|Year : 2016 | Volume
| Issue : 1 | Page : 6-13
Effects of radiation doses from different dental imaging modalities on cell-implant interaction: A comparison with cell culture study
Mehmet Hakan Kurt1, Bengi Oztas1, Arzu Atalay2
1 Department of Dentomaxillofacial Radiology, Faculty of Dentistry, Ankara University, Ankara, Turkey
2 Institute of Biotechnology, Ankara University, Ankara, Turkey
|Date of Web Publication||19-Feb-2016|
Mehmet Hakan Kurt
Department of Dentomaxillofacial Radiology, Faculty of Dentistry, Ankara University, Ankara
Source of Support: None, Conflict of Interest: None
Objectives: The effect of radiation after exposing with dental imaging devices on cell-implant material interaction has not been adequately studied. The aim of this study is to determine the effects of radiation by measuring proliferation and differentiation of the human fetal osteoblast (hFOB) cells using dental imaging techniques and evaluate the result in vitro conditions at cellular stage. Materials and Methods: hFOB were irradiated 1 day after planting on commercially pure titanium discs. Radiation was single dose in one exposure using five different dental imaging techniques as conventional dental and digital dental X-ray tubes, conventional panoramic, digital panoramic and cone beam computerized tomography (CBCT). In the 8 th day of experiment, exposured cells and nonexposured control group cells which planted on discs were compared and examined regarding their proliferations and alkaline phosphatase (ALP) activities. Results were evaluated using Student's Independent t-test in SPSS 15.0 (SPSS Inc., Chicago, IL, USA). Results: In exposure with digital dental imaging device, ALP activities of cells are increased according to the control group and statistically, with 0, 2 s of exposure, a significant increment was found (P < 0.05). The exposures with CBCT and conventional dental imaging devices decreased cell proliferation compared with the control groups, but it was found statistically insignificant. In exposure with the other dental imaging devices, cell proliferation increased insignificantly. Conclusions: Our in vitro study suggests that the ionizing radiation produced by digital dental imaging devices may result to certain increment of the cell number and activities. More controlled study should be made with cell cultures in order to verify the exact activity of digital dental imaging.
Keywords: Dental implant, osseointegration, osteoblast cell, radiation, spectrophotometer, X-ray
|How to cite this article:|
Kurt MH, Oztas B, Atalay A. Effects of radiation doses from different dental imaging modalities on cell-implant interaction: A comparison with cell culture study. J Oral Maxillofac Radiol 2016;4:6-13
|How to cite this URL:|
Kurt MH, Oztas B, Atalay A. Effects of radiation doses from different dental imaging modalities on cell-implant interaction: A comparison with cell culture study. J Oral Maxillofac Radiol [serial online] 2016 [cited 2023 Mar 25];4:6-13. Available from: https://www.joomr.org/text.asp?2016/4/1/6/177053
| Introduction|| |
Dental implants have become a favorable option in the treatment of edentulous patients in last decade. Numerous published studies related to the dental implant treatment have reported successful results. The successful outcome of implant osseointegration involves patient-originated and procedure-dependent variables. The quality of the bone and the type of surgical procedure are of primary factors for long-term survival of implants. 
After implant placement or initial healing of the bone site, radiographies (X-rays) are used to verify the presence of bone adjacent to the implant.  Bone quality and quantity are generally estimated from radiographs during/after implant placement session. For objective preoperative assessment of the bone, the ideal approach is the evaluating the relative distribution of compact and cancellous bone with radiographies. 
The initial interaction between ionizing radiation and matter occurs at level of the electron within the first 10-13 s after exposure.  Biologic molecules absorb the energy form ionizing radiation and form unstable free radicals. Free radicals are extremely reactive and quickly reforming into stable configurations. Generation of free radicals leads to cell damage.  Moreover, radiation scatter from high atomic number materials (such as implants) are tended to constitute affects both soft tissue and bony complications in the oral cavity after exposure.
