ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10015-2058 |
Periodontal In Vitro Cells Response on Zirconia Implant Surfaces Textured with Milled Machining Micropores
1,2Universidade 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, Lisboa, Portugal
3Universidade de Lisboa, Faculdade de Medicina Dentária, Unidade de Investigação em Ciências Orais e Biomédicas (UICOB), Rua Professora Teresa Ambrósio, Lisboa, Portugal
4-6Department of Mechanical Engineering, Center for Microelectromechanical Systems (CMEMS), University of Minho, Guimarães, Portugal
7Universidade de Lisboa, Faculdade de Medicina Dentária, Bone Physiology Research Group, Rua Professora Teresa Ambrósio, Lisboa, Portugal
8Universidade de Lisboa, Faculdade de Medicina Dentária, Cochrane Portugal,Instituto de Saúde Baseada na Evidência (ISBE), Avenida Professor Egas Moniz, Lisboa, Portugal
Corresponding Author: Mariana B da Cruz, Universidade 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, Lisboa, Portugal, Phone: +351-911-042-881, e-mail: mariana.cruz@campus.ul.pt
ABSTRACT
Aim: The aim of this in vitro study was to investigate the influence of milled micropores created on zirconia implant surfaces with different widths, depths, and spacing on osteoblasts and fibroblasts cells response.
Materials and methods: A total of 108 zirconia disks were produced using press-and-sintering techniques and randomly assigned in five groups textured with milled micropores with different dimensions of widths, depths, and spacings. All samples including control samples were sandblasted and acid-etched (SBAE). Fibroblasts and osteoblasts were cultured on disks for up to 14 days. Morphology and cellular adhesion were observed using scanning electron microscopy (SEM). Cell viability and proliferation were assessed using CellTiter-Blue® reagent and the alkaline phosphatase (ALP) activity was evaluated using a fluorometric enzyme assay. The levels of interleukin-1β, collagen type I, interleukin-8, and osteopontin were assessed using an appropriate enzyme-linked immunosorbent assay (ELISA) kit. The data was subject to statistical analysis performed using the IBM® SPSS® 24.0 software for Mac (SPSS, Chicago, USA). Group comparisons were tested using two-way ANOVA or Mann-Whitney U test with Tukey’s multiple comparisons (Tukey’s post hoc). Results were presented as mean ± standard deviation and the significance level was set at p < 0.05.
Results: Cell viability and proliferation increase over time in all groups, in both cell lines, without significant differences between them. SEM images reveal adherent cells after 1 day of culture. The production of interleukin-1β, collagen type I, interleukin-8, and osteopontin did not show statistically significant differences, as well as the ALP activity when all groups were compared.
Conclusion: Milled micro-pore dimensions between 10 μm and 100 μm on Zirconia implant surfaces with different widths, depths, and spacings did not improve periodontal cells behavior in SBAE surfaces.
Clinical significance: The production of milled micro-pore modified Zirconia implant surfaces may help us to improve their clinical behavior.
How to cite this article: da Cruz MB, Marques JF, Silva N, et al. Periodontal In Vitro Cells Response on Zirconia Implant Surfaces Textured with Milled Machining Micropores. World J Dent 2022;13(4):307-315.
