A Comparison of Zirconia and Titanium Abutments for Microleakage Mohamed I. Abdelhamed, DDS, MS;* Jeffrey D. Galley, BS;† Michael T. Bailey, PhD;‡ William M. Johnston, PhD;§ Julie Holloway, DDS, MS;¶ Edwin McGlumphy, DDS, MS;§ Binnaz Leblebicioglu, DDS, MS, PhD††

ABSTRACT Background: Microleakage through the interface has been documented in implant systems with titanium (Ti) abutments. There is a current increase in the use of zirconia (Zi) abutments especially in esthetic zone in where higher risk of visible metal color through the peri-implant tissues exists. Purpose: The aim of the present in vitro study is to evaluate the leakage at the implant fixture–abutment interface with two different screw-retained abutment systems at different torque values in a nonloading condition. Materials and Methods: In vitro study design included four groups (Ti and Zi torqued at 25 and 15 Ncm [N = 8/group]). Microcomputed tomography (micro-CT) was chosen to detect microgap. Microleakage from the implant chamber to the external milieu was evaluated using limulus amebocyte lysate (LAL) test, while microleakage from external milieu to the implant chamber was evaluated using toluidine blue dye (TBD) and colorimeter. Results: Micro-CT images did not reveal any microgap. LAL test showed that there is a time-, abutment-, and torquedependent increase in microleakage (p = .001) with Zi torqued at 15 Ncm having higher leakage with time compared with Ti torqued at 15 Ncm (p = .002), as well as Zi torqued at 15 Ncm having higher leakage with time compared with Zi torqued at 25 Ncm (p = .01). TBD test showed a nonsignificant increase in microleakage with higher leakage related to titanium abutment groups (p > .05). Repeated torque/antitorque handling differentially affected microleakage (p = .01). Conclusions: Within the limits of this study, there is a statistically significant difference in bidirectional microleakage with time, abutment type, and torque values being major players for leakage from internal implant chamber to external milieu, while the abutment type and time but not the torque value being important factors for leakage from external milieu into implant chamber in nonloading condition. Future studies are needed to determine peri-implant health around Zi abutments. KEY WORDS: abutments, implant, microleakage, titanium, zirconia

INTRODUCTION Peri-implant pathology exists and currently not well understood despite the high success rates for dental implant-supported prostheses.1–4 Among others, the presence of a microgap between implant fixture and abutment has been investigated as a possible etiological factor.5 Microgap is defined as the microspace that exists between the implant fixture and abutment.7,8 This gap is generally in micron size and is located at the alveolar crest level.5 The microgap acts as a reservoir for bacteria which leads to release of bacterial byproducts and induction of inflammatory reaction at both soft and hard tissue levels.6,9,10 Bidirectional microleakage (e.g., leakage from internal implant chamber to external milieu and from external milieu to internal implant chamber) has been

*Previously graduate student, Division of Prosthodontics, The Ohio State University, currently private office, Columbus, OH, USA; †PhD candidate, Division of Biosciences, The Ohio State University, Columbus, OH, USA; ‡associate professor, Division of Biosciences, The Ohio State University, Columbus, OH, USA; §professor, Division of Prosthodontics, The Ohio State University, Columbus, OH, USA; ¶ previously associate professor, Division of Prosthodontics, The Ohio State University, Columbus, OH; currently professor and head, Department of Prosthodontics, University of Iowa, Iowa City, Iowa, USA; ††associate professor, Division of Periodontology, The Ohio State University, Columbus, Ohio, USA Corresponding Author: Dr. Binnaz Leblebicioglu, Division of Periodontology, College of Dentistry, The Ohio State University, 305 West 12th Avenue, Columbus, OH 43210, USA; e-mail: [email protected] © 2015 Wiley Periodicals, Inc. DOI 10.1111/cid.12301

