43 minute read
Pre-Sintering Airborne Particle Abrasion Improves Surface and Biological Properties of Zirconia
AUTHORS
Tanjira Kueakulkangwanphol, DDS, is a postgraduate student in the Master of Science program in dentistry (prosthodontics) at Faculty of Dentistry, Thammasat University in Pathumthani, Thailand. Conflict of Interest Disclosure: None reported.
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Nichamon Chaianant, DDS, PhD, is a lecturer in oral epidemiology and statistics at Faculty of Dentistry, Thammasat University in Pathumthani, Thailand. Conflict of Interest Disclosure: None reported.
Yanee Tantilertanant, DDS, PhD, is a clinical lecturer in the department of operative dentistry at Faculty of Dentistry, Chulalongkorn University in Bangkok. Conflict of Interest Disclosure: None reported.
Weerachai Singhatanadgit, DDS, PhD, is an associate professor in oral biology at Faculty of Dentistry, Thammasat University in Bangkok. Conflict of Interest Disclosure: None reported.
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ABSTRACT
Background: Surface topography of a zirconia dental implant has a major impact on osseointegration and clinical success. Modification of a fully sintered zirconia surface can be problematic due to its high strength and bio-inert surface.
Methods: The present study therefore aimed to investigate the effect of airborne-particle abrasion of presintered zirconia on surface properties and biological performance of the resulting fully sintered zirconia.
Results: The results showed that surface topographies of fully sintered zirconia were influenced by abrasive particle size and blasting time used in repeated pre-sintering airborne particle abrasion. Well-optimized pre-sintering airborne-particle abrasion of zirconia surface could render a highly roughened and hydrophilic surface with increased fibronectin-to-albumin adsorption ratio and mesenchymal stem cell adhesion.
Practical implications: This technique may be suitable for modifying zirconia surface to facilitate osseointegration of zirconia-based dental implants.
Keyword: Pre-sintering airborne-particle abrasion, zirconia, surface roughness, surface hydrophilic, mesenchymal stem cells
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Dental implants provide aesthetic and functional tooth replacement due to their high survival rate and success rate. [1] The demand for use of implants has increased. A titanium dental implant is reliable for tooth replacement supported by numerous studies with long-term clinical success. [2–4] However, its natural gray color is an important concern in the aesthetic zone where the titanium color can show through thin peri-implant tissues. [5] Furthermore, titanium may cause metal allergy and metal toxicity produced by corrosion of titanium as reported in multiple titanium implant cases. [6–12]
To solve the aesthetic and metal particle problems, new materials should be tooth colored and nonmetallic materials such as ceramics. Zirconia, namely 3-mol% yttrium oxide-stabilized zirconia (3Y-TZP), has widely been introduced for dental implants. [13] In addition to good biocompatibility, zirconia has very high flexural strength and fracture toughness. Compared to a titanium implant, the zirconia implant demonstrated good performance in osseointegration in several studies that reported no significant differences in bone-to-implant contact and removal torque value or even better than a titanium implant. [14–20] Moreover, zirconia was superior to the titanium material in lower bacterial adhesion. [21–24]
The conventional surface of zirconia implants is usually a smooth surface. Surface topographies and surface roughness significantly affect osseointegration by increasing biological responses, enhancing osseointegration and increasing torque resistance. [25–31] It is then undoubtedly necessary to modify the surface of a zirconia implant. There are several ways to modify the zirconia implant’s surface roughness, including airborne-particle abrasion, acid etching, plasma spraying, bioactive material coating and UV-light treatment. [32] Airborne abrasion was most commonly used to increase the surface roughness of the zirconia because it is simple and inexpensive to use. Airborne abrasion also yielded a microroughness surface of the zirconia when compared to other techniques. [27,28,32–35]
Due to its high strength and bioinert property, increased surface roughness of a fully sintered zirconia surface can be problematic, requiring high blasting pressure for airborne-particle abrasion and strong acid with high temperature for acid etching. [28,36,37] Moreover, an airborne-particle abrasion of a fully sintered zirconia is associated with phase transformation from a tetragonal to a monoclinic phase that induced a negative effect on long-term clinical performance by increasing the rate of aging and zirconia fracture. [38–42] Additionally, airborne-particle abrasion of fully sintered zirconia can cause flaws, microcracks, plastic deformation or even embedding of abrasive particles on the zirconia surface. [38,43] These unfavorable results have also been reported to be associated with nonoptimized abrasive particle size and shape, blasting distance, blasting duration and blasting pressure. [38,43]
A number of airborne-particle abrasion methods to modify the zirconia surface before the final sintering step have recently been introduced. It is possible that airborne-particle abrasion of pre-sintered zirconia produces higher surface roughness compared with that of fully sintered zirconia. [38,39,44–51] Moreover, the modification of pre-sintered zirconia may contribute to greater durability of the implant because zirconia presents no monoclinic phase from reverse transformation after the sintering process. [49,50] However, the optimized airborne-particle abrasion protocol in pre-sintered zirconia has not yet been established, and nonoptimized abrasive particle size and shape, blasting distance, blasting duration and blasting pressure could induce a negative effect to the zirconia. Longer blasting duration and higher blasting pressure could produce volume loss and height loss of the zirconia surface. [43,47,48,51,52] The purpose of the present study was to investigate the use of airborne abrasion and its effect on zirconia surfaces. Different sizes of abrasive particles and various blasting times were used under controlled blasting pressure on fully sintered zirconia. The parameters measured included zirconia surface topographical characteristics, zirconia surface hydrophilicity and in vitro biological response of mesenchymal stem cells (MSCs).
