Human gingival fibroblast and osteoblast behavior on groove-milled zirconia implant surfaces

  Citation: da Cruz, M.B.; Marques, J.F.; Silva, N.; Madeira, S.; Carvalho, Ó.; Silva, F.S.; Caramês, J.; Mata, A. Human Gingival Fibroblast and Osteoblast Behavior on Groove-Milled Zirconia Implant Surfaces. Materials 2022,15, 2481. https://doi.org/ 10.3390/ma15072481 Academic Editors: Paolo Cappare, Satoshi Komasa, Yoshiro Tahara, Tohru Sekino and Joji Okazaki Received: 6 February 2022 Accepted: 24 March 2022 Published: 28 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). materials Article Human Gingival Fibroblast and Osteoblast Behavior on Groove-Milled Zirconia Implant Surfaces Mariana Brito da Cruz 1,* , Joana Faria Marques 1, Neusa Silva 2, Sara Madeira 3,Óscar Carvalho 3, Filipe Samuel Silva 3, João Caramês1,4 and António Mata 1,5 1Universidade de Lisboa, Faculdade de Medicina Dentária, Unidade de Investigação em Ciências Orais e Biomédicas (UICOB), LIBPhys-FTC UID/FIS/04559/2013, Rua Professora Teresa Ambrósio, 1600-277 Lisboa, Portugal; [email protected] (J.F.M.); [email protected] (J.C.); [email protected] (A.M.) 2Universidade de Lisboa, Faculdade de Medicina Dentária, Unidade de Investigação em Ciências Orais e Biomédicas (UICOB), Rua Professora Teresa Ambrósio, 1600-277 Lisboa, Portugal; [email protected] 3Center for Microelectromechanical Systems (CMEMS), Department of Mechanical Engineering, University of Minho, 4800-058 Guimarães, Portugal; [email protected] (S.M.); oscar[email protected] (Ó.C.); [email protected] (F.S.S.) 4Universidade de Lisboa, Faculdade de Medicina Dentária, Bone Physiology Research Group, Rua Professora Teresa Ambrósio, 1600-277 Lisboa, Portugal 5Universidade de Lisboa, Faculdade de Medicina Dentária, Cochrane Portugal, Instituto de Saúde Baseada na Evidência (ISBE), Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal *Correspondence: mariana.cr[email protected]; Tel.: +351-911-042-881 Abstract: Two type of cells representing periodontal hard tissues (osteoblasts) and soft tissues (fibroblasts) were evaluated in response to microgroove-milled zirconia surfaces. A total of 90 zirconia discs were randomly assigned to four width-standardized milling microgroove-textured groups and a control group without grooves (UT). The sandblast and acid-etch protocol were applied to all samples. Both cell lines were cultured on zirconia discs from 1 day up to 14 days. Cell morphology and adhesion were evaluated after 1 day of culturing. Cell viability and proliferation of the cells were measured. Alkaline phosphatase activity, collagen I, osteopontin, interleukin 1 β and interleukin 8 secretions were assessed at predefined times. The results obtained were presented in the form of bar graphs as means and standard deviations. Multi comparisons between groups were evaluated using two-away ANOVA or Mann–Whitney tests, and a p-value < 0.05 was established. Group comparisons with regard to cell viability, proliferation and secretion of collagen I, interleukin-1 β and interleukin 8 revealed no statistically significant differences. The alkaline phosphatase activity and osteopontin secretion were significantly higher in the group with a large groove compared to the small one and the control group. Nevertheless, the viability of gingival and bone cells did not appear to be affected by the milled microgroove texture compared to the conventional sandblasted and acid-etched texture, but they seem to influence osteoblasts’ cellular differentiation. Keywords: zirconia; dental implants; milling; osteoblasts; fibroblasts 1. Introduction Since their discovery, dental implants have revolutionized implantology and have become a routine treatment in everyday dental practice. Despite the high survival rate of implant treatment [ 1 ], inflammatory conditions around osteointegrated dental implant have been reported resulting in bone and soft tissue losses [ 2 – 5 ]. The frequent occurrence of these phenomena in daily oral rehabilitation underscores the need for new materials and improved implant surfaces that can promote osseointegration and preserve bone and gum tissue around dental implants. The first material successfully used in dentistry for dental implants was titanium, but the drawbacks identified over the years, such as the release of