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reported for various implant systems with different types of abutments.11–17 In addition, based on several in vitro and in vivo study models, this leakage has a potential role in bacterial growth and peri-implant pathogenesis.16,18 As the closing torque increases from 10 to 20 Ncm to manufacturer’s recommended closing torque value, microleakage decreases significantly.19 However, it is important to note that repeated tightening of the abutment screw has been shown to result in a progressive decrease in removal torque.20,21 Zirconia (Zi) abutments have been routinely preferred as the abutment of choice especially with increasing esthetic demands in patient with thin soft tissue biotype. In addition, these abutments are generally placed with closing torque values lower than what manufacturer recommends due to high risk of abutment fracture.22 Currently, there is very limited data available on possible microleakage problems related to Zi abutments23 although the clinical biocompatibility, measured through peri-implant crevicular fluid content following 6 months of loading, was reported as similar to titanium (Ti) abutments.24 Thus, the purpose of this study was to investigate microleakage through the implant-abutment interface using the same implant fixture with two different abutments of the same design. The working hypothesis was that torque value is the primary factor affecting bidirectional microleakage. The findings of this study is significant in that Zi abutmentsupported implant crowns are specifically indicated for esthetically challenged anatomical locations presenting thin soft tissue biotype, and microleakage can have more detrimental effects at such locations. MATERIALS AND METHODS Thirty-two dental implant fixtures,a 16 Ti abutmentsb and 16 Zi abutments,c were included. Implants were either fixed in an acrylic block (toluidine blue dye [TBD] test) or mounted into a sterilized holder (limulus amebocyte lysate [LAL] test). Abutments were connected to the implants and torqued using torque wrench to two different closing torque values (25 and 15 Ncm). These two values were selected based on manufacturer’s recommended torque values and what was reported in the literature for an average value with hand torquing.25 aOsseospeed 4.5 × 13 mm implants, AstraTech Dental, Molndal, Sweden. b TiDesign 4.5/∅5.5, 3 mm, AstraTech Dental. c ZirDesign 4.5/∅5.5, 3 mm, AstraTech Dental.

Study design included LAL test to study leakage from implant internal chamber into external milieu and TBD penetration experiment to investigate leakage from external milieu into implant internal chamber (modified from Guindy and colleagues13). Implants were divided into four experimental groups: group A (N = 8) in which Ti abutments were torqued to 25 Ncm, group B (N = 8) in which Ti abutments were torqued to 15 Ncm, group C (N = 8) in which Zi abutments were torqued to 25 Ncm, and group D (N = 8) in which Zi abutments were torqued to 15 Ncm. Radiographic and Micro-Computed Tomography Analysis The implant fixture–abutment interfaces were scanned in a micro-computed tomography (micro-CT)d at a resolution of 9.7 micron prior to and following paper dot placement inside implant chamber (Figures S1, A and B). This documentation was necessary to determine whether insertion of a filter paper dot into implant chamber is creating a microgap between implant fixture and abutment. The acquisition was a full 360-degree scan at a maximum power and filtration 120 KV and 1.5 mm Al, respectively. This was done to accommodate for the beam hardening artifact due to the rather dense subject material. The manufacturer’s own softwaree was used to visualize each assembly on three-dimensional pictures and confirm or deny the presence of gap, according to the resolution capabilities of the scanner. Similarly, high-resolution radiograph pictures were obtained to determine presence/absence of microgap at implant-abutment interference (Figures S2, A and B). The data acquisition was repeated three times for Zi and Ti abutments torqued at recommended values, with and without the inserted filter paper dots into implant chamber. Leakage from Implant Chamber into External Milieu Each implant fixture and abutment was unpacked under the biosafety level 2 ventilation hood in sterile conditions. Total of 1,500 EU Escherichia coli lipopolysacharide (LPS)f in 0.5 μl volume was placed d Siemens Inveon micro-CT, Siemens Medical Solutions USA Inc., Malvern, PA, USA. e Siemens Research Work Place®, Siemens Medical Solutions USA Inc. f Sigma Aldrich, St. Louis, MO, USA [Catalog number L2630, Lot number 118K4052].