Source of support: Nil
Conflict of interest: None
Keywords: Conventional milling, Human fetal osteoblasts, Human gingival fibroblast, Micro-pore, Zirconia
INTRODUCTION
Nowadays Yttria-stabilized Polycrystalline Tetragonal Zirconia (YTZP) has become a promising candidate to replace Titanium (Ti) as a dental implant material in dentistry.1 Due to its biocompatibility, high chemical resistance and fracture toughness, reliable flexural strength, low affinity for bacterial colonization, and aesthetic white color, which can mimic the natural color of teeth with comparable osseointegration, YTZP materials are now available for dental implants. In addition, the biologically inert properties of dental implants made of zirconia have also been reported.2-6
The success of dental implants depends on several factors. Both biological and mechanical properties play a key role in its longevity.6,7 It is well known that surface modifications can improve bone healing, resulting in greater interfacial interaction between bone and implant, but this is not fully understood for Zirconia dental implants. Therefore, research into the advantages of surface modification of Zirconia dental implants has received particular interest.8,9
Scientific knowledge on implant rehabilitation has focused on developing strategies that improve the interface between zirconia dental implants and the surrounding peri-implant tissue. Different surface strategies were used: aggregation of hydroxyapatite or calcium phosphate nanolayers, sandblasting with aluminum oxide particles, etching with hydrochloric or hydrofluoric acid, micro- and nano-topographies.2,9,10
The currently most widely used strategies for modifying Zirconia implant surfaces are SBAE.11,12 To perform a SBAE implant surface, alumina (Al2O3) particles ranging size from 250–500 μm have been used to sandblast the implant surfaces, which are later chemically treated with an acid such as hydrochloric acid, nitric acid, sulfuric acid, hydrofluoric acid, and others combined solutions. Topographically, this technique provides the implant surfaces an optimal roughness which is expected to increase the contact area and promote osseointegration. In addition, the biological enhancement that this combined treatment promotes on implant surfaces has been linked to reducing the risk of implant failure.13-15 An experimental study16 has reported that an SBAE surface can affect the phenotypic in vitro expression of human osteoblast-like cells, showing a significant increase in collagen I and α2-β1 expression levels compared to the smooth surface. An alternative approach to modifying implant surfaces, which has been explored in dental implantology, is the development of geometric patterns such as micropillar array, micro-grooves, pores structures, and other patterns, that´s are also described in the literature. These geometric patterns can be produced using different manufacturing technologies, such as laser or conventional milling.4,17,18 On zirconia dental implant surfaces, micro-grooves patterns with predefined and controlled dimensions4 are better described than micro-pore patterns for which there is no knowledge of predefined and controlled pore size in the literature. Most of the studies found in the literature in this area have been performed on implant surfaces made of titanium or other materials and none of them evaluated milled micropores with different dimensions in terms of widths, depths, and spacings on the same specimen. In addition, most of them do not provide information on the roughness of the examined surface, which is known to affect the phenotypic expression of osteoblasts.12,17,19,20 As zirconia has been highlighted as a potential substitute for titanium, this study evaluating hard and soft tissues cells in contact with milled micropores modified zirconia implant surfaces would be of additional value. Thus, the aim of this in vitro study was therefore to investigate the different dimensions (width, depth, and spacing) of milled zirconia micropores implant surfaces on human fibroblasts and osteoblasts and to assess whether these surface modifications affect the cell behavior compared to the SBAE-gold standard surface.
MATERIALS AND METHODS
Production of Zirconia Specimens
Commercial yttria-stabilized zirconia powder (YTZP) with a uniform dispersion of 3 mol % yttria (Tosoh Corporation©, Amsterdam, Netherlands) was used to prepare a total of 108 Zirconia disks with 8 mm in diameter and 2 mm thickness. Hot-pressing techniques were used as previously described by Cruz et al.1 and Fernandes et al.4 and their chemical compounds are described in Table 1.
| 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 |
To achieve the proposed objectives, all samples were randomly assigned in 6 groups according to the study design (Fig. 1). All samples, except the control group SBAE, was textured by conventional milling to create pores topography with the controlled size of the width, depth, and spacing differently labeled as groups A, B, C, D, and E according to micropores dimensions of Table 2.
| Sample Group | Width (μm) | Depth (μm) | Spacing (μm) |
|---|---|---|---|
| A | 79,85 ± 4,48 | 20,48 ± 2,32 | 22,10 ± 4,12 |
| B | 47,57 ± 1,50 | 10,63 ± 0,90 | 15,98 ± 0,87 |
| C | 77,60 ± 3,35 | 13,96 ± 2,18 | 77,88 ± 2,58 |
| D | 84,14 ± 3,84 | 13,64 ± 1,19 | 84,86 ± 3,70 |
| E | 66,43 ± 2,88 | 18,32 ± 0,97 | 98,97 ± 2,78 |
Fig. 1: Schematic representation of the study design detailing the methodologies used in the preparation of this research work. Replicates 3 independent times for each experiment
Then, the samples were sintered in an oven (Zirkonofen 700, Italy) and cleaned with isopropyl alcohol. Following 30 seconds blasted with alumina particles (Al2O3) at a pressure of 6 bar and washed. Each sample was immersed in hydrofluoric acid (HF with a concentration of 48%) for 30 minutes at room temperature and then immersed in isopropyl alcohol for 5 minutes. In the end, all the samples were ultrasonically cleaned with 100% ethanol and sterilized in the autoclave to carry out all biological tests (Fig. 2). All experimental tests were realized with three replicates.