nanoparticles, allergic Materials 2022,15, 2481. https://doi.org/10.3390/ma15072481 https://www.mdpi.com/journal/materials Materials 2022,15, 2481 2 of 15 reactions and poor aesthetic associated with a gray color, led to the search for new materials such as ceramics [ 1 , 6 – 8 ]. The recent technology advancement in dental materials led to an increase in the number of commercially available ceramic materials for clinical use, such as alumina ceramic, machinable glass ceramic, feldspathic porcelain, zirconia ceramic and others. Among them, yttria-stabilized polycrystalline tetragonal zirconia (YTZP) is the most popular because of its mechanical properties and similarity to titanium. In addition, due to its metal-free properties, coupled with its aesthetic white color and translucency like human teeth, YTZP could be the preferred material for dental implants in the future as an alternative to titanium implants [9–13]. The effectiveness of dental implants is mainly based on the bone-implant interaction [ 14 ]. However, several factors can influence this process such as the: chemical composition, surface wettability, roughness and topography of the dental implant surface [15–17]. Surface modifications of dental implants such as the one mentioned above, surface roughness, are often used on implants made of zirconia, as it has been recognized that they affect the in vitro cell response and implant integration into the bone in vivo. These micro irregularities conceived by sandblasting and acid etching (SBAE) improve the biocompatibility of the implants, increasing contact surface area and adhesion of osteogenic cells. The microroughness from 1 to 100 µ m it is obtained by blasting and acid etching resulting in the improvement of osteoblast biological behavior [13,18–20]. Therefore, the topography of the implant surface can be divided into three levels depending on its characteristics: macro (10 µ m to 1 mm), micro (1–10 µ m) and nano (1–100 nm) scale [ 15 ]. These characteristics have been mentioned in recent studies of titanium implants as having a significant impact on promoting bone ingrowth [ 21 ], but this has hardly been investigated in the case of YTZP dental implants. Overall, it has been described that microtopography is a critical determinant of human cells’ adaptation and differentiation [22], due to the production of directional physical signals in cell regulation and in the assembly and collagen matrix orientation [23]. In that regard, the standardization of implant surfaces by performing topographies on them has been described over the years [ 24 – 26 ]. Among the various types of topographies designed, microgroove topographies have been widely explored for their impact on cell alignment, as they can be generated relatively conveniently using a range of microfabrication techniques such as conventional milling. In vitro studies of osteogenic cell behavior showed that they are strongly oriented towards the direction of the grooves when compared to surfaces without texture, on which arbitrary alignment is usually noticed. However, the ideal texture and dimensions for optimal osteointegration are still being extensively researched, and most of these studies are performed on titanium implant surfaces. It is therefore important to know whether creating microgroove patterns on implants made of zirconia would be beneficial for hard and soft tissues [22,26]. Thus, the purpose of this investigation was therefore to understand whether cell behavior of osteoblasts and fibroblasts on width-standardized milling microgroove-textured zirconia surfaces is improved compared to sandblasted and acid-etched microtopography. 2. Materials and Methods 2.1. Substrates Ninety zirconia discs with 8 mm in diameter and 2 mm in thickness made of a commercially available 3 mol % yttria-stabilized zirconia powder (3Y-TZP) (3YSB-E Tosoh Corporation, Tokyo, Japan) with 40 nm in particle size and 60 µ m in average cluster size. Their chemical compounds are described in Table 1. Table 1. Description of commercial 3Y-ZTP powder chemical compounds in accordance with manufacture Tosoh (Tosoh Corporation©, Amsterdam, The Netherlands). Chemical Compounds Y2O3HfO2Al2O3SiO2Fe2O3Na2O Weight percentage (wt%) 5.2 ±0.5 <5.0 0.1~0.4 ≤0.02 ≤0.01 ≤0.04 Materials 2022,15, 2481 3 of 15 The production