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into implant chamber by using sterile pipette tips (Figures S3, A and B). Implant was then placed into a sterile holder, and abutment was torqued with wrench at 25 or 15 Ncm. Implant fixture-abutment apparatus was examined for any wetness and placed into sterile test tubes containing sterile Pyrotell waterg and incubated in 37°C incubator. From these tubes, 220 μl of supernatant was taken at 5 minutes, 25 hours, and 195 hours. The supernatant was diluted in half with sterile water a number of times to create a range of dilutions. The LAL testh was run on these dilutions. Briefly, reconstituted Pyrotell, with a sensitivity of 0.03 EU/ml, was added at a 1:1 (v:v) ratio with each dilution. Each tube was gently mixed and then placed in a 37°C water bath for 1 hour. After 1 hour, the tubes were removed and swiftly inverted. If a clot had formed and did not break upon inversion, the test was positive. If a clot had not formed or broke upon inversion, the test was negative. In order to calculate the amount of LPS in each sample, the ratio of the last positive sample was inverted and multiplied by the sensitivity of the Pyrotell water. The data were analyzed both as the actual concentration of LPS and as the ratio between test and control (no LPS inoculation). Leakage from External Milieu into Implant Chamber Toluidine dye penetration experiments were conducted to detect a time-dependent leakage from the external milieu into the implant chamber for different abutment types with various closing torque values. Leakage through screw access hole as compared with the implant fixture–abutment interface was also investigated. Finally, the effect of repeated torques and antitorques upon the leakage from external milieu into implant chamber was studied. For these experiments, implant fixture was mounted in a block made of orthodontic clear acrylici and fixed to a metal holder. Tissue punch 1.5 mm in diameter was used to cut absorbent filter paperj to make rounded dots to be placed inside of the implant chamber (Figure S4, A and B). Two dots of the filter paper were placed inside of the implants, and then, the abutments were connected. Abutments were torqued to assigned torque value for different groups. The implant fixtureabutment assembly was placed in a sterile test tube. TBD g

Associates of Cape Cod Inc., East Falmouth, MA, USA. Associates of Cape Cod Inc. i OrthoJet, Lang Dental Mfg Co., Inc. South San Francisco, CA, USA. j Periopaper, Oraflow Inc., Smithtown, NY, USA.

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was placed first into screw access hole, and then, the remaining of implant fixture-abutment apparatus was embedded into dye. In some experiments, screw access hole was sealed with composite plug to differentiate microleakage from microgap and from screw access hole. For the same purpose, a separate experiment was also conducted by placing 20-μl TBD only into the screw access hole after the abutment was torqued in place. The rest of implant fixture/abutment assemblies was placed in distilled water up to the implant-abutment connection for this experiment. The test tubes were incubated in a water bath at temperature 37°C for 3 days, 1 week, 2 weeks, or 3 weeks. Finally, a third experiment was performed to determine whether repeated torques/ antitorques would affect microleakage from the external milieu into the implant chamber. The incubation time for this experiment was chosen as 2 weeks as more variations in microleakage were previously detected at the 2-week time interval. Experiment was repeated for 2-week time period after the abutments were unscrewed and screwed 10 times. At the end of each test period, the implant fixture-abutment assemblies were removed from the test tube and washed under running water. Screw access hole was rinsed with distilled water using disposable injector.k All implant fixture-abutment apparatus were left to dry at room temperature for 2 days. To evaluate the occurrence and the degree of fluid penetration, the abutments were unscrewed, and the two filter paper dots were retrieved from the inside of the implants. These paper dots were then mounted on a glass slab and covered with glass cover (Figures S4, A and B). Colorimeter Analysis Color change on paper filters (e.g., the difference between dark blue to bright white) was determined using Colorimeter.l The meter measures both incidents and reflected light through high-sensitivity silicon photocells. The color was measured after the colorimetric has been calibrated according to the manufacturer’s recommendation using a standard white reflecting plate. Two paper filter dots mounted on glass slab were used as baseline. The baseline measurement was followed by the sample measurements, and the color difference was calculated using the Commission Internationale de L’Eclairage 1976 (1*a*b*) color difference formula

h

k

Monojet, Choice Medical Supply, East Hampton, NY, USA. Minolta Chroma Meter CR 221-b, Konica Minolta, Tokyo, Japan.