The possibility of the negative effect of ionizing radiation on the healing and remodeling of bone is well documented.  However, only limited researches were performed in head and neck region especially regarding the head and neck radiotherapy. The dose enhancement from scattered radiation at bone-dental implant interfaces during simulated head and neck radiotherapy was examined and concluded that dose enhancement factor that may contribute to osteoradionecrosis.  In a recent review, the risks of irradiation regarding dose levels, timing of radiation, implant location, and materials were searched. They concluded that irradiated bone has a greater risk of implant failure than nonirradiated bone.  Although limited information is available on the effects of the ionizing radiation on the initial responses of osteoblast cells, including the tissue-implant interface zone in the literature. 
The effect of ionizing radiation on early stages of the proliferation and differentiation of the osteoblast cells on and around dental implant surfaces are not well known.  To the best of our knowledge, the first step of osseointegration depends on proliferation and after that differentiation of the osteoblast cells. No study was found regarding the effects of radiation on osteoblast cell activity (proliferation and differentiation) at single exposure by using dental imaging techniques.
Hence, it was considered worthwhile to evaluate the effect of the ionizing X-ray irradiation that was emitted from dental imaging devices using in vitro model with cellular stages.
| Materials and Methods|| |
Study design and sample
The present study was an in vitro cell cultured study. The cell culture was made by HK under supervision of AA who is a 20-year experienced on molecular biology and genetics. Custom made titanium (Ti) disks were prepared in order to simulate the implant material. For this purpose, commercially available pure Grade IV Ti was used. 1.0 mm thick with a 22 mm diameter disks were prepared. The disks were sand-blasted with 50-150 μm size alumina (Al 2 O 3 ) particles, blown under 5 bars of pressure to develop smooth or rough surfaces and cleaned mechanically.
All disks were washed in ultrasonicated bath with 99.8% ethanol 2 times and then washed for another 15 min in double distilled H 2 O. All disks were autoclaved. The disks were sterilized again using ultra violet (UV) for 15 min before of the experiments. Randomly selected Ti disks were evaluated with a JSM-6400 electron microscope (JEOL, Japan) equipped with the NORAN 6 X-ray Microanalysis System and Semafore Digitizer for surface characterization [Figure 1]. Energy-dispersive X-ray spectroscopy (EDS) analysis was conducted for the surface properties [Figure 2].
|Figure 1: Sand-blasted titanium surface (×1000, magnification with electron microscopy)|
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Osteoblast cell cultures
Human fetal osteoblast (hFOB 1.19) cells (ATCC, US) were cultured on 100 mm dishes (Sarstedt, Germany) in α-Mem media (Hyclone, Thermo Scientific, US), containing penicillin (+10,000 U/ml) and streptomycin sulfate (10,000 μg/ml) and 10% fetal bovine serum (Hyclone, Thermo Scientific, US) at 37°C in the presence of 5% carbon dioxide. The media was changed twice weekly. At confluence, cells were trypsinized using 0.25% Trypsin-EDTA (Hyclone, Thermo Scientific, US). The cells were counted using hemocytometer (Sigma Bright-line, US) and were seeded as 50,000 cells/well on Ti disks in 6-well dishes (Sarstedt, Germany). The day of planting was considered as the day 0 of the experiment [Figure 3].
|Figure 3: The microscopic appearance of seeded human fetal osteoblast 1.19 cells around titanium discs|
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Day 1: The experiment cells were irradiated using five different dental imaging devices including conventional dental and digital dental X-ray tubes, conventional panoramic, digital panoramic and cone beam computerized tomography (CBCT). The specifications and exposure factors (kVp, Ma, seconds) were noted. Various kVp, mA, and exposure times were used for conventional and digital imaging modalities. For CBCT imaging, various field-of-view and voxel sizes were used.
Radiation dose measurements of the each exposure parameters were confirmed by placing ionization chamber at the level of the platform together 6-well dishes. Nonirradiated cells which seeded on disks in the 6-well dishes were set as control groups for each test on the day 0 of the experiment.
On the day 8 of experiment like Ahmad et al. did in their study,  exposured cells and nonexposured control group cells were examined regarding proliferations and alkaline phosphatase (ALP) activities. Wells (n = 3 per group) were washed twice with α-Mem media (Hyclone, Thermo Scientific, US) to remove nonattached cells. The adherent cells were trypsinized using 0.25% Trypsin-EDTA (Hyclone, US), and then counted using hemocytometer (Sigma Bright-line, US). All samples were investigated and the average number of cells was calculated.