Fig. 2: Zirconia specimen’s production using hot-pressing technique, followed by texturization with conventional milling to created micropores topography with the controlled size of width (w), depth (D) and spacing (S). Human osteoblast and fibroblast were evaluated in a controlled condition
Before the biological test, the final appearance of all the tested samples was observed and evaluated by SEM JSM-6010 LV (JEOL Ltd., Japan). The images were acquired at 100 x magnification, at an acceleration voltage of 10 kV. Atomic contrast images were obtained using a backscattering electron detector (BSED), at an acceleration voltage of 15 kV (Fig. 3). The images confirmed similar micro-pore topography in all surface groups under the treatment.
Fig. 3: SEM images after blasting and acid etching of all study groups. Images at 100 x magnification and cross-sectional images at 100 x magnification at the same 90°angle
Cell Culture
Human Gingival Fibroblasts (HGF) (HGF; Applied Biological Materials Inc., Richmond, BC, Canada) was cultured in a Dulbecco’s modified Eagle’s medium–DMEM (Lonza®, Switzerland) supplemented with 10% Bovine Fetal Serum (Biowest®, France) and 1% Penicillin with streptomycin (G255 Applied Biological Materials Inc., Richmond, BC, Canada).
Human Fetal Osteoblasts hFOB 1.19 (CRL-11,372TM; American Culture Collection, Manassas–ATCC®, VA, USA) was cultured in a culture medium composed of a mixture (1:1 v/v) of Dulbecco’s modified Eagle’s medium-DMEM (BiowhittakerTM, LonzaTM, Basel, Switzerland) and Ham’s F-12 Medium (Sigma-Aldrich® 51,651C, St. Louis, Missouri, USA) supplemented with 0.3 mg. ml-1 of G418 (InvivoGgen, Toulouse, France) and 10% of Bovine Fetal Serum (Biowest®, Nuaillé, France).
Both cells’ lines were incubated at 37ºC in an atmosphere of 5% CO2 and 98% humidity. After reaching 80% confluence, they are detached using Trypsin, centrifuged at 800 rpm, and re-suspended in their respective culture media. To perform each cell culture assay, cells were seeded in 48-well plates (Corning®, United States) containing sterile disks, randomly assigned as shown in Figure 2.
Cell Viability and Proliferation
Cell viability and proliferation were evaluated using a resazurin-based viability assay—CellTiter-Blue® reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol. N = 15 of osteoblast and fibroblast cultures were analyzed per group. The conversion rate was measured as fluorescence intensity in arbitrary fluorescence units (AU) after 1, 3, 7, and 14 days of culture. Fluorescence intensity was detected at excitation/emission wavelengths of 560/590 nm using a Luminescence spectrometer (PerkinElmer LS 50B, Waltham MA, USA).
Cell Morphology
To determine cell morphology, specimens with fibroblasts and osteoblasts were observed after one day of culture. After being washed, all cell samples were fixed with 1.5% glutaraldehyde and dehydrated with increasing concentrations of ethanol (70%, 80%, 90%, and 100%). Samples were incubated in Hexamethyldisilazane—HMDS ( 440,191 Aldrich Chemistry, United States) and then covered with gold by the sputtering method (LEICA EM ACE600, Switzerland). Scanning Electron Microscope—SEM JSM-6010 LV (JEOL Ltd., Japan) were performed at different magnifications (100, 200, 500 x), at an acceleration voltage of 10 kV. Atomic contrast images were obtained using a Backscattering Electron Detector (BSED), at an acceleration voltage of 15 kV. N = 3 of SEM images were analyzed per group.
