Abstract
Objectives
The etiology of the reduced marginal bone loss observed around platform-switched implant-abutment connections is not clear but could be related to the release of variable amounts of corrosion products. The present study evaluated the effect of different concentrations of metal ions released from different implant abutment couples on osteoblastic cell viability, apoptosis and expression of genes related to bone resorption.
Methods
Osteoblastic cells were exposed to five conditions of culture media prepared containing metal ions (titanium, aluminum, vanadium, cobalt, chromium and molybdenum) in different concentrations representing the amounts released from platform-matched and platform-switched implant-abutment couples as a result of an earlier accelerated corrosion experiment. Cell viability was evaluated over 21 days using the Alamar Blue assay. Induction of apoptosis was measured after 24 h of exposure using flow cytometry. Expression of interleukin-6, interleukin-8, cyclooxygenase-2, caspase-8, osteoprotegerin and receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblastic cells were analysed after exposure for 1, 3 and 21 days using real-time quantitative polymerase chain reaction assay
Results
Metal ions in concentrations representing the platform-matched groups led to a reduction in cell viability ( P < 0.01) up to 7 days of exposure. Stimulated cells showed higher rates of early apoptosis ( P < 0.01) compared to non-treated cells. Metal ions up-regulated the expression of interleukin-6, interleukin-8, cyclooxygenase-2 and RANKL in a dose dependent manner after 1 day of exposure ( P < 0.05). The up-regulation was more pronounced in the groups containing the corrosion products of platform-matched implant-abutment couples.
Conclusion
Osteoblastic cell viability, apoptosis, and regulation of bone resorbing mediators were significantly altered in the presence of metal ions. The change in cytokine levels expressed was directly proportional to the metal ion concentration.
Clinical significance
The observed biological responses to decreased amounts of metal ions released from platform-switched implant-abutment couples compared to platform-matched couples may partly explain the positive radiographic findings in respect to crestal bone level when utilising the “platform-switching” concept, which highlights the possible role of corrosion products in the mediation of crestal bone loss around dental implants
1
Introduction
Dental implants have been widely used for the replacement of missing teeth in fully and partially edentulous patients. According to the American Academy of Implant Dentistry, 3 million people in the United States have dental implants and that number is growing by 500,000 a year . The use of endosseous dental implants was initiated by the discovery that these implants could be anchored in the jawbone with direct bone contact . In 1991, Zarb and Albrektsson described the osseointegration phenomena as “a process in which a clinically asymptomatic rigid fixation of alloplastic material is achieved and maintained in bone during functional loading” For proper osseointegration, several factors must be controlled , including biocompatibility of the implant material, design and surface of the implant, the condition of the tissues in the implant site, the surgical techniques, and loading procedures . Biocompatibility of an implant material is closely related to its susceptibility to corrosion . Therefore, titanium (Ti) has been the material of choice for dental implants due to its superior corrosion resistance behaviour and desirable mechanical properties .
An important parameter in the long-term success of dental implants is the stability of the peri-implant bone. Previous literature has showed that alterations in the connection geometry of the dental implant-abutment interface, such as platform-switching, may lead to a decrease in peri-implant bone loss that occurs through time . Platform-switching is defined as a protocol that includes smaller diameter restorative components that have been placed onto larger diameter implant restorative platforms – the outer edge of the implant-abutment interface is horizontally repositioned inwardly and away from the outer edge of the implant platform . Nevertheless, the etiology for this difference is still questioned. A recent study demonstrated an increase in the amount of metal ions released through accelerated corrosion from platform-matched compared to platform-switched implant-abutment couples.
The role of implant corrosion products in peri-prosthetic osteolysis has been extensively demonstrated in the orthopedic literature . This phenomenon may occur as corrosion and wear products can influence the metabolic pathways of various cells including macrophages, lymphocytes, fibroblasts, osteoclasts, and osteoblasts . Osteoblasts exposed to cobalt (Co) and chromium (Cr) ions undergo a dose dependent reduction in proliferation . Titanium (Ti) ions at concentrations of 10 ppm or higher for 24 h were found to be toxic . Additional past studies have demonstrated that nontoxic concentrations of metal ions influence the differentiation and function of osteoblastic cells in vitro .