was based on the pressing and sintering technique. After performing the pressing process (210 MPa) in a steel mold that has 10 nm of internal diameter and 50 nm in height, all samples except for the control group (UT) were textured with widthstandardized milling microgroove defined as groups A, B, C and D, according to Table 2. Table 2. Schematic table of groove dimensions in µm obtained by SEM (N = 10). Sample Group Width (µm) Depth (µm) A 126.93 ±3.76 12.26 ±1.46 B 47.83 ±2.05 10.42 ±1.76 C 67.06 ±2.04 8.41 ±1.51 D 72.32 ±2.77 17.07 ±1.26 Once the surfaces were textured, all samples were sintered in an oven (Zirkonofen 700, South Tyrol, Italy) and then cleaned with isopropyl alcohol using ultrasonic equipment. The samples were then sandblasted for 30 s with particles of alumina (Al 2 O 3 ) with an average size of 250 µ m, 6 bars of pressure and 12 cm of distance, and washed again with ultrasonic equipment and isopropyl alcohol. After this step, each sample was immersed in hydrofluoric acid (48% HF) at room temperature for half an hour and cleaned once more with ultrasonic equipment, where it was immersed in isopropyl alcohol for 5 min, resulting in a surface roughness of 1.45 µ m. In the final step, an ultrasonic bath process was once again performed for all the samples with absolute ethanol and sterilized in the autoclave in order to carry out all biological tests (Figure 1). All the biological tests were repeated in three independents experiments. Materials 2022, 15, x FOR PEER REVIEW 3 of 15 Table 1. Description of commercial 3Y-ZTP powder chemical compounds in accordance with manufacture Tosoh (Tosoh Corporation©, Amsterdam, Netherlands). Chemical Compounds Y2O3 HfO2 Al2O3 SiO2 Fe2O3 Na2O Weight percentage (wt%) 5.2  0.5 <5.0 0.1~0.4 0.02 0.01 0.04 The production was based on the pressing and sintering technique. After performing the pressing process (210 MPa) in a steel mold that has 10 nm of internal diameter and 50 nm in height, all samples except for the control group (UT) were textured with widthstandardized milling microgroove defined as groups A, B, C and D, according to Table 2. Table 2. Schematic table of groove dimensions in μm obtained by SEM (N = 10). Sample Group Width (μm) Depth (μm) A 126.93 ± 3.76 12.26 ± 1.46 B 47.83 ± 2.05 10.42 ± 1.76 C 67.06 ± 2.04 8.41 ± 1.51 D 72.32 ± 2.77 17.07 ± 1.26 Once the surfaces were textured, all samples were sintered in an oven (Zirkonofen 700, South Tyrol Italy) and then cleaned with isopropyl alcohol using ultrasonic equipment. The samples were then sandblasted for 30 s with particles of alumina (Al2O3) with an average size of 250 μm, 6 bars of pressure and 12 cm of distance, and washed again with ultrasonic equipment and isopropyl alcohol. After this step, each sample was immersed in hydrofluoric acid (48% HF) at room temperature for half an hour and cleaned once more with ultrasonic equipment, where it was immersed in isopropyl alcohol for 5 min, resulting in a surface roughness of 1.45 μm. In the final step, an ultrasonic bath process was once again performed for all the samples with absolute ethanol and sterilized in the autoclave in order to carry out all biological tests (Figure 1). All the biological tests were repeated in three independents experiments. Figure 1. Study design detailing the methodologies used to develop this research work. The samples were divided in two groups and then biological tests were carried out, which was repeated in three independents experiments. Figure 1. Study design detailing the methodologies used to develop this research work. The samples were divided in two groups and then biological tests were carried out, which was repeated in three independents experiments. The final aspect of the samples from each group after the surface treatment was observed and evaluated by SEM JSM-6010 LV (JEOL Ltd., Tokyo, Japan). SEM JSM-6010 LV images were obtained at 500 × magnification at 10 kV acceleration voltage. Backscattering Electron Detector (BSED) images were taken at 15 kV, (Figure 2). FEG-SEM images reveal similar topographies in all textured samples. Materials 2022,15, 2481 4 of 15 Materials 2022, 15, x FOR PEER REVIEW 4 of 15 The final aspect of the samples from each group after the surface treatment was observed and evaluated by SEM JSM-6010 LV (JEOL