Improving Osteoblast Response In Vitro by a Nanostructured Thin Film with Titanium Carbide and Titanium Oxides Clustered around Graphitic Carbon

Abstract

Introduction: Recently, we introduced a new deposition method, based on Ion Plating Plasma Assisted technology, to coat titanium implants with a thin but hard nanostructured layer composed of titanium carbide and titanium oxides, clustered around graphitic carbon. The nanostructured layer has a double effect: protects the bulk titanium against the harsh conditions of biological tissues and in the same time has a stimulating action on osteoblasts.

Results: The aim of this work is to describe the biological effects of this layer on osteoblasts cultured in vitro. We demonstrate that the nanostructured layer causes an overexpression of many early genes correlated to proteins involved in bone turnover and an increase in the number of surface receptors for α3β1 integrin, talin, paxillin. Analyses at single-cell level, by scanning electron microscopy, atomic force microscopy, and single cell force spectroscopy, show how the proliferation, adhesion and spreading of cells cultured on coated titanium samples are higher than on uncoated titanium ones. Finally, the chemistry of the layer induces a better formation of blood clots and a higher number of adhered platelets, compared to the uncoated cases, and these are useful features to improve the speed of implant osseointegration.

Conclusion: In summary, the nanostructured TiC film, due to its physical and chemical properties, can be used to protect the implants and to improve their acceptance by the bone.

Fig 1. Primer sequences used in Q-RT-PCR.

Fig 2. Viability, ALP and TGFβ1 production and gene expression of osteoblast cells cultured on uncoated and TiC coated titanium disks.

Panel a: Cellular viability, assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyltetrazolium bromide) method. The human primary osteoblasts (hOB) (2 x 10⁴) were cultured for 3 and 7 days on uncoated titanium (Ti) disks (light grey bars) and on disk coated with nanostructured TiC layer (dark grey bars). Formazan extracted from cultures was determined spectrophotometrically at 570 nm. Panel b: ALP activity was quantitatively determined by colorimetric assay into cell lysate and TGFβ1 was quantitatively determined by ELISA into culture medium obtained from hOB cultured on uncoated Ti and on TiC coated disks. Panel c: Gene expression of osteocalcin (OC), collagen1a2 (COL1), paxillin (PAX), integrin α3β1 (ITGA-3), Four and Half LIM domains protein (FHL1) and Runt related transcription factor 2 (RUNX-2) obtained from the mRNA extracted from Saos-2 cells cultured 3, 6 and 15 hours on uncoated Ti and on TiC coated disks and analyzed by Q-RT-PCR. Panel d: Same analysis on mRNA obtained from hOB. All the presented results are reported as relative mRNA levels and expressed as fold change compared to mRNA extracted from cells cultured on uncoated titanium disks. Results represent the mean +/- Standard Error of the mean of data obtained by five independent experiments, performed using five cell samples, and each sample was analyzed in triplicate with a standard deviation among triplicate ranging between 0,38 and 0,04. * Indicates P-value < 0.05.

Fig 3. Immunofluorescence images of integrin α3β1, talin and paxillin in Saos-2 cells and in human primary osteoblasts (hOB).

Panel a: The Saos-2 (left panels) and the hOB cells (right panels) were grown for 96 h on glass slides coated with 10.5 nm of titanium or the nanostructured TiC layer, treated with primary monoclonal antibodies against integrin α3β1 (10 μg/ml). Panel b: The cells were treated with primary monoclonal antibodies against talin (10 μg/mL). Panel c: Cells were treated with primary monoclonal antibodies against paxillin (10 μg/ml). In all cases, the treatment was followed with Alexa Fluor 568 goat anti-mouse secondary antibodies, diluted 1:500 and the nuclei were stained with DAPI. The images were collected with a magnification of 63X for ITGA and PAX and of 100x for TAL, and the bar represents 100 μm.

Fig 4. Immunofluorescence images of tubulin and actin in Saos-2 cells and in human primary osteoblasts.

Panel a: The Saos-2 (left panels) and the hOB cells (right panels) were grown for 96 h on glass slides coated with 10.5 nm of titanium or the nanostructured TiC layer, treated with primary monoclonal antibodies against tubulin (tubulin mouse monoclonal antibody 10 μg/ml) and Alexa Fluor 568 goat anti-mouse secondary antibodies, diluted 1:500. Panel b: The cells were treated with Phalloidyn Alexa Fluor 488-conjugated diluted 1:10. In all images, the nuclei were stained with DAPI. The images were collected with a magnification of 63X, and the bar represents 100 μm.

Fig 5. Adhesion of Saos2 cells on the nanostructured TiC surface.

Panel a: Optical microscopy images of cells grown for 6h on Ti (left) and TiC coated (right) glass slides: the former exhibit cells with elongated form and fewer adhesion structures compared to those on TiC coated glass slides. The optical images were obtained using a 40x objective in phase contrast mode, the scale bar represents 100 μm. Panel b: AFM images of cells grown for 6h on Ti (left) and TiC coated titanium disks (right). The cells grown on the Ti coated substrates were rod-like with few large adhesion structures, highlighted in the inset. The cells grown on the TiC coated substrates appeared to have a stronger attachment with a larger amount of smaller filopodia and lamellipodia (shown in the inset) and with a more flattened form. The scale bar represents 10 μm.

Fig 6. Investigation of cell morphology by SEM.

Panel a: SEM micrographs showing the morphology of Saos-2 cells grown for 6h and 24h on uncoated (Ti) and TiC coated (TiC) titanium disks. Panel b: similar analysis on hOB cells. The images reveal that both types of cells are richer in philopodia and lamellipodia and better adhered to the substrate when grown either for 6 or 24 hrs on the TiC coated titanium disks compared to the uncoated. In each figure, the bars represent 5 μm.

Fig 7. Adhesion strength of Saos-2 cells to the nanostructured TiC film.

Panel a: Estimation of the detachment of Saos-2 cells grown on the titanium (Ti) and TiC coated titanium disks (TiC) for 6h upon exposure to the detaching buffer. Panel b: Optical image of a cantilever used for a SCFS experiment with an osteoblast cell firmly attached to the apical area of the sensor. Panel c: Comparison of the adhesion forces between the Saos-2 cells and bare, titanium and TiC treated glass substrates. For these experiments, the cells were placed in growing medium and placed in contact with the substrates using a maximum applied force of 6 nN for 20 seconds.

Fig 8. SEM analyses of micrographs of hWB and hPRP on differently treated substrates.

Panel a: uncoated (left) and TiC coated titanium disks (right) treated with human Whole Blood (hWB) for 4 minutes. Panel b: uncoated (left) and TiC coated titanium disks (right) treated with human Platelet-Rich Plasma (hPRP) for 90 minutes. In both cases, the TiC coated titanium disks present a more efficient formation of blood clots and a higher number of adhering platelets, compared to the uncoated cases. In each figure, the bars represent 10 μm.