Metal ions/particles may also stimulate osteoblasts to produce pro-inflammatory mediators that contribute to the overall inflammatory process involved in peri-prosthetic osteolysis . It has been shown that cobalt ions stimulate increased prostaglandin E2 (PGE2) secretion in primary human osteoblasts . This was preceded by up-regulated cyclooxygenase COX-1 and COX-2 gene expression . Secretion of interleukins 6 and 8 (IL-6 and IL-8) by osteoblasts in response to Ti and other experimentally derived wear particles/ions has also been previously reported . Receptor activator of nuclear factor kappa-B ligand (RANKL) is another important protein in peri-prosthetic osteolysis and acts by stimulating osteoclastogenesis . Osteoprotegerin (OPG) is an inhibitor of RANKL. Mine et al. revealed that Ti ions enhanced the expression of RANKL in osteoblast-like cells, suggesting that Ti ions may have adverse effects on bone remodelling at the interface of dental implants and tissues .
Although several investigations have documented the potential toxicity and the ability of metal ions/particles to stimulate cytokine production in cultured cell systems , little is known regarding cell apoptosis, or programmed cell death . It has been suggested that biocompatibility testing should include assessment of apoptosis which is featured by the stimulation of cysteine proteases called caspases. An in vitro study showed that Ti particles could induce apoptosis in osteoblasts which may lead to suppressed bone formation.
Although the orthopedic literature is replete with studies regarding the influence of corrosion products on the peri-prostheic tissues and cells , the dental literature, however, contains little information about the direct interaction between metal ions released from dental implants and osteoblasts from peri-implant tissues. This interaction may provide important insights into the pathogenesis of the observed marginal bone resorption around dental implants . In highly corrosive environments, such as the oral cavity, metals, including those of the implant and abutment materials, are prone to degradation . The combination of an acidic medium, due to inflammation, presence of acidogenic bacteria, fluorides or food intake, and the micromotion, resulting from occlusal forces, can lead to disruption of the oxide layer protecting the titanium surface . A recent study demonstrated that Ti implants connected to platform-matched abutments released significantly larger amounts of corrosion elements compared to implants connected to platform-switched abutments, following an accelerated corrosion process. The authors’ hypothesis was that this difference in corrosion may be significant on a cell metabolic level in order to change the peri-implant bone homeostasis .
The aim of the present study was to investigate the effect of such differences in metal ion concentrations on cell viability, apoptosis, and inflammatory gene expression of human osteoblastic cells cultured within conditioned culture media containing the different concentrations of metal ions obtained from the earlier study . The null hypothesis was that there would be no difference between groups regarding the aforementioned variables.
2
Materials and methods
2.1
Preparation of culture media containing metal ions
Five different conditions of culture media solutions were prepared containing different levels of metal ions obtained from the respective 5 groups of a recent study that evaluated the levels of metal ions released from different implant abutment couples as a result of accelerated corrosion . The metal ions corresponded to the following groups : implants connected to platform-matched titanium (Ti6Al4V) abutments (TM), implants connected to platform-switched titanium (Ti6Al4V) abutments (TSW), implants connected to platform-matched cobalt-chrome (CoCr) abutments (CM), implants connected to platform-switched cobalt-chrome abutments (CSW) and unconnected titanium implants (UI). The amount of mismatch was 0.5 mm between the platform-switched and platform-matched abutments . The concentrations of the measured elements which were used in this study are presented in Table 1 . To prepare culture medium containing these concentrations, single element standard solutions for ICP-MS for each measured element (titanium (Ti), vanadium (V), aluminum (Al), cobalt (Co), chromium (Cr) and molybdenum (Mo)) were utilized (TraceCERT ® , Sigma-Aldrich Company Ltd.,Dorset, England). Each single standard solution of each element was sterilized by passing through 0.22 μm membrane filters (Millex, Merck Millipore Ltd., Germany) before diluting in culture medium (Clonetics™ OGM™ BulletKit™, Lonza, Walkersville, MD, USA). To reach the desired concentrations of the test solutions, the single element standard solutions were diluted with the serum-added culture medium, under pH monitoring, according to the method described by Taira et al. . No visual precipitation was formed after adding the standard elements and the pH of the prepared solutions was measured immediately after preparation. Metal ion-free culture medium was used as a reference solution (REF) and served as the control group.
| Test Groups | Code | Levels of Metal Ions (ppb) | ||||||
|---|---|---|---|---|---|---|---|---|
| Ti | Al | V | Co | Cr | Mo | Total | ||
| Unconnected implant | UI | 998 | 998 | |||||
| Connected platform matched titanium alloy abutment (6 mm) | TM | 1250 | 67 | 60 | 1377 | |||
| Connected platform switched titanium alloy abutment (5 mm) | TSW | 1080 | 57 | 36 | 1137 | |||
| Connected platform matched cobalt-chrome abutment (6 mm) | CM | 678 | 219 | 27 | 10 | 934 | ||
| Connected platform switched cobalt-chrome abutment (5 mm) | CSW | 623 | 122 | 11 | 6 | 762 | ||
2.2
Cells and cell cultures
Osteoblastic cells were purchased from Lonza (Clonetics™ Normal Human Osteoblast Cell System, NHOst, Lonza, Walkersville, MD, USA). Cells were cultured in monolayer in osteoblast basal medium (OBM™ , Clonetics™ OGM™ BulletKit™, Lonza, Walkersville, MD, USA) containing 10% fetal bovine, 0.1% Gentamicin Sulfate/Amphotercin-B and 0.1% Ascorbic acid (OGM™ SingleQuot™, Lonza, Walkersville, MD, USA) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air. The culture medium was changed every two days. At 70–80% confluency, adherent cells were detached using 0.25% trypsin/EDTA solution (Trypsin EDTA, Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA). Osteoblastic cells of passages 4–6 were used for the experiments. All experiments were performed three times.