Materials and Methods Specimen Preparation
All specimens in this study were prepared from pre-sintered yttriastabilized zirconia blocks (Prettau zirconia, Zirkonzahn, Gais, Italy). The blocks were cut into 5 x 5 x 2 mm 3 rectangular pieces using a precision high-speed lathe machine (CL4070, YAM, Taiwan) under copious water by setting the rotational speed at 1200 rpm. To standardize the initial surface roughness and create a finishing smooth surface, all specimens were polished with silicon-carbide grit papers for 10 vertical strokes in each grit value from #600, #800, #1000 and #2000 manually. The surfaces of the specimens were then cleaned to eliminate remnants of the powder produced through the cutting and polishing process using a strong stream of air and dried under room temperature. The specimens were randomly allocated into nine groups as shown in FIGURE 1.
An airborne-particle abrasion was performed using a sandblasting machine (Basic eco, Renfert, Hilzingen, Germany). Fine (25 µm) and coarse (110 µm) aluminum oxide particles were used to create submicron-scale and micronscale surface roughness, respectively, under the conditions used in the present study. These roughness scales have been shown to have a positive impact on osseointegration. [25–31] Airborne abrasion was used 25 µm and 110 µm aluminum oxide particles (Cobra, Renfert) with varying blasting times under control 1 bar pressure and 20 mm in perpendicular distance from nozzle tip to specimen surface. One-second blasting specimens were blasted in one direction with a single motion and the blasting was repeated two, three and four times in the same manner in two-, three-, and foursecond blasting specimens respectively. Our preliminary study suggested that for most of the roughness parameters studied, longer blasting duration (e.g., five to eight seconds) was found to produce similar roughness values observed on the samples abraded for one to four seconds. Thus, a maximum blasting duration of four seconds was used. After blasting, all specimens were cleaned ultrasonically in 99% isopropanol for three minutes to remove residual Al2O3 particles then sintered to the final temperature at 1600 degrees C in a furnace (Zirkonofen 700 Ultra-Vakuum, Zirkonzahn) as recommended by the manufacturer.50,51 Specimens were cleaned again in an ultrasonic bath of acetone for one minute and absolute ethanol for three minutes then stored in a desiccator for 24 hours before further tests.[53]
Surface Roughness and Topography
Surface roughness was characterized under a confocal laser scanning microscope (OLS4500, Shimadzu, Kyoto, Japan). Four different areas of each of the three specimens were used to measure surface roughness parameters (n = 12). The evaluating parameters include vertical roughness profiles (Ra, Sa, Rq, Rz, Rt), horizontal roughness profiles (Sm, S) and surface area as previously defined by Gadelmawla. [54] Ra is defined as the average absolute deviation of the roughness irregularities from the mean line over one sampling length. Sa is defined as the average absolute deviation of the roughness irregularities compared to the arithmetical mean of the surface. Rq, root mean square roughness (RMS), represents the standard deviation of the distribution of surface height. Rq is more sensitive than the arithmetic average height (Ra) to large deviation from the mean line. Rz, 10-point height, is defined as the difference in height between the average of the five highest peaks and the five lowest valleys along the assessment length of the profile. Rmax or Rt is defined as the vertical distance between the highest peak and the lowest valley along the assessment length of the profile. Sm is defined as the mean spacing between profile peaks at the mean line. S is defined as the average spacing of adjacent local peaks of the profile measured along the assessment length.
Surface topography and elemental compositions were characterized by scanning electron microscope (SEM) (JCM-6000, Jeol, Tokyo) and energydispersive X-ray spectroscopy (SU5000 FE-SEM, Hitachi, EDS HORIBA, Kyoto, Japan). In this test, three representative areas were randomly selected for each group and SEM surface topography was also analyzed.