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0.6

1

ΔE = [(ΔL*)2 + (Δa *)2 + (Δb*)2 ] 2 ,

Statistical Analysis The LPS concentration and ratio data were each analyzed using a three-way anova, with the factors being the material (Ti or Zi), the torque (15 or 25 Ncm), and the time (5 minutes, 25 hours, or 195 hours). For each analysis, all interactions were incorporated into the model. The sas procedure MIXEDm using the maximum likelihood estimation method was chosen as this method allows for deviations from normality and homogeneity of variances. When an anova indicated statistical significance involving more than one degree of freedom, pairwise comparisons of interest were analyzed using Student’s t-tests adjusted with the step-down Bonferroni correction method. For each dependent variable, the experiment-wise type I error rate was set at α = 0.05. The color differences were analyzed using a fourway anova using basically the same analysis method, but with the additional factor being the mode of obtaining the specimen (Standard leakage, leakage from screw entrance, or leakage after repeated torques). RESULTS Radiographic and Micro-CT Analysis There was no detectable separation at the implant fixture–abutment interface when filter paper dots were present at the bottom of the abutment screw hole. Confirmatory high-resolution radiographic analysis revealed similar results by observing the density difference between Zi and Ti abutments with no detectable separation following filter paper dot insertion (Figures S1 and S2). Leakage from Implant Chamber into External Milieu A time-dependent increase in LPS concentration was observed (p = .0004). Figure 1 represents the actual LPS concentration within supernatant. The results related to m

sas Proprietary Software 9.2, SAS Institute Inc., Cary, NC, USA.

Ti 15 Ncm Zi 25 Ncm

0.4

Zi 15 Ncm EU/ml

where ΔE is color difference, ΔL is lightness variable and describes the achromatic characteristics of color, and a* and b* are coordinates describing chromatic characteristics of color.26

Ti 25 Ncm

0.5

0.3 0.2 0.1 0 5 min

25 hrs

195 hrs

Figure 1 LPS leakage from implant chamber into external milieu. Data are expressed as mean LPS concentration 1 standard error (SE) detected within the supernatant. Time factor, p = .0004. Abutment type × time interaction, p = .02. Torque value × time interaction, p = .03. Abutment type × torque value × time interaction, p = .001. ★p = .002 the difference between Ti 15 Ncm and Zi 15 Ncm at 195 hours. ★★p = .01 the difference between Zi 25 Ncm and Zi 15 Ncm at 195 hours.

the ratio of LPS concentration leakage observed in test samples (e.g., implants inoculated with E. coli LPS) to negative controls (e.g., implants with no E. coli LPS inoculation) were similar to LPS concentration data and were not included in this manuscript. Data analysis based on concentration revealed differential response based on abutment type as well as abutment type and torque values (p = .02 and p = .03, respectively). At 195 hours, the difference between Zi and Ti abutments torqued at 15 Ncm was statistically significant (p = .002). Similarly, the difference in microleakage between Zi abutments torqued at 15 and at 25 Ncm was statistically significant (p = .01). Baseline concentration readings (measured for the 5-minute time period) were 0.1 1 0.02; 0.06 1 0; 0.12 1 0; 0.04 1 0.01 EU/ml for Ti 25 Ncm, Ti 15 Ncm, Zi 25 Ncm, and Zi 15 Ncm groups, respectively (see Figure 1). Concentrations calculated for the 195-hour time period were 0.18 1 0.06, 0.08 1 0.05, 0.14 1 .11, and 0.36 1 0.12 EU/ml for the same groups, respectively (see Figure 1). Leakage from External Milieu into Implant Chamber The leakage from external milieu into implant chamber was studied through three different experiments: leakage from implant fixture–abutment interface (Figure 2), leakage through screw access hole (Figure 3), and leakage following repeated torques and antitorques (Figure 4).

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ΔE (mean±se)

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10

Ti 15 Ncm

8

Zi 25 Ncm Zi 15 Ncm

6 4 2 0 3 days

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Figure 2 TBD penetration from external milieu into implant chamber. Data are expressed as mean ΔE 1 SE of color difference (from white to dark blue). Each time period was studied as a separate experiment due to difficulty in preventing dye contamination. ★p = .05; the difference between 3 days and 3 weeks for Ti abutments only.