Ltd., Tokyo, Japan). SEM JSM-6010 LV images were obtained at 500× magnification at 10 kV acceleration voltage. Backscattering Electron Detector (BSED) images were taken at 15 kV, (Figure 2). FEG-SEM images reveal similar topographies in all textured samples. Figure 2. FEG-SEM images after blasting and acid etching of all study groups. Images at 100× magnification and cross-sectional images at 100× magnification at the same 90° angle. D—depth, W— width, S—spacing. Groups A, B, C and D were textured with width-standardized milling microgroove and control group (UT) were only sandblasting and acid etching. The scale bars are 100 μm. 2.2. Cell Culture Human Gingival Fibroblasts—HGFs (HGF; Applied Biological Materials Inc., Richmond, BC, Canada) were cultured in a supplemented Dulbecco’s Modified Eagle’s Medium—DMEM (Lonza®, Switzerland) with 10% Bovine Fetal Serum (Biowest®, France) and 1% Penicillin with streptomycin (G255 Applied Biological Materials Inc., Richmond, BC, Canada). Human Fetal Osteoblasts—hFOBs 1.19 (CRL-11372TM; American Culture Collection, Manassas-ATCC®, VA, USA) were cultured in a mixture (1:1 v/v) of DMEM (Dulbecco’s modified Eagle’s Medium-DMEM) from (BiowhittakerTM, LonzaTM, Basel, Switzerland) and Ham’s F-12 Medium (Sigma-Aldrich® 51651C, St. Louis, MO, USA), in which was added 0.3 mg. ml−1 of G418 (InvivoGgen, Toulouse, France) and 10% of Bovine Fetal Serum (Biowest®, Nuaillé, France). Both cell lines were incubated at 37 °C with 5% CO2 and 98% humidity. When the cells reached approximately 80% confluence, trypsin-EDTA (Lonza, Veners, Belgium) was added to detach them, they were later centrifuged, and the pellet was resuspended in the respective medium. To perform each cell culture assay 1 × 104 cells/mL cells at a 4th passage were seeded in 48-well plates containing sterile sample (Corning, NY, USA). 2.2.1. Fibroblast and Osteoblast Cells Viability and Proliferation The viability and proliferation of both cells was assessed with—Cell-Titer Blue® reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions. After 1, 3, Figure 2. FEG-SEM images after blasting and acid etching of all study groups. Images at 100 × magnification and cross-sectional images at 100 × magnification at the same 90 ◦ angle. D—depth, W—width, S—spacing. Groups ( A – D ) were textured with width-standardized milling microgroove and control group (UT) were only sandblasting and acid etching. The scale bars are 100 µm. 2.2. Cell Culture Human Gingival Fibroblasts—HGFs (HGF; Applied Biological Materials Inc., Richmond, BC, Canada) were cultured in a supplemented Dulbecco’s Modified Eagle’s Medium—DMEM (Lonza ® , Basel, Switzerland) with 10% Bovine Fetal Serum (Biowest ® , Nuaillé, France) and 1% Penicillin with streptomycin (G255 Applied Biological Materials Inc., Richmond, BC, Canada). Human Fetal Osteoblasts—hFOBs 1.19 (CRL-11372 TM ; American Culture Collection, Manassas-ATCC ® , Manassas, VA, USA) were cultured in a mixture (1:1 v/v) of DMEM (Dulbecco’s modified Eagle’s Medium-DMEM) from (BiowhittakerTM, LonzaTM, Basel, Switzerland) and Ham’s F-12 Medium (Sigma-Aldrich ® 51651C, St. Louis, MO, USA), in which was added 0.3 mg · mL −1 of G418 (InvivoGgen, Toulouse, France) and 10% of Bovine Fetal Serum (Biowest®, Nuaillé, France). Both cell lines were incubated at 37 ◦ C with 5% CO 2 and 98% humidity. When the cells reached approximately 80% confluence, trypsin-EDTA (Lonza, Veners, Belgium) was added to detach them, they were later centrifuged, and the pellet was resuspended in the respective medium. To perform each cell culture assay 1 × 10 4 cells/mL cells at a 4th passage were seeded in 48-well plates containing sterile sample (Corning, NY, USA). 