Surface Hydrophilicity
Surface hydrophilicity was used in the present study to demonstrate the importance of zirconia surface topographical changes by different airborne-particle abrasion protocols. Hydrophilicity has been shown to significantly influence cell adhesion and subsequent osseointegration of the implanted biomaterials because water molecules are rapidly adsorbed within seconds on the implant surface following implant placement. [53,54] Surface hydrophilicity was determined by static contact angles using the sessile water drop method under atmospheric conditions at room temperature. [55] A drop (0.5 µl) of water was gently deposited on the specimen surface using a droplet dispenser and computer-controlled stage by dispensed 0.5-µl of water so a drop freely hung at the dispenser tip, then moved the stage upward until the drop contacted with the specimen surface and moved the stage downward to detach the drop from the dispenser tip. The water contact angle was measured by a video-based optical contact angle measuring system (OCA 40 Micro, Data Physics Instruments GmbH, Filderstadt, Germany). Four drops were obtained per group. Water contact angles were analyzed 60 seconds after the droplet contacted the surface of specimens. [56]
Measurements of Fibronectin and Albumin Adsorption
Solutions of human plasma fibronectin (Fn, Harbor Bio-Products, Norwood, Mass.) and bovine serum albumin (Sigma-Aldrich, St. Louis) were prepared at a concentration of 0.5 mg/ml (pH 7.4), and the fully sintered zirconia samples (control, 110 µm-2s and 110 µm-4s) were incubated in the protein solutions at 37 degrees C for 60 minutes. The samples were then washed with PBS to eliminate nonadsorbed protein molecules and then air dried at 37 degrees C. The adsorbed Fn and Alb were extracted and measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, Mass.) according to the manufacturer’s instructions. The amounts of protein adsorbed were calculated as ng/cm 2 and the ratio of adsorbed Fn/Alb was calculated accordingly, with the four replicates being performed for two independent experiments with similar results.
Cell Culture
In the present study, human MSCs (Lonza Biologics plc, Cambridge, U.K.) were maintained in a standard medium consisting of the α-minimum essential medium (α-MEM, Gibco Life Technologies Ltd, Paisley, U.K.) containing 15% heat-inactivated fetal calf serum (FCS, PAA Laboratories, Yeovil, U.K.) supplemented with 200 U/ml penicillin, 200 μg/ml streptomycin and 2 mM L-glutamine (all from Gibco) at 37 degrees C in a humidified atmosphere of 5% CO 2 in air. The media were changed every two to three days. Cells between passages 5–6 were used.
Cell Adhesion
MSCs were seeded on tested zirconia surfaces at a density of 1.5 x 10 4 cells/ cm 2 , and after six hours, the samples were fixed with 4% paraformaldehyde for 10 minutes and stained with rhodamine phalloidin (Thermo Fisher Scientific) for actin and the nucleus counterstained with DAPI (Sigma-Aldrich). The morphology and number of adhered MSCs were examined under a confocal fluorescence microscope (Nikon Ti Eclipse, Nikon Instruments Inc., Melville, N.Y.). The results were expressed as the mean number of attached MSCs ± SD per mm 2 derived from three experiments.
Cell Proliferation Index
Flow cytometric analysis was performed using the CytoFLEX Flow Cytometer and the CytExpert software (both from Beckman Coulter, Brea, Calif.) for data acquisition and analysis, respectively. Cells were collected and the cell pellets were resuspended in ice cold 70% ethanol for 30 minutes. Cells were then rinsed twice in PBS and resuspended in 20 μg/ ml propidium iodide (PI) in PBS with 50 μg/ml RNase A (both from Sigma) at 4 degrees C for 30 minutes and subjected to flow cytometry. The distribution of cells in three major phases of the cycle (G0/G1 versus S versus G2/M) was analyzed and the cell proliferation was calculated by the following equation:
Proliferation index (%) = (S + G2/M) / (G0/G1 + S + G2/M) x 100%
Proliferative index is expressed as mean percentage and the data are presented as the mean percentage ± SD from three independent experiments.
Determination of Osteogenic Differentiation and Mineralization by MSCs
For osteogenic differentiation induction, MSCs were seeded onto the tested surfaces at a density of 1.5 x 10 4 cells/cm 2 and allowed to grow with the standard medium for the first 48 hours until the cells reached 80% confluence. Then, the cells were incubated for 14 to 21 days with an osteogenic medium (OM) (standard medium with 100 nM dexamethasone, 50 μM ascorbate-phosphate and 10 mM β-glycerolphosphate, all from Sigma- Aldrich). The expressions of runt-related transcription factor 2 (Runx2), type-I collagen (COL-I), alkaline phosphatase (ALP) and osteocalcin (OC) mRNAs were determined on day 14 using a quantitative real-time PCR, and the formation of mineralization on day 21 was examined using alizarin red S staining.