Toluidine dye penetration experiments revealed no statistically significant time effect on the microleakage from the external milieu into the internal chamber (p > .05; see Figure 2). Mean ΔE values were similar at 3 weeks compared with 3 days for Zi abutments with closing torque values at 25 and 15 Ncm (9 1 0.2 vs 8 1 2 for Zi 25 Ncm and 9.2 1 1.3 vs 9 1 1.2 for Zi 15 Ncm,

16

respectively). A time-dependent increase in mean ΔE values was observed for Ti abutments torqued at 25 Ncm and at 15 Ncm (9 1 1 vs 13 1 2 and 10.4 1 1.4 vs 12.3 1 5, respectively). This increase was statistically significant (p = .05; see Figure 2). Ti and Zi abutments torqued at 15 Ncm had slight but not statistically significant increase in timedependent leakage from screw access compared with from abutment connection (mean ΔE was 11 1 2 vs

Ti 25 Ncm

14 Ti 15 Ncm

25

Zi 25 Ncm

10

Zi 15 Ncm

8

20

6 ΔE (mean±se)

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screw access

Figure 3 TBD penetration from screw access hole versus from implant fixture-abutment connection into implant chamber following 3-day incubation. Data are expressed as mean ΔE 1 SE of color difference (from white to dark blue). TBD was placed directly into screw access hole following assigned torqueing, and the rest of the implant fixture/abutment assembly was placed into distilled water to study possible leakage from screw access hole. Similarly, implant/abutment assembly was torqued, screw access hole was sealed with composite plug, and all assembly was placed into TBD to study possible leakage from implant-abutment connection. Three-day incubation time was chosen as a faster leakage was hypothesized through screw access hole.

5

0 single torque Ti 15 Ncm

mulple torques Ti 25 Ncm

Zi 15 Ncm

Zi 25 Ncm

Figure 4 TBD penetration – comparison between single torque versus multiple torques following 2-week incubation. Data are expressed as mean ΔE 1 SE of color difference (from white to dark blue). Group assigned for multiple torques received 10 separate torqueing/antitorqueing sessions prior to incubation within TBD. ★p = .01; the difference between single torque and repeated torques for Titanium abutments only.

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10.4 1 1.4 for Ti abutments and, 12.2 1 2.3 vs 9.2 1 1.3 for Zi abutments, respectively; p > .05) (see Figure 3). There was no difference in microleakage from screw entrance or abutment connection for abutments torqued at 25 Ncm (see Figure 3). The results of third set of experiments conducted to determine whether repeated torques/antitorques would affect microleakage from the external milieu into the implant chamber revealed that mean ΔE values were higher in titanium groups with multiple torque/ antitorque application compared with single torque application independent of closing torque value (16.8 1 0.8, 16.5 1 4.7 for multiple torque vs 10.8 1 0.3, 7.4 1 0.6 for single torque, for Ti 15 Ncm and Ti 25 Ncm, respectively) (p = .01) (see Figure 4). The differences observed in Zirconia abutment groups were negligible (p > .05).

DISCUSSION The aim of this study was to investigate microleakage at the implant fixture–abutment interface using the two different abutments of the same design (Ti and Zi abutments) and at varying torque values. An internal connection, locking taper implant system, was chosen for two reasons: Less microleakage has been reported with tapered systems,8,11,27 and the implant fixture of the specifically chosen system has a significant internal chamber even after abutment screw connection which allowed for insertion of filter paper specimens without interfering with abutment seating. The implant fixture– abutment interface was studied using micro-CT and high-resolution radiography to verify seating. Similar studies have chosen a scanning electron microscopy (SEM) observational technique over micro-CT and radiography to detect microgaps. The average microgap was found to be 2 to 7 micron for screw-retained abutments as investigated using the SEM technique.11,16 The micro-CT technique has a maximum resolution of 9.7 micron and would not be sensitive enough to detect the gap size found by SEM studies. The goal in using micro-CT technique in the current study was to detect any intentional space created by inserting filter paper specimens into the implant internal chamber. Also, this technology enables to study implant fixture-abutment connection in three dimensions, which is not possible with SEM and conventional radiographic techniques. High-resolution radiography was chosen as an addi-