Alkaline Phosphatase (ALP) Activity
Alkaline phosphatase activity was measured at 7 and 14 days of osteoblast culture, using a fluorometric enzyme assay (ab 83,371 ALP assay Fluorometric, Abcam, Cambridge, UK) following manufacturer instructions. A standard curve was performed at each measurement to calculate enzymatic activity. Standards and samples were measured using a Fluorescence spectrometer (PerkinElmer LS 45, Waltham MA, USA) with a fluorescent intensity at excitation/emission wavelengths of 360/440 nm. N = 5 of osteoblast culture was analyzed per group.
Interleukin 1β (IL-1β)
Quantification of interleukin IL-1ß was measured in osteoblast cell cultures at 1 and 3 days of culture, using the Human IL-1b Chemiluminescent ELISA Kit (LumiAB TM, United States) and the results were measured by a luminescence technique with Victor Nivo Multimode Plate Reader (PerkinElmer® Inc., United States). N = 4 of osteoblast culture suspension was analyzed per group.
Collagen I
The levels of collagen I in each fibroblast and osteoblast cell culture were measured at 3 and 7 days of culture with HumanPro-Kit Collagen I alpha 1 Duo Set Elisa (DY6220 05 R & D Systems, Inc., USA). All samples’ fluorescence intensity was detected at excitation/emission wavelengths of 540 nm using Victor Nivo Multimode Plate Reader (PerkinElmer® Inc., United States). N = 4 of osteoblast and fibroblast culture suspension was analyzed per group.
Interleukin 8
Interleukin 8 quantification was performed at 1 and 3 days of fibroblast culture with Human IL-8 Chemiluminescent ELISA kit (Lumi AB TM, United States) by a luminescence technique with Victor Nivo Multimode Plate Reader (PerkinElmer® Inc., United States). N = 4 of fibroblast culture suspension was analyzed per group.
Osteopontin
Human Osteopontin Chemiluminescent ELISA kit (LumiAB TM, United States) was used to quantify osteopontin by a luminescence technique with Victor Nivo Multimode Plate Reader (PerkinElmer® Inc., United States) at 3 and 7 days of osteoblasts culture. Samples fluorescence intensity was detected at excitation/emission wavelengths of 700 nm. N = 4 of osteoblast culture suspension was analyzed per group.
Statistical Analysis
Statistical analyzes were performed using IBM® SPSS® 24.0 software for Mac (SPSS, Chicago, USA). The data were tested for normality (Kolmogorov-Smirnov test). Comparison between groups to assess different dimensions of widths, depths and spacings were performed using two-way analysis of variance (two-way ANOVA) or Mann-Whitney U test with Tukey’s multiple comparisons (Tukey’s post hoc). All data are presented as mean ± standard deviation (SD) and the level of significance was set at p < 0.05.
RESULTS
Cell Viability and Proliferation
Cell viability and proliferation results for fibroblast and osteoblast were obtained after 1, 3, 7, and 14 days, as shown in Figure 4. Fibroblast and osteoblast cell behavior revealed cell growth over time in all groups, without statistically significant differences between them at any time of culture (p > 0.05). Similar cell proliferation of fibroblast was found for all groups, with no statistically significant differences between them (p > 0.05). As for the osteoblast cell proliferation, in the 1st and 2nd week of culture, it was similar in all groups as well as over time, without statistically significant differences between all groups (p > 0.05).
Figs 4A to D: Bar graphs representing the viability of fibroblasts (A) and osteoblasts (B) and osteoblast proliferation ratios (C) expressed in values of fluorescence intensity (AU) as mean and standard deviation; fibroblasts (D) calculated as the average of the fluorescence intensity results, obtained between 7 days /1 day, 14 days / 7 days and 14 days/1 day (N = 15). A repeated-measures ANOVA was performed, with Tukey’s post hoc test, for comparison between study groups. Error bars represent standard deviation
Cell Morphology
Figure 5 shows FEG-SEM images of adherent fibroblast (A) and osteoblast (B) cells after 1-day culture. The cells found, had similar morphology according to the cell’s lines. However, Group B and SBAE groups presented more numerous flattened cell bodies and cell extensions. Additionally, the distribution of adhesion of fibroblast cell bodies seems to reveal greater adhesion outside the pores. On the other hand, osteoblast cell morphology reveals to be less elongated than the typical morphology of these cells, with less spreading of their cell bodies. Milled micro-pore groups seemed to reveal a higher concentration of osteoblast cell distribution in the areas between the pores than inside when compared to SBAE group with more regular and homogeneous cell distribution.