To determine the expression of osteogenic genes, total RNA was isolated and first strand cDNA was synthesized from 1 μg RNA. The first strand cDNA was subjected to Q-PCR using SYBR Green I dye performed in an iQ5 iCycler (Bio Rad, Bradford, U.K.), with specific primers for the RUNX2, COL-I, ALP, OCN and GAPDH mRNA. GAPDH was used as an endogenous control. SYBR Green PCR reaction mixtures using SYBR Green I Master kit (Roche Diagnostic Co., Basel, Switzerland) were set up as suggested by the manufacturer. The amplification conditions consisted of 40 cycles at 95 degrees C for 15 seconds, followed by 60 degrees C for 30 seconds and subsequently 72 degrees C for 30 seconds. The specificity of the PCR products was verified by melting curve analysis. The PCR reactions were performed in six replicates, and each of the gene signal was normalized to the GAPDH signal in the same reaction. The mRNA expression is expressed as mean fold-change of control (1.0). Data are presented as the mean fold-change ± SD from three independent experiments. Primer sequences were as follows:
RUNX2 F 5′‐TGGTTACTGTCATG- GCGGGTA-3′, R 5′‐TCTCAGATC- GTTGAACCTTGCTA-3′; COL-I F 5′‐GAGGGCCAAGACGAAGA- CATC-3′, R 5′‐CAGATCACGTCATC- GCACAAC-3′; ALP F 5′‐ACTG- GTACTCAGACAACGAGAT-3′, R 5′‐ACGTCAATGTCCCTGATGT- TATG-3′; OC F 5′‐ CACTCCTC- GCCCTATTGGC-3′, R 5′‐ CCCTCCTGCTTGGACACAAAG-3′; GAPDH F 5′‐ CTGGGCTACACT- GAGCACC-3′, R 5′‐ AAGTGGTC- GTTGAGGGCAATG-3′. 57,58
For the alizarin red S staining, the samples were fixed with cold methanol for 30 minutes at 4 degrees C and washed with distilled water. The 1% alizarin red S solution (pH 4.2, Sigma) was added and incubated for 10 minutes at room temperature, and the samples were rinsed twice with methanol to remove unbound alizarin red S. Alizarin red S-stained mineralized matrices observed as bright-red deposits were photographed under a Nikon digital camera, and the amount of stained dye was eluted by 10% cetylpyridinium chloride and measured spectrophotometrically at 560 nm. The mineralization is expressed as mean foldchange of the absorbance of control (1.0).
Statistical Analyses
To compare means of surface roughness and water contact angles between experimental groups and the control (25 µm Al 2 O 3 blasting group versus control and 110 µm Al 2 O 3 blasting group versus control), the one-way analysis of variance (ANOVA) was used followed by Tukey’s post-hoc test for multiple comparisons. Pearson’s correlation coefficient was used to evaluate the relationship between surface roughness and water contact angle. In addition, the linear regression model was performed to assess the association between particle size, blasting duration and water contact angle after adjusting for covariates (SPSS software, version 23, IBM, Armonk, N.Y). A value of p < 0.05 was considered statistically significant for all analyses.
Results: Surface topographies of fully sintered zirconia were influenced by abrasive particle size and blasting time used in repeated airborne-particle abrasion in the pre-sintering stage.
We first examined the shape and size of the commercial Al 2 O 3 particles, and the SEM results showed that both 25 µm and 110 µm Al 2 O 3 particles appeared to be polygonal and vary in size with average sizes of approximately 25.5 ± 5.7 µm and 102.9 ± 17.7 µm, respectively (FIGURE 2A). In FIGURE 2B, SEM micrographs showed different topographical appearances of pre-sintered zirconia after airborne-particle abrasion. The control surface was relatively smooth with characteristics of polished surface with silicon-carbide grit papers, which could also be observed only in the 25 µm-1s group (FIGURE 2B). In general, the surface of all pre-sintered samples became roughened with increasing blasting time in both 25 µm and 110 µm groups, except the 110 µm-4s group, the surface of which appeared smoother than the 110 µm-3s group (FIGURE 2B). The results also demonstrated that the samples in 110 µm groups were rougher than the corresponding samples in the 25 µm groups. All the surface topography of fully sintered zirconia previously receiving pre-sintered zirconia airborneparticle abrasion (FIGURE 2C) seemed to have similar patterns that were observed in the corresponding presintered specimens. This suggests that the surface characteristics of the fully sintered zirconia surface were influenced by particle size and blasting time that were used in the pre-sintered zirconia airborne-particle abrasion protocol.