2.3
Cell viability assay
For viability experiments, the osteoblastic cells were transferred to 24-well plates and were seeded in triplicate at a density of 3000 cells/well. The cells were allowed to attach for 24 h, then the metal ion-free medium was replaced by the respective metal ion-containing medium. Cells incubated with metal ion-free medium served as test controls. The cell viability assay was conducted at time points of 1, 4, 7, 10, 14 and 21 days. After 21 days, RNA was extracted for later gene expression analysis. Cell viability at each time point was determined using Alamar Blue™ (AB) bioassay (AbD Serotec, UK). Absorbance measurements were performed at 560 nm and 590 nm using a microplate reader (FLx800, BioTek Instruments Ltd, UK). Reduction of AB by cells was calculated as the percentage reduction from the blue oxidized form of AB to red reduced form according to the following equation:
Percentage reduction o f alamar Blue = Fl 590 of test agent − Fl 590 untreated control F l 590 of 100 % redu c ed alamarBlue − Fl 590 untreated control × 100
Where: Untreated control is a cell-free culture media subjected to similar incubation conditions as the test groups and control (REF)
Fl 590 = Fluorescent intensity at 590 nm emission (560 excitation)
The resultant AB reduction percentages represented the percentage of cell viability and was used in statistics for viability comparison between test groups and the metal ion-free control (REF).
2.4
Flow cytometric analysis of early apoptosis
For apoptosis experiments, the cells were transferred to 24-well plates and were seeded in triplicates at a density of 50 000 cells/well. The cells were allowed to attach for 24 h, then the metal ion-free medium was replaced by the respective metal ion-containing medium for a period of 24 h. Cells incubated with metal ion-free medium served as test controls. At the end of the 24 h exposure period, the cells were collected by centrifugation and washed twice with phosphate buffer solution (PBS, BioWhittaker, Lonza, Belgium). The cells were then re-suspended in Annexin Binding Buffer and aliquots (5 μL) of Phycoerythrin Annexin V and Propidium Iodide Staining Solution (FITC Annexin V Apoptosis Detection Kit II, BD Pharmingen™, BD Bioscience, UK) were added to each test tube following manufacturer instructions. The samples were placed into a fluorescence activated cell sorting (FACS) flow cytometer (EPICS XL ® , Coulter Corporation, Florida, USA) for analysis. A minimum of 10,000 events in the target area was recorded for each sample.
2.5
Gene expression analysis
RNA levels of IL-6, IL-8, COX-2, Caspase-8, OPG and RANKL expressed by osteoblastic cells were analysed after incubation with metal ion-containing media for 24 h, 72 h and 21 days. Cells incubated with metal ion-free medium served as test controls. Cells were seeded onto 24-well plates in triplicates at different densities based on the exposure period to the metal ion-containing media. For the 24 h and 72 h exposure periods, cells were seeded at a density of 50 000 cells/well or 25 000 cells/well respectively. For the 21-day exposure period, RNA was extracted from the same cells that were initially seeded for the viability assay after conducting the 21 day time point viability analysis.
2.5.1
RNA extraction
After each exposure period, culture medium was removed from the wells, cells were washed twice with phosphate buffered saline solution (PBS, BioWhittaker, Lonza, Belgium) and were immediately lysed using the lysis buffer of an RNA extraction kit (RNeasy ® Plus Mini Kit, QIAGEN GmbH, Hilden, Germany). Total RNA was extracted according to the protocol of the manufacturer. Quantity and purity of the RNA were determined by 260/280 nm absorbance measurements using TECAN plate reader (Infinite M200, TECAN, GmbH, Austria) The remaining RNA was stored at −80° C until complementary DNA (cDNA) synthesis was performed.
