Gingival fibroblasts behavior on bioactive zirconia and titanium dental implant surfaces produced by a functionally graded technique

Abstract Adding a biological apatite layer to the implant surface enhances bone healing around the implant. Objective This study aimed to characterize the mechanical properties and test human gingival fibroblasts behavior in contact with Zirconia and Titanium bioactive-modified implant materials. Methodology 6 groups were considered: Titanium (Ti6Al4V), Ti6Al4V with 5% HA and 5% ßTCP, Zirconia (YTZP), YTZP with 5% HA and 5% ßTCP. For each group, we produced discs using a novel fabrication method for functionally graded materials, under adequate conditions for etching and grit-blasting to achieve equivalent surface microroughness among the samples. Surface roughness (Ra, Rz), water contact angle, shear bond strength, and Vickers hardness were performed. Human gingival fibroblasts immortalized by hTERT gene from the fourth passage, were seeded on discs for 14 days. Cell viability and proliferation were assessed using a resazurin-based method, and cellular adhesion and morphology using field emission gun scanning electron microscopy (FEG-SEM). After 3 days of culture, images of fluorescent nucleic acid stain were collected by confocal laser scanning microscopy (CLSM). Results Results were presented as mean ± standard deviation (SD). We compared groups using one-way ANOVA with Tukey’s post-hoc test, and significance level was set at p<0.05. After 14 days of culture, cell viability and proliferation were significantly higher in YTZP group than in other groups (p<0.05). Samples of YTZP-ßTCP presented significantly higher wettability (p<0.05); yet, we observed no improvement in cell behavior on this group. Fibroblast spreading and surface density were more evident on YTZP specimens. Adding calcium-phosphate bioactive did not alter the tested mechanical properties; however, Ti6Al4V material shear bond strength was statistically higher than other groups (p<0.05). Conclusion Adding bioactive materials did not improve soft-tissue cell behavior. When compared to other zirconia and titanium groups, pure zirconia surface improved adhesion, viability and proliferation of fibroblasts. Cell behavior seems to depend on surface chemical composition rather than on surface roughness.


Introduction
Titanium or titanium alloys (Ti6Al4V) presents excellent biocompatibility and mechanical properties, being the material of choice for producing dental implants. 1,2 Yet, metal-free restorations provide a viable option to meet the increasingly higher aesthetic standards. 3,4 Yttria-stabilized Zirconia (YTZP) was introduced as an alternative for titanium implants due to its favorable biological, mechanical, and aesthetic properties. 5 Clinical evidence suggests that these two materials exhibit comparable osseointegration behavior, 6,9 and soft-tissue favorable response to zirconia implants is widely reported. 10,11 Osseointegration is the core of a successful endosseous oral implant, depending on the chemical, physical, mechanical, and topographic characteristics of the surface. 1,12 Zirconia surface have been modified to increase roughness and bioactivity, improving function and cellular responses. 13,14 According to the literature, adding a biological apatite layer enhances bone healing around the implant. 1 For years, hydroxyapatite (HA) or beta tricalcium phosphates (βTCP) have been used, achieving promising outcomes. 15,16 Various methods have been employed into coating metal implants, 1 within which one of the major concerns is possibly delaminating the surface of the titanium implant and failing at the implant-coating interface. 17 Considering that, we developed a fabrication method, which combines Functionally Graded Materials (FGM) with hot pressing, to produce composite materials with the advantages of the biological bioactive calcium phosphate layer, and without the potential risk of delamination. 18,19 The FGM technique comprises the formation of gradients of chemical composition, and phases distribution or microstructure. 20 The idea was creating an implant which outer layer had a percentage of bioactive compounds integrated into the implant matrix, whereas the inner layer was composed solely by YTZP or Titanium; this would guarantee both the mechanical properties within the implant core, and the bioactive properties (in contact with the surrounding tissue) in its outside area. Titanium and zirconia materials -modified with these bioactive compounds, and using this strategy -have already been observed to enhance osteoblast activity. 21 Backscattering Electron (BSED), images were acquired at an acceleration voltage of 15 kV. 24 Shear tests were carried out with composite samples to measure the maximum stress that the material was able to support before rupturing: samples were positioned half fixed in a metallic support and half exposed -portion in which the cutting insert acted. This test was conducted in a servo hydraulic machine (Instron 8874) with a 25 kN capacity load cell and a 0.02 mm/s crosshead speed at room temperature. The maximum shear stress was determined by the ratio between maximum load and cross-section area (n=3). 22 For the vickers hardness tests, a Vickers micro-hardness tester (DuraScan, emcotest, Germany) was used on 4.9N (500g) loading for 15s; the average hardness was calculated from five indentations on each of three different groups. 21,22 Roughness was measured by determining Ra and Rz values, according to ISO 4287-1997, using a contact profilometer (Surftest SJ 201 from Mitutoyo, Japan), at a 4 mm evaluation length, a 0.8 mm cut-off wavelength, and a 0.25 mm/s scan speed (n=3).
Roughness average (Ra) was determined as average length between peaks and valleys and deviation from mean line, and the peak-to-valley roughness (Rz) as average vertical distance between the highest and the lowest peak.
Wettability was assessed by contact angle evaluation, using the drop shape analysis : a water droplet was deposited on the surface and, after stabilizing it, we determined four different measurements for each group by image processing

Statistical analysis
Shapiro-Wilk test was used for checking the normal distribution of data. For determining significant differences among groups for mechanical and biological tests, a factorial analysis of variance (one-way ANOVA) or Mann-Whitney tests were used when appropriate.
Tukey's post-hoc test was applied to identify significant differences among groups, considering p < 0.05 as significance level. Data is presented as mean ± standard deviation (SD). All statistical analyses were performed using IBM® SPSS® 24.0 statistics software for Mac (SPSS, Chicago, USA).

Mechanical Properties
As Figure 1 shows, before mechanical and biological tests, we observed all specimens using FEG-SEM.
Preliminary micrographs confirmed similar surface roughness in all study groups, resulting from surface treatment.

Discussion
This study is pioneer in characterizing soft-tissue cell behavior in contact with a new FGM-based dental implant design that uses either Zirconia or Titanium substrates embedded with bioactive calcium-phosphate   within a given sample; thus, extreme points are blended into the average, and the method is unable to discriminate them. 16,30 Surface hydrophilicity is also an important feature associated with cell response. Strong hydrophilic surfaces are deemed more appropriate for a favorable biological response, considering their enhanced affinity with biological fluids, cells, and surrounding tissues.
In titanium implant surfaces, contact angle measures ranged from 0º (hydrophilic) to 140º (hydrophobic). 28,31 However, our results showed that hydrophilicity does not affect fibroblast behavior in the same way as it does for osteoblasts: cell adhesion, viability, and proliferation were higher on pure YTZP, although YTZP-ßTCP was the most hydrophilic material. Even though, YTZP-based groups showed a higher hydrophilicity and improved cell behavior than titanium samples. We found that surface hydrophilicity may be an important characteristic for implants, but chemical composition seems more important in fibroblast cell modulation. As in other studies, we encountered some difficulties in isolating surface variables for independently studying their effects, as most of these parameters are related to cell modulation. [32][33][34] Cell behavior