2.2.1. Fibroblast and Osteoblast Cells Viability and Proliferation The viability and proliferation of both cells was assessed with—Cell-Titer Blue ® reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions. After 1, 3, 7 and 14 days of the cells culturing, fluorescence intensity was measured in arbitrary fluorescence units (AU). The detection range used was excitation/emission wavelengths of 560/590 nm, measured with a luminescence spectrometer (PerkinElmer LS 50B, Waltham, MA, USA). N = 15 samples cultured with osteoblasts and fibroblasts were analyzed per group for the viability and proliferation of the two cell lines. Materials 2022,15, 2481 5 of 15 2.2.2. Fibroblasts and Osteoblasts Morphology To determine the cell morphology, samples with osteoblasts and fibroblasts were observed after one day of culturing. After washing, all cell samples were fixed with 1.5% glutaraldehyde and dehydrated with increasing ethanol concentrations (70%, 80%, 90% and 100%). The samples were incubated in Hexamethyldisilazane—HMDS (440191 Aldrich Chemistry, Milwaukee, WI, USA) and then covered with gold by the sputtering method (LEICA EM ACE600, Heerbrugg, Switzerland), with a 15 nm ultrathin gold-palladium film 80–20% in weight using high-resolution sputter coater (208HR Cressington Company, Watford, UK) coupled with an MTM-20 Cressington High Resolution Thickness Controller. Scanning Electron Microscopy—SEM JSM-6010 LV (JEOL Ltd., Akishima, Japan) was carried out at different magnifications (500, 2000, 5000 × ) at 10 Kv. Backscattering Electron Detector (BSED) images were taken at 15 kV. Two researchers performed the observations within the focus on morphology, spreading and early cell contact establishment with the materials. N = 3 samples cultured with osteoblasts and fibroblasts were analyzed for SEM images. 2.2.3. The Activity of Alkaline Phosphatase (ALP) The activity of ALP was evaluated on osteoblasts at 7 days of culturing with fluorometric enzymatic tests from (ab83371 ALP Assay Fluorometric, Abcam, Cambridge, UK) following the instruction by the manufacturer. To evaluate the enzyme activity, a standard curve was elaborated. Both samples and standards were evaluated at excitation/emission wavelengths of 360/440 nm utilizing a Fluorescence spectrometer (PerkinElmer LS 45, Waltham, MA, USA) The ALP values were transformed into mU/ µ L according to the regression equation from the standard curve. N = 4 samples cultured with osteoblasts were analyzed per group for ALP activity. 2.2.4. Quantification of Interleukin 1β(IL-1β) by ELISA Method IL-1ß was quantified in osteoblast cell cultures after 3 days using an ELISA kit, the Human IL-1b Chemiluminescent (LumiAB TM , San Francisco, CA, USA), according to the manufacturer‘s protocol. The samples were measured by a luminescence technique with a Victor Nivo Multimode Plate Reader (PerkinElmer ® Inc., Waltham, MA, USA). The results are obtained in absorbance units (nm) and converted into pg/mL by correlation with the linear regression equation obtained in the standard curve. To quantify IL-1 β N = 4 were analized per group 2.2.5. Quantification of Collagen I by ELISA Method The quantification of collagen in each osteoblast and fibroblast cell culture was carried out after 7 days of culturing. HumanPro-Kit Collagen I alpha 1 DuoSet Elisa ( DY6220 05 R&D Systems, Inc., Minneapolis, MN, USA) was used according to the manufacturer‘s protocol. The fluorescence intensity of all the samples was detected at excitation/emission wavelengths of 540 nm using a Multimode Plate Reader (PerkinElmer ® Inc., Waltham, MA, USA). Results were acquired in units of absorbance (nm) and transformed into pg/mL according to the standard curve. N = 4 osteoblasts and fibroblasts with culture suspension were analyzed per group to quantify collagen I. 2.2.6. Quantification of Osteopontin by ELISA Method The quantification of osteopontin was carried out using the ELISA Chemiluminescent Human Osteopontin kit (LumiAB TM , San Francisco, CA, USA) using a luminescence technique with Multimode Plate Reader (PerkinElmer ® Inc., Waltham, MA, USA ) after 3 days of osteoblast culturing. The fluorescence intensity of the samples was detected at excitation/emission wavelengths of 700 nm. Results were acquired in units of absorbance (nm) and transformed into concentration (pg/mL) based on the standard curve. N = 4 osteoblasts with culture suspension were analyzed per group to quantify osteopontin. Materials 2022,15, 2481 6 of 15 2.2.7. Quantification of Interleukin 8 by ELISA Method Interleukin 8 was quantified with the Human IL-8 Chemiluminescent ELISA Kit (Lumi AB TM , San Francisco, CA, USA) using Multimode Plate Reader (PerkinElmer ® Inc., Waltham, MA, USA), at 1 day of fibroblast culturing. Results were acquired in units of absorbance (nm) and transformed into concentrations (pg/mL) based on the standard curve. N = 4 fibroblasts with culture suspension were analyzed per group to quantify IL-8. 