Nanometer scale titanium surface texturing are detected by signaling pathways involving transient FAK and Src activations

Abstract

Background: It is known that physico/chemical alterations on biomaterial surfaces have the capability to modulate cellular behavior, affecting early tissue repair. Such surface modifications are aimed to improve early healing response and, clinically, offer the possibility to shorten the time from implant placement to functional loading. Since FAK and Src are intracellular proteins able to predict the quality of osteoblast adhesion, this study evaluated the osteoblast behavior in response to nanometer scale titanium surface texturing by monitoring FAK and Src phosphorylations.

Methodology: Four engineered titanium surfaces were used for the study: machined (M), dual acid-etched (DAA), resorbable media microblasted and acid-etched (MBAA), and acid-etch microblasted (AAMB). Surfaces were characterized by scanning electron microscopy, interferometry, atomic force microscopy, x-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy. Thereafter, those 4 samples were used to evaluate their cytotoxicity and interference on FAK and Src phosphorylations. Both Src and FAK were investigated by using specific antibody against specific phosphorylation sites.

Principal findings: The results showed that both FAK and Src activations were differently modulated as a function of titanium surfaces physico/chemical configuration and protein adsorption.

Conclusions: It can be suggested that signaling pathways involving both FAK and Src could provide biomarkers to predict osteoblast adhesion onto different surfaces.

Figure 1. SEM micrographs taken at 3 kX magnification of (top left) M, (top right) AAMB, (bottom left), MBAA, (bottom right) DAA.

Figure 2. SEM micrographs taken at 200 kX magnification of (top left) M, (top right) AAMB, (bottom left) MBAA, and (bottom right) dual acid etched.

Figure 3. Multiparametric cytotoxicity assay.

Cytotoxic effects of extracts of Titanium Discs with different treatments, as measured by mitochondrial dehydrogenase activity (XTT assay), membrane integrity (NR: Neutral Red assay) or density of cells (CVDE: Crystal Violet Dye Elution Assay), and represented as a percentage of control (tissue plastic) cell viability. *Statistically significant differences between groups (p<0.01). ** Statistically different from all other groups (p<0.001). 2% Phenol and Polystyrene were used as positive and negative controls for cytotoxicity, respectively.

Figure 4. Ti-modified surfaces promote osteoblast proliferation.

The cells were cultured on the different kind of Ti-modified surfaces in order to estimate cellular proliferation. A) Graph shows cell growth at 24 and 48 hours. Cells were cultured on different titanium discs and thereafter stained with crystal violet, as detailed in material and methods. B) Cells were cultured on Titanium surfaces and after 48 hours they were lysed in order to analyze CDK6 expression by performing western blotting approach. The results showed that Ti surfaces are able to stimulate CDK6 expression, a signaling protein known to control cell cycle progression, and which has been associated to conditions of increased proliferation of mouse osteoblasts. In graphs (A), letters mean significant differences (ANOVA with Bonferroni corrected post-test).

Figure 5. Signaling proteins during pre-osteoblast attachment on nanometer scale titanium surface texturing.

To assess signaling proteins involved in initial osteoblast response on titanium discs, we checked the phosphorylation profile of proteins related to focal adhesion components [A: Src (Y416) and B: FAK (Y925, Y397)]. In all experiments, the pre-osteoblasts cells were seeded on different Ti-surfaces or polystyrene and after 3 h the samples were collected to perform western blotting approach. Briefly, the results showed that there is a balance of phospho-proteins, at Y-residue, in response to nanoscaled Ti-surfaces. However both FAK and Src have been activated by all Ti-surfaces evaluated in this study, it was clear that MBAA provoked a bigger phosphorylation of FAK at Y397 when compared with others; the same phosphorylation profile was found to Src; C) Schematization of signaling proteins proposed in this work. Upon integrin activation e Integrin binding results in the recruitment of focal adhesion kinase (FAK), a cytoplasmic protein-tyrosine kinase. Focal adhesions are often the most prominent sites of tyrosine phosphorylation in eukaryotic cells, and FAK is one of the major tyrosine-phosphorylated proteins found at these sites. The clustered FAK molecules cross-phos- phorylate each other on a specific tyrosine (Y), thereby creating a phosphotyrosine docking site for members of the Src family of cytoplasmic tyrosine kinases. These kinases then phosphorylate FAK on additional tyrosines, creating docking sites for a variety of intracellular signaling proteins; they also phosphorylate other proteins in focal adhesions. In general, these intracellular events will culminate to cell morphological changes favoring adhesion, migration or differentiation by rearranging their cytoskeleton molecules.

Figure 6. Different Ti-based surfaces are able to promote preosteoblast differentiation.

Preosteoblasts were seeded on discs of Ti for 7 (A) and 14 days (B). Afterward, the samples were collected, and alkaline phosphatase (ALP) activity was measured using pNPP as substrate. Our data showed that all surfaces tested were able to guarantee osteoblast differentiation, cellular mechanisms expected to promote implant osseointegration. It is important to mention that the relative percentages of data were normalized from mean of polystyrene at 7 days. Significant differences were considered when p<0.05 (*, ANOVA with Bonferroni corrected post-test).

Figure 7. Protein adsorption onto different Ti-surfaces.

The Ti-discs were incubated in cell medium containing 10% bovine serum. The adsorbed proteins were extracted from titanium discs and the samples then prepared and digested with trypsin. Then, identified by LC/MS in a nanoacquity nanoUPLC system coupled toa Synapt G1 Q-TOF mass spectrometer. A) The total protein adsorption at per square centimeter; B) Relative amount of vitronectin: in this regard, it was possible to verify that MBAA's surface properties provided a better adsorption of vitronectin when it was compared with others; C) Graphical presentation of focal adhesion pathway obtained from KEGG database. Considering the role of vitronectin on osteoblast adhesion, it is possible that the impact of these nanosurfaces on cell adhesion may be mediated by protein adsorption.