tional technique to evaluate the implant fixture– abutment interface to overcome the density differences between the Zi and Ti materials. In this study, bidirectional microleakage was studied by using two experimental methods. Microbial penetration between implant components has been reported in the literature both by using in vitro study models and in clinical studies.11,13,14,16,27,28 The internal part of the implant can be contaminated during implant placement and/or abutment placement.5 Thus, bacteria can grow inside the implant fixture or within the peri-implant sulcus.5,29 In this aspect, penetration of bacterial byproducts instead of the bacteria themselves may play a more important role in peri-implant pathogenesis.12 Among those byproducts, lipopolysaccharide (LPS) is well established.30 LPS was chosen in this microleakage study as the reported microgap is small enough to stop most whole bacteriae. E. coli LPS was preferred compared with other oral flora-related bacterial LPS as it is easily accessible and it is still relevant for oral cavity.31 Baseline LPS contamination is a commonly reported problem related to similar in vitro study models as LPS can be easily found in the surrounding environment. Most of the published in vitro studies report several cycles of autoclaving and/or chemical processing to remove this type of baseline LPS prior to the actual experiment.12,13 This procedure by itself may cause various problems as it may change structural integrity of materials and especially implant fixtureabutment fit. Thus, in this study, the implant fixtures and abutments were unpacked under a ventilation hood, and LPS inoculation and abutment placement were conducted under sterile conditions. Also, implants in control group were not inoculated with LPS and were used to determine baseline levels of LPS contamination due to the environment. This study design helped to detect actual time-dependent LPS leakage from inoculated implants and compared it with LPS levels detected from noninoculated implants without extra sterilization procedures. Similarly, LPS overflow due to torque forces is a possibility during abutment placement. The volume chosen for this study (0.5 μl) was very small and was previously reported for the type of implant and experimental design used in this study.13 Following each abutment placement and torque procedure, the implant fixture/abutment assembly was examined for any wetness prior to incubation in the Pyrotell

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water. In other studies, approximately 60% of the samples have been reported as contaminated immediately after inoculation with the same volume of LPS.11,13–15 However, their inoculation technique was different than the present study as they inoculated the tip of the screw11 compared with inoculating the deepest part of implant internal chamber as performed in the current study. The results of the LPS leakage study showed that leakage from the inside of the implant to the outside is time dependent which is in agreement with results reported by Harder and colleagues.19 do Nascimento and colleagues17 compared microleakage using two different abutment systems (premachined and cast abutment) and showed that both systems have low percentage of microleakage and that there was no significant difference between different abutment systems in an unloaded condition. Similar studies on bacterial leakage from external milieu into implant chamber report that leakage occurs independent from material or screw torque value.23 Some pilot studies using in vitro loading conditions33 as well as some long-term clinical outcome studies34 report increased problems in marginal accuracy and wear in relation to Zi abutments. Based on the current study results, it appears that there is an obvious timeand abutment/torque-dependent increase in LPS leakage especially for Zi abutments (see Figure 1). A time-dependent increase was also noted for Ti abutments torqued at 25 Ncm and Zi abutments torqued at 25 Ncm, but this did not reach statistically significant levels. Zi abutments torqued at 15 Ncm initially showed very small amount of leakage. However, the increase in leakage for this group was almost ninefold by 195 hours compared with 5 minutes incubation. This may be due to lower torque value as well as Zi materials properties under low torque pressure. Nevertheless, de Silva-Neto and colleagues35 in their critical review of the similar in vitro study models report that the bacterial concentrations used in published similar work are ranging from 2.41 × 106 to 8 × 108 colony-forming units/ml. Also, the type of bacteria varies from oral bacterial flora, mixtures of bacteria, toluidine blue, and gentian violet; and LPS of Salmonella enterica bacterial toxins. They conclude that the methodological variation observed within literature may explain different conclusions. They also recommend using inoculation in lower concentrations at a specific volume.35 Similarly, long-term biocompatibility of the implant materials requires

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further research especially in relation to dental implants. Although some recent work report no difference in how soft tissue response to abutments made in Ti and in Zi,24 fatigue, wear, and related microleakage may negatively affect tissue integrity and health. Microleakage from the external milieu into the implant internal chamber was investigated by placing two 1.5-mm diameter filter paper specimens inside the chamber. Similar studies using similar technique have been already published.13,36 The main difference for the current experimental protocol was that a colorimetric analysis of the retrieved paper specimens allowed generation of calibrated numeric data which would be impossible with naked eye even under magnification. Piattelli and colleagues studied dye leakage into the internal part of implant with two different abutment systems (screw-retained vs cement-retained abutments) following 30-hour incubation.36 In the case of cemented abutments, a sectioning procedure had to be used in order to retrieve the filter paper. Stereomicroscopy was used to evaluate the internal threads of the implant for possible dye penetration. Dye penetration into most apical portion of the internal implant chamber instead of just the internal threads was investigated in the current study. However, internal threads were also examined under magnification and lighting. It was noted that leakage from the screw access hole to the implant fixture–abutment interface (most apical portion of abutment recovered with TBD) was a general finding with faster leakage observed in abutments torqued at 15 Ncm. There was no detectable contamination on internal threads (data not shown). The results of the current study report a statistically significant effect of repeated torquing on microleakage specific to titanium abutment groups (see Figure 4). The effect of repeated torquing was attributed to the decrease in the preload value decreasing the clamping force between implant fixture and Ti abutment.21,32 This in vitro study aimed to investigate bidirectional microleakage in a nonloaded environment. It has been reported that loading the implant fixture-abutment assembly induces micromotion between these two pieces.14 It has been hypothesized that this micromotion depends on the structural strength of the implantabutment connection.14 Thus, loading may have a significant effect on bidirectional microleakage results reported in the current study.