Figs 5A and B: (A) SEM images with fibroblasts cultured on the surfaces of specimens from all groups under study at 1 day of culture at 100 x, 200 x, and 500 x magnification; (B) SEM images with osteoblasts cultured on the surfaces of specimens from all study groups at 1 day of culture with a magnification of 100 x, 200 x, and 500 x (N = 3)
Biological Markers
Osteoblast Markers: Figure 6A
Figs 6A and B: Bar graphs representing osteoblasts (A) and fibroblasts biomarkers results; (B) (A): alkaline phosphatase activity in (mU/μL) in osteoblast cultures in all study groups at 7 and 14 days respectively, presented as mean and standard deviation (N = 5). Collagen I concentration was obtained in (pg/ml) in the culture in all groups under study at 3 days and 7 days (N = 4). The concentration of 1ß interleukin (pg/ml) in the culture of osteoblasts in all groups under study at 1 day and 3 days (N = 4). Osteopontin concentration was obtained in (pg/ml) in all study groups at 3 days and 7 days (N = 4). A repeated-measures ANOVA was performed, with Tukey’s post hoc test, for comparison between study groups. Error bars represent standard deviation. (B): collagen I concentration obtained in (pg/ml) in the culture in all groups under study at 3 days and 7 days (N = 4). The concentration of interleukin 8 (pg/ml) in the culture (E) in all groups under study at 1 day and 3 days (N = 4). A repeated-measures ANOVA was performed, with Tukey’s post hoc test, for comparison between study groups. Error bars represent standard deviation
Alkaline phosphatase activity
ALP activity was tested on osteoblast cell suspension at 7 and 14 days of cultures. The results were very similar for all tested groups at 7 days of culture. Therefore, the SBAE group was found to be superior, but only statistically significant when compared to the B group (p < 0.05). At 14 days of culture, the C group proved to be superior to the other groups, without statistically significant differences (p > 0.05).
Interleukin 1β
Similar results of interleukin 1ß were obtained at 1 and 3 days of osteoblast culture and, when all groups were compared, no statistically significant differences were found between them (p > 0.05).
Collagen I
The results were very similar between the groups and no statistically significant differences were observed in the levels of collagen I produced at 3 and 7 days of culture between all groups presented (p > 0.05).
Osteopontin
In most groups, an increase in the concentration of osteopontin was observed from 3–7 days of osteoblast culture. However, in each culture period, when all groups were compared, there were no statistically significant differences between them (p > 0.05).
Fibroblast Markers: Figure 6B
Collagen I
Similar results were found between the groups and no statistically significant differences were observed in the levels of collagen I produced at 3 and 7 days of culture between all groups presented (p > 0.05).
Interleukin 8
The concentration of interleukin 8 determined in the fibroblast culture decreased in all groups from 1–3 days. However, when all groups were compared to each other, there were no statistically significant differences at each time (p > 0.05).
DISCUSSION
In recent years, Zirconia has emerged as a versatile dental material with unique properties that can overcome Titanium implant surfaces.21,22 Implant surface modifications have gathered special attention due to their improvement in the cell’s biological behavior. However, most of the studies in the literature have mainly focused on titanium surface modifications.23-25
Thus, in this in vitro study, milled micropores Zirconia implant surfaces with different dimensions of width, depth, and spacing were extensively explored to improve human osteoblast and fibroblast biological behavior when compared to SBAE-gold standard surface.