2.3. Statistical Evaluation The statistical evaluation was performed with IBM ® SPSS ® 24.0 for MacBook (SPSS, Chicago, IL, USA). The normality analyses were carried out for all data. Comparisons among groups in terms of viability, proliferation, ALP, interleukin 1 β , collagen I, osteopontin and interleukin 8 were based on two-way ANOVA or Mann–Whitney. Tukey’s post-hoc analysis was used to find the statistical differences between the groups, and the significance level was stablished at p-value < 0.05. The obtained data were presented in terms of mean ±standard deviation (SD). 3. Results 3.1. Fibroblast and Osteoblast Viability and Proliferation The viability and proliferation results for the two cell lines, fibroblast and osteoblast, were attained for 1, 3, 7 and 14 days of culturing (Figure 3). Materials 2022, 15, x FOR PEER REVIEW 7 of 15 Figure 3. Fibroblast and osteoblast viability and proliferation measured as means and presented as bar graphs, with error bars representing the standard deviation. They were obtained in fluorescence intensity (AUs) and proliferation ratios after up to 14 days of culturing (N = 15). The group comparisons were based on one-way ANOVA with post-hoc Tukey. 3.2. Fibroblast and Osteoblast Morphology Images obtained from FEG-SEM after 1 day of osteoblast culturing (Figure 4) showed adherent cells with similar morphologies in all groups, with the exception of the C group, in which the cells showed an elongated phenotype with larger cytoplasmatic extensions. The cell bodies appear to be distributed in the direction of the groove in all samples, and no differences in cell numbers were observed between any of the groups. In the culture with fibroblast cells (Figure 4), the images showed cell adhesion after 1 day of culturing. Fibroblasts showed a characteristic morphology in all groups, with many cell extensions and also flattened cell bodies According to the pattern of the samples, no cell distribution could be seen. Figure 3. Fibroblast and osteoblast viability and proliferation measured as means and presented as bar graphs, with error bars representing the standard deviation. They were obtained in fluorescence intensity (AUs) and proliferation ratios after up to 14 days of culturing (N = 15). The group comparisons were based on one-way ANOVA with post-hoc Tukey. The viability of osteoblasts increased over the 14 days of culturing, although without significant differences when comparing all groups (p> 0.05). Likewise, the rate of proliferation of the osteoblast cells showed greater cell growth in the first seven days of culturing. However, there are no statistically significant differences between any of the groups ( p> 0.05 ). Regarding the fibroblasts’ behavior, the groups comparison revealed Materials 2022,15, 2481 7 of 15 no statistically significant differences ( p> 0.05 ). Over the culturing time, the cell proliferation rate was higher from 3 to 7 days of fibroblast culturing, but not statistically different between groups however (p> 0.05). 3.2. Fibroblast and Osteoblast Morphology Images obtained from FEG-SEM after 1 day of osteoblast culturing (Figure 4) showed adherent cells with similar morphologies in all groups, with the exception of the C group, in which the cells showed an elongated phenotype with larger cytoplasmatic extensions. The cell bodies appear to be distributed in the direction of the groove in all samples, and no differences in cell numbers were observed between any of the groups. In the culture with fibroblast cells (Figure 4), the images showed cell adhesion after 1 day of culturing. Fibroblasts showed a characteristic morphology in all groups, with many cell extensions and also flattened cell bodies According to the pattern of the samples, no cell distribution could be seen. Materials 2022, 15, x FOR PEER REVIEW 8 of 15 Figure 4. Images taken with FEG-SEM of fibroblast and osteoblast cultured on milled microgroove zirconia implant surfaces at 1 day (N = 3) with 100×, 200× and 500× magnification, respectively. The scale bars are 50 μm and 100 μm. 3.3. ALP Activity ALP results based on the suspension of osteoblasts are shown in Figure 5. The results show the greatest ALP activity from the A group, but only with significant differences when compared to the B group (p < 