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CONCLUSIONS Within the limits of this in vitro study, it can be concluded that there is a statistically significant difference in microleakage from internal implant chamber to external milieu between Ti and Zi abutments in nonloading conditions. In addition, a general but statistically nonsignificant time-dependent microleakage from external milieu into implant chamber was noted for both abutment types. Further in vitro and in vivo studies are necessary to determine the effect of this bidirectional microleakage on peri-implant tissues around Zi abutments. Also, this in vitro study should be repeated under loading conditions to determine differential effect of loading on bidirectional microleakage around Zi abutments.

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9.

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11.

12.

ACKNOWLEDGMENTS The authors would like to thank Dr. M. Knopp and Ms. M. Carlton from Microimaging Laboratory, Department of Radiology, Wexner Medical Center, The Ohio State University, for their assistance in micro-CT and radiographic scanning. Implant materials used in this study was provided by Astra Tech Dental (currently Dentsply) through a grant (Grant #D-2009-27). This study was also supported by Divisions of Periodontology and Prosthodontics, OSU.

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REFERENCES 1. Adell R, Lekholm U, Rockler B, et al. A 15 year study of osseointegrated implants in the treatment of the edentulous jaw. Int J Oral Surg 1981; 10:387–416. 2. Adell R, Eriksson B, Lekholm U, Branemark PI, Jemt T. Long term follow up study of osseointegrated implants in treatment of totally edentulous jaw. Int J Oral Maxillofac Implants 1990; 5:347–359. 3. Albrektsson T, Zarb GA, Worthington P, Eriksson AR. The long-term efficacy of currently used dental implants: a review and proposed criteria of success. Int J Oral Maxillofac Implants 1986; 1:11–25. 4. Albrektsson T. On long-term maintenance of osseointegrated response. Aust Prosthodont J 1993; 7(Suppl):15–24. 5. Quirynen M, Van Steenberghe D. Bacterial colonization of the internal part of two stage implants. An in vivo study. Clin Oral Implants Res 1993; 4:158–161. 6. Zambon JJ. Periodontal diseases: microbial factors. Ann Periodontol 1996; 1:879–925. 7. Esposito M, Hirsch JM, Lekholm U, Thomsen P. Biological factors contributing failures of osseointegrated oral implants

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figures S1 Micro-computed tomography (CT) analysis: Pictures presented in this figure represent an example of studied details related to abutment-implant interface in zirconia (A) and titanium (B) abutmentimplant fixture apparatus at cross-section in three dimension. Micro-CT documentation was repeated prior to and following placement of paper filter dot placement inside implant chamber. Figures S2 High-resolution radiographic analysis: Pictures presented in this figure represent an example of studied details related to abutment-implant interface in zirconia (A) and titanium (B) abutment-implant fixture apparatus in two dimension. Figures S3 LPS microleakage study: Implants and all related components were unpacked under the flow hood using sterile conditions. The 0.5-μl LPS was placed inside implant chamber for test group using a pipette (A). Implant was placed inside a sterile metal holder and abutment was torqued at assigned torque value (B). C and D are representing zirconia abutment/implant fixture and titanium abutment/fixture assemblies during incubation. Figures S4 Determination of toluidine blue dye leakage with Colorimeter. Pictures presented in this figure represent paper filter dots retrieved from implant chamber following incubation within TBD. Colorimeter was used to determine color differences between samples. A is representing no detection of leakage (white color). B is representing significant leakage (dark blue).