Surface patterns from 10 μm to 1 mm have been little studied on zirconia surfaces and most invitro studies contradict each other as to the best shapes and dimensions of these micropatterns.21 In order to minimize the gap observed in most of the studies found in the literature, each tested structure’s pores’ dimensions were determined, allowing a detailed assessment of all modification parameters generated on the zirconia implant surfaces under study.26
Prior to samples preparation, a preliminary analysis was performed to determine the real diameter of the texturing drill. The initially programed dimensions showed dispersion in the different parameters of width, depth, and spacing. It should be noted that these patterns were made with milling cutters, which over time show wear that can influence the type of pattern created. This is rarely mentioned in the literature, but it is a limitation that can affect the comparison of surface topographies.27,28 In addition, the processing of Zirconia leads to a distortion of the structure due to the high-temperature sintering, and it has been found that changing the sintering parameters changes the microstructural, mechanical, and optical properties of Zirconia.29
Nevertheless, we were able to evaluate the generated patterns and estimate their actual effect on cell behavior. However, when comparing the three parameters (width, depth, and spacing) of the micro-pore between the groups examined, the results were statistically different, width from 47–84 μm, depth from 10–20 μm, and spacing from 15–98 μm, what limited the assessment of the influence of each parameter separately.
According to the findings in the literature, surface topography increases cell adhesion and regulates the growth and organization of tissues.8,11,12 However, cell behavior can vary according to cell lines23 and there is no consensus as to which physical topographical measurements are reflected in a surface with relevant biomodelling properties.22,30 According to the results obtained, it should be noted that all milled micro-pore groups show a higher concentration of cell distribution of fibroblasts and osteoblasts. Most cells, however, were found in the areas between the pores than inside, as opposed to what was observed on the control surface only SBAE, in which there is a more even and homogeneous distribution of cells. Similar results were reported by Torres-Sanchez et al. when they examined the unimodal pore size range of Titanium scaffolds and found a higher concentration of cells in pore sizes less than 212 μm with cells spanned across these pores without colonizing them.19 On the other hand, Murphy et al. found different results when they report higher cell adhesion in collagen-glycosaminoglycan scaffolds with a larger mean pore size of 325 μm.17 However, these studies were carried out on implant surfaces made of different materials and with different manufacturing techniques. Besides that, none of these studies provide information on the dimensions of the pores spacing and depths as well as the roughness of the surfaces tested.
In addition to the milled micro-pore pattern created on the Zirconia implant surface with different widths, depths, and spacings, a microtopography was also created by sandblasting in combination with acid etching. Sandblasting and acid etching are the most widely used method for Zirconia implant surface modification, which is fundamental to modeling cell behavior and one of the most relevant approaches to promoting the early osseointegration process.31,32 However, there is a lack of information about the in vitro response of fibroblasts and osteoblasts to modified Zirconia implant surfaces, with different dimensions of width, depth, and spacing.
The evaluation of cellular viability and proliferation was then carried out, but there does not appear to be any influence of micropores with different parameters of width, spacing, and depth on the cellular behavior of osteoblasts and fibroblasts. These results are consistent with an in vitro study by Holthaus et al. from 2012 that reported that the depth of the microchannel created by micro modeling on ceramic surfaces had virtually no significant impact on the behavior of the osteoblasts.12 An in vitro study by Stangl et al. in 2001 demonstrated that a pore geometry of 25–200 μm seems to have a positive influence on osteoblast cell behavior with increased production of ALP compared with pores of 25–500 μm. However, the spacing and shape of the textures presented differ between the groups examined, and the authors do not disclose the roughness of the surfaces examined either.20 Contrary, all samples in our study were performed with SBAE, ensuring similar roughness between all micropores surfaces and control.
