Summary Statement

O-terminated nanocrystalline diamond films proposed as bone implant coatings are promising for adhesion and growth of osteoblasts, as well as for osteogenic cell differentiation and extracellular matrix production.

Nanocrystalline diamond (NCD) films are promising materials for tissue engineering, especially for bone implants coating, due to their biocompatibility, chemical resistance and mechanical hardness. Nanostructure and morphology of the NCD films can efficiently mimic the properties of natural tissues, and thus they support the cell adhesion, proliferation and differentiation. In addition, the NCD wettability can be tailored by grafting specific atoms and functional chemical groups (e.g., oxygen, hydrogen, amine groups, etc.) which influence the adsorption and final geometry of proteins, and thus the behaviour of cultivated cells. Therefore, the NCD films are proposed as multifunctional materials for fundamental studies on the growth and adhesion of osteoblasts on bone implants, which is particularly our interest.

The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition. The quality of the grown NCD films was investigated by Raman spectroscopy, scanning electron microscopy and atomic force microscopy. In order to control the hydrophobic or hydrophilic character, the NCD film surfaces were grafted by hydrogen (H-termination) or oxygen (O-termination) atoms. The influence of surface termination on the surface wettability (wetting contact angle) was characterised by reflection goniometry using droplet of deionised water. The primary human osteoblasts and osteoblast-like Saos-2 cells were used for biological studies on H- and O-terminated NCD films. The cell adhesion and spreading was analysed by the visualisation of focal adhesion proteins (talin, paxillin) and actin fibers. Expression of markers of osteogenic cell differentiation (alkaline phosphatase, osteocalcin, collagen I) was monitored by the reverse transcription and Real-time PCR method, and also by immunostaining of expressed proteins and image analysis. The extracellular matrix production and composition, i.e. collagen content, calcium content and activity of alkaline phosphatase, were also quantified. Native type I collagen fibres were visualised by two-photon excitation microscopy and second harmonic generation imaging, together with immunostaining and fluorescence microscopy.

We found that primary human osteoblasts cultivated on the O-terminated NCD films exhibited better adhesion compared to the H-terminated NCD films. Also the expression of osteogenic cell markers such as collagen and osteocalcin was higher on the O-terminated films. The mature collagen fibers were detected in Saos-2 cells on both H- and O-terminated NCD films; however, the quantity of collagen in extracellular matrix was higher on O-terminated NCD films. The amount of calcium and alkaline phosphatase activity were also significantly higher in Saos-2 cell layers on O-terminated NCD films. In conclusion, the higher wettability of the O-terminated NCD films (contact angle < 20°) is promising for adhesion and growth of osteoblasts. Besides, the O-terminated surface also seems to support the osteogenic differentiation of the cultivated cells, production of extracellular matrix proteins and subsequent extracellular matrix mineralization.

This work was supported within the project “The Centre of Biomedical Research” (CZ.1.07/2.3.00/30.0025). This project is co-funded by the European Social Fund and the state budget of the Czech Republic. Other supports were provided by the Grant Agency of the Czech Republic (grant No. P108/11/0794).