To determine whether ultrasonic cleansing before the sintering process could eliminate any possible Al 2 O 3 particle remnants, ultrasonic cleansing as described in the materials and methods was performed. The SEM-EDS was used to examine the presence of any possible Al element remaining on the fully sintered zirconia. The results showed that the main composition of both control and tested surfaces consisted of Zr and O (approximately 95 wt%) and very little (0.1 wt%) of Al residues was detected (FIGURE 2D). This indicated that ultrasonic cleansing was an effective procedure to clean the surface before the sintering process.
Abrasive particle size and blasting duration in repeated pre-sintering airborne-particle abrasion affected surface roughness cycle.
Surface topographical characteristics of the fully sintered zirconia following repeated pre-sintering airborne-particle abrasion were analyzed in terms of vertical roughness parameters (Ra, Sa, Rq, Rz and Rt) and horizontal roughness parameters (S and Sm) and surface area. Almost all the tested groups possessed higher values of all the roughness parameters and surface area compared with the control group (p < 0.05) (FIGURE 3A). In the 25 µm-1s group, however, only the Ra, Rq and Rz were statistically higher than those of the control group (Ra and Rq p = 0.003; Rz p = 0.002) whereas the Sa and Rt showed a nonsignificant difference (p > 0.05). Peak values of vertical roughness parameters (FIGURE 3A) in the 25 µm and 110 µm groups were at two seconds and three seconds repeated blasting duration, respectively, whereas the peak values of horizontal roughness parameter (FIGURE 3B) in the 25 µm and 110 µm groups were at three to four seconds and two seconds repeated blasting duration, respectively. In FIGURE 3C, repeated pre-sintering airborne-particle abrasion using 25 µm abrasive particles did not increase the surface area of the fully sintered samples regardless of the blasting duration (p < 0.05), but 110 µm abrasive particles statistically significantly increased the surface area at all blasting times used, with the peak surface area being observed with three seconds repeated blasting duration (p < 0.05). Taken together, using 110 µm abrasive particles in pre-sintering airborne-particle abrasion of zirconia was more effective in creating increased surface roughness and surface area, and optimal, but not the longest, duration was required to obtain fully sintered zirconia with maximal values of these parameters, suggesting the presence of surface roughness cycles following repeated pre-sintering airborne-particle abrasion. One cycle of surface roughness profiles thus included mild roughness at one second of blasting duration, maximum roughness during repeated blasting for two to three seconds and subsequently lesser roughness in the last phase of the cycle (i.e., repeated blasting for four seconds).
Increased surface hydrophilicity of a fully sintered zirconia was obtained by an optimal pre-sintering airborne-particle abrasion protocol with maximum surface roughness.
In the present study, surface hydrophilicity was determined by water contact angle and under the conditions used here, only the 110 µm-2s group was found to have increased surface hydrophilicity by statistically significantly decreased water contact angle from approximately 70 degrees in the control group to 60 degrees (p = 0.005) (FIGURE 4).
Statistical analyses further suggested that surface hydrophilicity was correlated with blasting time and horizontal, but not vertical, roughness profiles (Ra, Sa, Rq, Rz and Rt, p > 0.05). Pearson’s correlation coefficient revealed that water contact angle was moderately negatively correlated with horizontal roughness profiles, S (r = –0.36; p = 0.039) and Sm (r = –0.30; p=0.085). However, only S value, indicating average spacing of adjacent local peaks of the profile measured along the assessment length, was statistically significantly correlated with water contact angle (TABLE 1).
A linear regression model concurrently assessed associations between water contact angle, particle size and blasting time after being adjusted for horizontal roughness profile. The results showed that only blasting time and S value were statistically significant predictors for water contact angle. A blasting time at four seconds increased water contact angle (β = 15.21; p < 0.001) while an increasing of S value decreased water contact angle (β = –2.47; p < 0.001) (TABLE 2).
In vitro biological responses of 110 µm-2s and 110 µm-4s surfaces.