2.5.2
cDNA synthesis
RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Thermo Fisher Scientific, NY, USA) according to the manufacturer instructions in a final reaction volume of 20 μL. cDNA was synthesized with total RNA (100 ng) and was amplified by polymerase chain reaction (PCR) in a thermal cycler (PTC-100™ Programmable Thermal Controller, MJ Research Inc., MA, USA). Thermal cycling conditions were as follows: 10 min at 25 °C, 120 min at 37 °C, 5 min at 85 °C after which temperature gradually drops to 4 °C. The resulting cDNA was stored at −20° C until further analysis.
2.5.3
Real-time quantitative polymerase chain reaction (RT-qPCR) assays
For RT-qPCR, 5 μL of the abovementioned diluted cDNA was added to TaqMan Fast Universal PCR Master Mix and TaqMan Gene Expression Assay primer/probe mixes (TaqMan ® Gene Expression Assays, Applied Biosystems™, Thermo Fisher Scientific, NY, USA) according to the manufacturer’s instructions to achieve a final reaction volume of 25 μL. Gene expression was measured using primer–probe sets specific for human IL–6 (Hs00985639_m1), IL–8 (Hs00174103_m1), COX-2 (PTGS2) (Hs00153133_m1), Caspase 8 (Hs01018151_m1), RANKL (TNFSF11) (Hs00243522_m1), OPG (TNFRSF11B) (Hs00900358_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Hs03929097_g1) by means of RT-qPCR using 7300 Real Time PCR System (Applied Biosystems™, Thermo Fisher Scientific, NY, USA). Gene specific primers and the TaqMan qPCR mastermix for FAM™ reporter dye were purchased from TaqMan ® (TaqMan ® Gene Expression Assays, Applied Biosystems™, Thermo Fisher Scientific, NY, USA). Each cell sample was assayed for each gene a minimum of three separate times in 96-well optical plates with primer concentrations of 0.8 mM. The PCR protocol consisted of: initiation at 1 cycle at 50 °C for 2 min and 1 cycle at 95 °C for 10 min, followed by amplification for 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Ct data were collected via Sequence Detection Software 1.4 (7300 System SDS software RQ Study Application, Applied Biosystems). Gene expression was normalized to housekeeping gene (GAPDH) and expressed relative to the reference control group (REF) for each incubation time using the 2 −ΔΔCt method . The ratio of RANKL/OPG was calculated by dividing the normalized fold expression of both genes within the same sample. Since the experiment was performed in triplicate and the PCR reactions were also performed in triplicate, there were 54 data points collected
2.6
Statistical analysis
All data were expressed as mean and standard deviation. For viability and apoptosis analysis, statistically significant differences were tested by univariate analysis of variance (ANOVA) using SPSS version 22.0 (IBM SPSS Statistics, IBM, Tokyo, Japan) ( P < 0.05). For gene expression analysis, statistically significant differences were tested by multivariate repeated measures (ANOVA) statistical model where all 54 data points were used to fit this model. Comparisons were performed between each test group and the control (REF) within each incubation period and between the platform-matched groups and the platform-switched groups within each abutment material and within each incubation period. Levene’s test of homogeneity of variance was employed (α = 0.05), following the assumption of equal variances. When equal variances were assumed ( P > 0.05) the Bonferroni post hoc test was used to analyze significant differences between test groups. Whereas when equal variances were not assumed ( P < 0.05) the Dunnett’s T3 post hoc test was used to analyze significant differences between the test groups. To confirm statistically significant differences between the platform-matched groups and the platform-switched groups within each abutment material, t -test for two independent samples was used ( P < 0.05).
3
Results
3.1
Effect of metal ions on cell viability
The percentages of reduction reaction from the blue oxidized form of AB to red reduced form over the 21-day time period, which represented the percentages of cell viability, are presented in Fig. 1 . There was statistically significant lower cell viability in the TM ( P < 0.001), TSW ( P < 0.001) and CM ( P < 0.01) groups compared to the control (REF) after 24 h of exposure as well as on day 4 ( P < 0.001) and day 7 (TM and TSW ( P < 0.001), CM ( P < 0.05)). The platform-matched CoCr abutment group (CM) also showed lower cell viability compared with the platform-switched group of the same material (CSW) on day 4 ( P < 0.01). On day 10, the TM was the only group that showed less cell viability when comparing with the control (P < 0.05). After 14 days of exposure, all test groups did not differ in their cell viability from the control ( P > 0.05). However, on day 21, the CM and CSW groups had significant lower cell viability than the control ( P < 0.001).
3.2
Effect of metal ions on early apoptosis
All groups of osteoblastic cells exposed to metal ion-containing media showed significantly higher percentage of apoptosis after 24 h compared to the control (UI, TM, CM, and CSW ( P < 0.001), TSW ( P < 0.005)) ( Fig. 2 ). The percentage of apoptotic cells did not differ significantly between the different ion concentration groups ( P > 0.05)