0.05). When compared to all the study groups in regard to alkaline phosphatase activity, there were no statistically significant differences between all groups (p > 0.05). Figure 5. The activity of ALP was assessed on osteoblast cultures at 7 days (N = 4), presented in the form of bar graphs representing mean enzyme activity (mU/uL) and error bars representing the standard deviation. The group comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. Level of significance:  p < 0.05. Figure 4. Images taken with FEG-SEM of fibroblast and osteoblast cultured on milled microgroove zirconia implant surfaces at 1 day (N = 3) with 100 × , 200 × and 500 × magnification, respectively. The scale bars are 50 µm and 100 µm. 3.3. ALP Activity ALP results based on the suspension of osteoblasts are shown in Figure 5. The results show the greatest ALP activity from the A group, but only with significant differences when compared to the B group (p< 0.05). When compared to all the study groups in regard to alkaline phosphatase activity, there were no statistically significant differences between all groups (p> 0.05). Materials 2022,15, 2481 8 of 15 Materials 2022, 15, x FOR PEER REVIEW 8 of 15 Figure 4. Images taken with FEG-SEM of fibroblast and osteoblast cultured on milled microgroove zirconia implant surfaces at 1 day (N = 3) with 100×, 200× and 500× magnification, respectively. The scale bars are 50 μm and 100 μm. 3.3. ALP Activity ALP results based on the suspension of osteoblasts are shown in Figure 5. The results show the greatest ALP activity from the A group, but only with significant differences when compared to the B group (p < 0.05). When compared to all the study groups in regard to alkaline phosphatase activity, there were no statistically significant differences between all groups (p > 0.05). Figure 5. The activity of ALP was assessed on osteoblast cultures at 7 days (N = 4), presented in the form of bar graphs representing mean enzyme activity (mU/uL) and error bars representing the standard deviation. The group comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. Level of significance:  p < 0.05. Figure 5. The activity of ALP was assessed on osteoblast cultures at 7 days (N = 4), presented in the form of bar graphs representing mean enzyme activity (mU/uL) and error bars representing the standard deviation. The group comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. Level of significance: ∗p< 0.05. 3.4. Interleukin 1β Figure 6shows interleukin 1 β production, and the results are comparable between the groups, with no statistically significant differences among all groups (p> 0.05). Materials 2022, 15, x FOR PEER REVIEW 9 of 15 3.4. Interleukin 1β Figure 6 shows interleukin 1β production, and the results are comparable between the groups, with no statistically significant differences among all groups (p > 0.05). Figure 6. Interleukin 1β concentration (pg/mL) was assessed on osteoblast cultures at 3 days (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. Group comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. 3.5. Collagen Type I The level of collagen type I in the extracellular medium was measured after 7 days in osteoblast and fibroblast cultures (Figure 7). The results were identical between all study groups and for both cultures, with no statistically significant differences between them (p > 0.05). Figure 7. Concentration of collagen I (pg/mL) was assessed on fibroblast and osteoblast cultures at 7 days (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. The group comparisons were based on Kruskall–Wallis with Dunn’s posthoc tests. 3.6. Osteopontin The osteopontin concentration was assessed after 3 days of the osteoblast culturing (Figure 8). There was a marked increase in osteopontin production for all the samples, without statistically significant differences when all groups are compared (p > 0.05). Figure 6. Interleukin 1 β concentration (pg/mL) was assessed on osteoblast cultures at 3 days (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. Group comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. 