To test whether the most hydrophilic surface, which showed strong correlation with the S roughness value, possessed good in vitro biological responses, the effects of 110 µm-2s (higher hydrophilic and S value) and 110 µm-4s (lesser hydrophilic and S value) surfaces on protein adsorption and adhesion, proliferation, osteogenic differentiation and mineralization of MSCs were examined. The results showed that compared with the control machined surface, only the 110 µm-2s, but not 110 µm-4s, surface showed a significantly higher Fn/Alb adsorption ratio (FIGURE 5A). A significantly higher MSC adhesion to the 110 µm-2s surface was observed when compared with that in the control surface (FIGURE 5A), whereas only less than a 20% increase in MSC adhesion was evident on 110 µm-4s (FIGURE 5B). In addition, only limited cell cytoplasmic spreading was seen in the control surface; both 110 µm-2s and 110 µm-4s surfaces appeared to be better substrates for MSC spreading (FIGURE 5B). In contrast to their stimulatory effect on cell adhesion, 110 µm-2s and 110 µm-4s surfaces showed only a comparable effect to the control surface regarding MSC proliferation, osteogenic differentiation of MSCs and formation of mineralization (FIGURES 5C–5E, respectively).
Discussion
In the present study, the surface roughness of zirconia treated by pre-sintered airborne-particle abrasion was improved significantly compared with the control surface. This increase in roughness is within the range reported by several previous studies that demonstrated that zirconia treated by pre-sintered airborneparticle abrasion increased the surface roughness (Ra) by three to 17 times compared to the unmodified control surface. [38,39,44,46,51,59] Due to the lack of standardized methods, surface roughness values reported in the literature generally vary tremendously. It has been suggested that a moderate rough surface with Ra or Sa of 1-2 µm is optimal for dental implants and possess higher bone responses over a smoother or rougher surface. [25,30] However, a rougher implant surface might have a greater risk to develop peri-implantitis due to bacterial accumulation. [60,61] In the present study, the roughness obtained from presintering airborne-particle abrasion with 110 µm Al 2 O 3 showed surface roughness with Sa = 0.94-1.28 µm and Ra = 0.73-0.99 µm while 25 µm Al 2 O 3 blasting resulted in surface roughness with Sa = 0.38-0.51 µm and Ra = 0.23-0.40 µm. Therefore, the method carried out in the present study provided surface roughness within the suitable range for enhancing performance of a dental implant. A number of considerations should be weighed to obtain optimal conditions for pre-sintered FIGURE 5Dairborne-particle abrasion. These considerations are discussed below.
Extensive surface damage in presintered Y-TZP produced in the CNC milling process has been reported. [62] It has been suggested that this machininginduced damage cannot be naturally removed during the subsequent sintering process, and thus can cause stress concentrations under mechanical loads. [63] This damage causes weakly interconnected porous structures in the pre-sintered state, thus resulting in intragranular and transgranular fractures. [63] The currently studied method of pre-sintering airborneparticle abrasion is therefore suitable for eliminating such damage after the milling process. Although some scratches from the polishing process remained on the surface of the 25 µm-1s group as shown in FIGURE 2C, the other conditions showed no evidence of those scratches, suggesting that optimized pre-sintering airborne-particle abrasion conditions could effectively remove the milling process-induced damage of the pre-sintered surface of the zirconia.
Under the conditions tested, the repeated blasting for four seconds using 110 µm Al 2 O 3 particles constituted one cycle, which demonstrated mild roughness at one second, maximum roughness during repeated blasting for two to three seconds and subsequently less roughness in the last phase of the cycle (i.e., repeated blasting for four seconds). It is possible that increasing blasting time to five to six seconds could add a more complete profile of one cycle and that a longer blasting duration may produce another cycle of surface roughness profile with reduced thickness of the zirconia due to the loss of material from the surface. It has been shown that a high number of grits hitting the substrate modified the uppermost roughen surface, thus resulting in a flattened surface. [64] Compared with 110 µm abrasive particles, the use of abrasive particles sized 25 µm also demonstrated a similar profile of surface roughness but possibly with a longer blasting duration constituting of one complete cycle and lower values of all of the roughness parameters. These present results, therefore, indicated that the surface roughness of fully sintered zirconia was markedly dependent on abrasive particle size and blasting duration used in presintering airborne-particle abrasion. Moreover, our unpublished data showed that initial surface roughness (i.e., created by machining) influenced the roughness cycle profile. It is possible that different manufacturing systems and different types of zirconia-based material may produce pre-sintered zirconia implants with different surface roughness, suggesting the need