization of a bioactive nanotextured surface created by controlled chemical oxidation of titanium. Surf. Sci. 2006, 600, 4613–4621.

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