3.5. Collagen Type I The level of collagen type I in the extracellular medium was measured after 7 days in osteoblast and fibroblast cultures (Figure 7). The results were identical between all study groups and for both cultures, with no statistically significant differences between them (p> 0.05). Materials 2022,15, 2481 9 of 15 Materials 2022, 15, x FOR PEER REVIEW 9 of 15 3.4. Interleukin 1β Figure 6 shows interleukin 1β production, and the results are comparable between the groups, with no statistically significant differences among all groups (p > 0.05). Figure 6. Interleukin 1β concentration (pg/mL) was assessed on osteoblast cultures at 3 days (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. Group comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. 3.5. Collagen Type I The level of collagen type I in the extracellular medium was measured after 7 days in osteoblast and fibroblast cultures (Figure 7). The results were identical between all study groups and for both cultures, with no statistically significant differences between them (p > 0.05). Figure 7. Concentration of collagen I (pg/mL) was assessed on fibroblast and osteoblast cultures at 7 days (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. The group comparisons were based on Kruskall–Wallis with Dunn’s posthoc tests. 3.6. Osteopontin The osteopontin concentration was assessed after 3 days of the osteoblast culturing (Figure 8). There was a marked increase in osteopontin production for all the samples, without statistically significant differences when all groups are compared (p > 0.05). Figure 7. Concentration of collagen I (pg/mL) was assessed on fibroblast and osteoblast cultures at 7 days (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. The group comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. 3.6. Osteopontin The osteopontin concentration was assessed after 3 days of the osteoblast culturing (Figure 8). There was a marked increase in osteopontin production for all the samples, without statistically significant differences when all groups are compared (p> 0.05). Materials 2022, 15, x FOR PEER REVIEW 10 of 15 Figure 8. Bar charts showing mean concentration of osteopontin (pg/mL) measured at 3 days of osteoblast culturing (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. The groups comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. Level of significance:  p < 0.05. 3.7. Interleukin 8 Fibroblast interleukin 8 production was analyzed after 1 day (Figure 9). No statistically significant differences were found after 1 day (p > 0.05). When the repeated measures were performed to compare the effect of all study groups on the concentration of interleukin 8, no statistically significant differences were found (p > 0.05). Figure 9. Mean concentration of Interleukin 8 (pg/mL) was assessed on fibroblast and at 1 day (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. The groups comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. 4. Discussion Yttria-stabilized polycrystalline tetragonal zirconia is a promising alternative to titanium, as noted in current literature reviews, based on its biocompatibility, better soft tissue performance linked to aesthetic outcomes and similar tissue response to osteointegration. [27–30] In order to improve their biological behavior, several modifications are made to the surface of zirconia implants. [5,31] In this context, this in vitro study aimed to gain a deeper understanding of the effect of milled microgroove implants made of zirconia with width-standardized milling microgroove textures on the biological behavior of human fetal osteoblasts and human gingival 7 days 0 1000 2000 3000 Fibroblasts Collagen I Collagen Level (pg/mL) A B C D UT 1 day 0 2×105 4×105 6×105 8×105 Interleukin 8 Interleukin 8 Level (pg/mL) A B C D UT Figure 8. Bar charts showing mean concentration of osteopontin (pg/mL) measured at 3 days of osteoblast culturing (N = 4), measured as means presented in the form of bar graphs, with error bars representing the standard deviation. The groups comparisons were based on Kruskall–Wallis with Dunn’s post-hoc tests. Level of significance: ∗p< 0.05. 3.7. Interleukin 8 Fibroblast interleukin 8 production was analyzed after 1 day (Figure 9). No statistically significant differences were found after 1 day (p> 0.05). When the repeated measures were performed to compare the effect of all study groups on the concentration of interleukin 8, no statistically significant differences were found (p> 0.05).