for optimization of the pre-sintering airborne-particle abrasion in order to achieve the optimal surface roughness and hydrophilicity with minimum loss of abraded material for each of the systems used. A representative profile of a surface roughness cycle consisting of up and down surface roughness changes and the surface roughness trendline during the repeated blasting for one to four seconds using abrasive particles sized 110 µm under 1 bar of blasting pressure is proposed in FIGURE 3D. This shows the changes of surface roughness with an increasing abrasion time under indicated conditions, suggesting that for obtaining maximum surface roughness, optimal blasting time is pivotal and depends on conditions used in the pre-sintering airborne-particle abrasion protocol. It is important to note that the pre-sintering airborne-particle abrasion protocol was well defined and controlled to ensure precise effect of increasing times for repeated blasting on the surface roughness. To the best of our knowledge, the present study is the first to demonstrate the profile of the surface roughness cycle of fully sintered zirconia following repeated pre-sintering airborne-particle abrasion. Undefined blasting times reported in previous studies makes comparison with our results impossible. [38,39,44–47,49–51,59,65–67]
Volume loss of zirconia during the pre-sintering abrasion may affect the mechanical properties of the resulting sintered zirconia. A small volume loss of zirconia in the present study may be estimated from the surface roughness, as previously described. [68] Previous studies suggest that air-abrasion pressure and blasting duration have positive correlation to the amount of surface loss. [51,52] Using low air-abrasion pressure (1 bar) and short blasting durations (one to four seconds), the protocols used in the present study are thus unlikely to cause a significant amount of zirconia loss during the abrasion process. It is noteworthy that air abrasion may initiate some flaws or microcracks in zirconia structure, although we did not investigate this aspect in the current study. Abi-Rached and colleagues reported in 2015 that the flexural strength of abraded and nonabraded sintered materials showed no difference. [49] The study also confirmed by XRD analysis that abraded and nonabraded sintered materials contained no monoclinic phase (0.0%wt) because of the sintering process. This can imply that the grain of sintered materials may rearrange after air abrasion from phase toughening and reverse to a tetragonal phase as reported by Monaco and colleagues in 2013. [38] We assumed that with low pressure of air abrasion, a very small amount of microcrack can occur, but it may not be significant when the air abrasion was performed before the sintering process. However, the aging degradation of the material and different sintering moments should be further investigated.
Previous studies suggest that residual aluminum oxide particles on a dental implant surface following airborneparticle abrasion could negatively affect osseointegration and diminish the clinical success. [69,70] In the present study, elemental composition analysis showed that no residual alumina particles on a fully sintered surface receiving presintering airborne-particle abrasion were found. This is consistent with previous studies that reported that no byproduct or contamination of residual alumina particles were found on a modified surface after receiving pre-sintering airborne-particle abrasion and ultrasonically cleaned with 99% isopropanol for 10 minutes. [45,65] In contrast, Monaco suggested that blasting zirconia in a pre-sintered state induced surface contamination from alumina embedding after airborneparticle abrasion. [38] Martins also found alumina residue remained on a fully sintered surface after blasting presintered zirconia with Al 2 O 3 under 0.5 bar pressure and a working distance of 10 mm. [50] The explanation of this discrepancy may be the differences in air pressure and the working distance used. We again emphasize the importance of the optimization of each parameter used in pre-sintering airborne-particle abrasion. It is also noteworthy that the cleansing method used in our study was ultrasonic cleansing with isopropanol to remove any possible remaining Al 2 O 3 particles. This also shows that the cleansing agent is one of the important factors.
Among several surface roughness parameters tested in the present study, only the S value, which is defined as the average spacing of adjacent local peaks of the profile measured along the assessment length, was found to be statistically correlated with the surface hydrophilicity of fully sintered zirconia from the linear regression model (p < 0.001). The strong correlation of these two surface properties can be explained by the Wenzel theory, which stated that adding surface roughness can enhance hydrophilicity based on the assumption that the liquid penetrates into the roughness grooves. [71] Although several previous studies reported the use of Ra and Sa to exhibit surface roughness that represents only vertical roughness profiles, [38,39,46,47,50,51] we propose that other surface roughness values, especially the horizontal parameter, e.g., S values, must be included when performing analysis of surface roughness and hydrophilicity.