The biological process behind new bone formation is a complex cascade of events that involves an interaction between signaling molecules such as cytokines with the peri-implant space, resulting in the recruitment of mesenchymal stem cells, differentiation, and maturation of osteoblast cells.16,33 In this study we also analyzed the effect of milled micro-pore with different parameters of width, depth, and spacing on the expression of ALP, IL-1β, Collagen I, IL-8 and Osteopontin. ALP, IL-1β, Collagen I, and Osteopontin are major signaling molecules of hard tissue and have been used to assess osteoblast phenotype development. As described in the literature, ALP is a marker of mid-late differentiation19 and plays an important role in the mineralization of the extracellular matrix, which is mainly formed by collagen.32 Osteopontin is a non collagenous protein that has been suggested to regulate many physiological processes such as collagen organization, and it also interacts with several molecules that are present in the bone matrix.34 For all these markers to access the phenotype of the osteoblast on the milled micro-pore with different width, depth, and spacing, their expression levels showed no significant values compared to the SBAE group, apart from ALP after 7 days of culture. Significant levels of expression of ALP were found in the SBAE group compared to the B group, this could indicate that the B group was at an early stage of maturation due to its smaller pore size (47 μm). This finding is in line with an in vitro study that investigated a suitable micro-pore structure of physical modification for osteoblast adhesion, proliferation, and differentiation, and found that the osteoblast gene expression pattern was downregulated as the pore size increased from 300–800 μm.35 Furthermore, Teixeira and co-workers examined the influence of pore size on osteoblast phenotype expression in cultures grown on porous titanium and they found that a smaller pore size (62 μm) indicates higher gene expression.32 Both studies were carried out on titanium implant surfaces with different technologies of production and none of them describe the different dimensions of the pores, such as depth and spacing. Collagen I and IL-8 were assessed as a major marker of inflammation in the human gingival fibroblast. According to the literature, IL-8 is produced by several cell types including gingival fibroblasts in response to markers of inflammation such as IL-1β.36 All of the fibroblast markers evaluated in this study do not seem to be influenced by the different widths, depths, and spacings of the milled micro-pore compared to the SBAE groups. These results were also described by Barbeck et al. in an in vitro study, which they examined the cytokine expression patterns of monocytes or macrophages based on the different physical properties, pore sizes from 0.1–500 μm and they found that the expression levels did not change with the pore size created on the implant surfaces.37 However, the study was carried out with different cell types on pure-phaseβ - tricalcium phosphate (β-TCP) and there are no data on the roughness of the surface, or the dimensions of parameters such as depth of the pores created. When comparing the results of the two cells lines in this present study, it is noteworthy that, as expected, fibroblasts have much higher cell adhesion, growth, and proliferation than osteoblasts, which is characteristic of the type of tissue they mimic-gingival tissue and bone tissue, respectively. Because of these characteristics, it can be much more difficult to identify differences in their cell behavior. Regarding cell markers, neither of these two cell lines showed a better biological behavior.
The study presents as an asset in the zirconia implant surfaces scientific area. Nowadays implant materials research area it is based on surface modifications that improve clinical performance not only for osseointegration phenomena but to soft tissue management. It is crucial to evaluate cellular behavior on these zirconia surface modifications by the creation of micro-pore patterns with different widths, depths, and spacings by conventional milling at both levels of human periodontal tissue: osteoblasts that influence osseointegration process and fibroblasts soft tissue adhesion and stability. So, our study allows evaluating these two cellular lineages’ behavior on standardized micro topographies zirconia surface modifications, contrary to most existing studies that evaluate on titanium surfaces. These surface modifications improve in vitro cellular behavior and implant performance, in the future, they can be extrapolated to the clinical setting and asset to the long-term maintenance of dental implant and prosthetic rehabilitation.
Although the significance of the present study, is as an in vitro study, the main limitation of this project is based on the difficulty of immediate extrapolation to clinical practice without carrying out more in vitro and animal studies. Moreover, the results presented must be complementary with a bacteriological evaluation, since any implant surface designed, considering the state and art of osseointegration phenomena, only become innovative when it models and influences adhesion and proliferation bacterial behavior.
CONCLUSION
Micro-pore patterns with different widths, depths, and spacings produced by conventional milling on zirconia implant surface may not influence the human osteoblast and fibroblast cell responses when compared to SBAE-gold standard surface. As this is an in vitro study, despite allowing the evaluation of the cellular behavior of the two types of cell cultures that are fundamental for the process of osseointegration and soft tissue regeneration, knowing the cellular behavior in vivo, can reveal itself to be very different at the same time. To validate these results, it is essential to carry out animal studies as well as bacterial adhesion and biofilm formation, considered one of the main current causes of implant failure in the long term.
ACKNOWLEDGMENT
This work was supported by FCT project POCI–01–0145–FEDER–030,498–Portugal, by FEDER funds through the COMPETE2020–Programa Operacional Competitividade e Internacionalizacçaão (POCI).
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