Following implant placement, it is generally accepted that MSCs play a key role in bone healing and osseointegration of dental implants. Certain serum proteins adsorbed on the implant surface may regulate MSC adhesion, proliferation and differentiation toward an osteoblast lineage. [72] Responses of MSCs to the modified zirconia surfaces were thus investigated to assess the biological significance of the zirconia surface modification technique. The present results also showed that the most hydrophilic 110 µm-2s surface, which correlates well with the S roughness value, had enhanced in vitro biological responses by promoting Fn-to-Alb adsorption ratio and initial MSC adhesion compared with either the control machined surface or 110 µm-4s surface. The levels of Fn-to-Alb adsorption ratio, initial MSC adhesion, surface hydrophilicity and S value of the three surfaces appeared in the following order (from high to low): 110 µm-2s > 110 µm-4s > control, adding more evidence to support the importance of the S value. Although, MSCs grown on the 110 µm-2s surface showed comparable ability to proliferate, differentiate and form mineralization compared to those grown on the 110 µm-4s and control surfaces, the 110 µm-2s surface-enhanced Fnto-Alb adsorption ratio and initial MSC adhesion could hasten the osseointegration process. The adhesive protein fibronectin enhances osteoblast adhesion whereas the nonadhesive proteins, such as albumin, appear to be an inhibitor for osteoblast adhesion, [72] consistent with the finding in this study that high Fn-to-Alb adsorption ratio of the 110 µm-2s surface is associated with increased MSC adhesion. This is supported by previous studies. [59,73] However, a lack of effect on osteoblast differentiation and mineralization by MSCs shown in the present study is inconsistent with a previous report. [74] This could be due to a difference in the resulting surface roughness, cell type used and type and chemistry of the zirconia used. Further studies to test these hypotheses would help gain more knowledge about cellular responses to zirconia. These data suggest that well-optimized pre-sintering airborneparticle abrasion of zirconia, such as the 110 µm-2s surface tested in the present study, may improve osseointegration of a zirconia dental implant by increasing MSC adhesion through increased surface roughness and hydrophilicity.
Zirconia dental implants have been shown to be a successful alternative to titanium dental implants in the aesthetic zone, where a deficiency in alveolar bone volume is common. Adequate peri-implant tissues in the aesthetic zone is thus important for long-term maintenance of bone levels and aesthetic outcomes. An immediate restoration protocol to replace a natural tooth in the aesthetic zone may require simultaneous MSC-based bone tissue engineering for reconstruction of alveolar bone deficiency. [75] The improved surface properties of modified zirconia demonstrated in the present in vitro study might help maintain the number of MSCs (simultaneously loaded while placing dental implants) that are sufficient to regenerate bone in vivo. Further studies on the optimal surface characteristics of zirconia that can also stimulate MSC proliferation and osteogenic potency will enable the advancement and better understanding of stem cell research and implant dentistry.
A key strength of the present study is the well-defined and well-controlled pre-sintering airborne-particle abrasion protocol. The present study is the first to demonstrate the profile of the surface roughness cycle of fully sintered zirconia following repeated pre-sintering airborne-particle abrasion. The key findings of the study suggest some potential clinical implications. First, over-repeating airborne-particle abrasion leads to lower surface roughness. Maximum surface roughness could be achieved only by optimal blasting duration. Second, hydrophilicity could be predicted by certain surface roughness parameters. There is a significant association between the surface roughness parameters, especially horizontal roughness profiles (S and Sm) and the zirconia surface hydrophilicity. Thus, it might be possible to evaluate the surface roughness and approximate for biological responses of the dental implant surface. The present study has some limitations, as only one type of zirconia was used. Optimization of this method for other types of zirconiabased dental implants remains challenging. In addition, this study lacks the evaluation of key mechanical properties of the modified zirconia samples. This has a significant impact on the success of a dental implant.
Conclusion
In conclusion, the present study has shown, for the first time, that repeated pre-sintered zirconia airborne-particle abrasion with increasing blasting times resulted in a range of surface roughness with up and down changes of roughness, constituting one roughness cycle. The abrasive particle size and blasting duration significantly influenced surface roughness, hydrophilicity and certain in vitro biological responses of fully sintered zirconia. Well-optimized presintering airborne-particle abrasion of the zirconia surface could accomplish the highly roughened and hydrophilic surface of a fully sintered zirconia dental implant with improved Fn-to-Alb adsorption ratio and MSC adhesion. This inexpensive and simple airborne-particle abrasion technique may be suitable for modifying the surface of zirconia in order to facilitate osseointegration and enhance the performance of zirconia-based dental implants.
ACKNOWLEDGMENTS This study was financially supported by the National Metal and Materials Technology Center (Grant number P-18-50741 (MT-B-61-BMD-13-231-G)), Thailand, and Research Unit in Mineralized Tissue Reconstruction, Thammasat University, Thailand. We thank Terawat Tosiriwatanapong, Anucha Wannagon, Pattarawan Choeycharoen and Kannaporn Pooput for their technical assistance.
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THE CORRESPONDING AUTHORS, Weerachai Singhatanadgit, DDS, PhD, and Yanee Tantilertanant, DDS, PhD, can be reached at s-wrch@tu.ac.th and littlengi@hotmail.com.