Alkaline phosphatase assay
In the 8 th day intracellular ALP activity was measured for each sample with a commercially available ALP kit (Abcam, US). 10 5 washed cells were homogenized in the 200 μl Assay Buffer and centrifuged to remove insoluble material at 13,000 g-force for 3 min. For the ALP activity, 50 μl of the samples were added in a 96-well dish, after that 50 μl of the 5 mM pNPP solution were added on to each well containing the test samples and the dish was mixed gently. The mixture was incubated for 60 min at 25°C and protected from light. The optic density at 405 nm was measured with a spectrophotometer (Spectramax M2, US).
All of the experiments were made triplicate and statistical analysis was conducted using Student's Independent t-test using SPSS software version 15.0 (SPSS, Inc., Chicago, IL, USA), and significance was considered when P < 0.05.
| Results|| |
Radiation dose measurements
The radiation dose measurement results obtained using ionization chamber of different dental X-ray imaging devices were shown in [Table 1].
|Table 1: The radiation values of the dental imaging devices with different exposure parameters measured by ionization chamber|
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Effects of radiation dose on osteoblast proliferation
The Ti disks were found biocompatible and hFOB 1.19 cells grew on their surfaces. The results of cell proliferation after irradiation were cultured as shown in [Figure 4]. Proliferation assays revealed that exposure with CBCT and conventional dental imaging devices decreases the cell proliferation compared with nonirradiated control groups. This reduction was not statistically significant. Digital dental imaging, digital panoramic, and conventional panoramic devices were increased cell proliferation, without a significant difference (P > 0.05) in [Table 2].
|Figure 4: Results of the irradiated cell proliferation with different dental imaging modalities compared with nonirradiated control groups|
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|Table 2: P values of the irradiated hFOB 1.19 cell proliferation compared with nonirradiated cells (P < 0.05)|
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Effects of radiation dose on alkaline phosphatase activity
[Figure 5] summarizes the effects of radiation on ALP activity which measured by using ALP kit (Abcam, US) and spectrophotometer (Spectramax M2, US). ALP activities of irradiated hFOB 1.19 cells are decreased on the 8 th day of the experiments (but statistically insignificant, P < 0.05) in all imaging devices except digital dental imaging procedures compared to the nonirradiated control groups shown in [Table 3]. In exposure with digital dental imaging device, the increased enzyme activities of cells were observed according to the control group. While a statistically significant increment has found (P < 0.05) with 0.2 s of exposure, the other two parameters' increment were negligible.
|Figure 5: Results of the irradiated cells' alkaline phosphatase activity using different dental imaging modalities compared with nonirradiated control groups|
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|Table 3: P values of the irradiated hFOB 1.19 cells' alkaline phosphatase activity compared with nonirradiated cells (P < 0.05)|
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| Discussion|| |
The present study demonstrated the effects of X-ray radiation doses produced by dental imaging devices at a single exposure on hFOB 1.19 osteoblast cells. The proliferation and ALP activity of the irradiated and nonirradiated osteoblast cells were evaluated for this purpose.
During past decades, many studies reported immature bone and healing bone affected negatively after exposed with ionizing irradiation (2-5 Gy) but no significant effect on mature bone tissues. ,, This situation supports the classic theory that immature osteoblasts are extremely radiosensitive than mature osteoblast cells.  The term osseointegration is used for mandatory interaction of the bone cells with implant surface. It is necessary that mature bone is deposited directly on implant materials for osseointegration. 
In recent years, some investigators reported the effects of therapeutic radiation doses on osseointegration; however, the doses were extremely high. The success rates of endosseous implants were high, however approximately 5-7% implants failed in patients who underwent radiotherapy, ,, mainly due to the lack of osseointegration. Although the knowledge of the adverse effect of ionizing radiation on preosteoblasts, the importance of the radiologic examination after implant surgery should not be undeniable. Brägger  emphasized the importance of the radiologic examination for evaluating dental implants' stability and success. Batenburg et al.  and Urban et al.  investigated the bone resorption around loaded dental implants with intraoral radiographs. Tyndall et al.  reported that CBCT imaging should not be used for postoperative periodic controls of clinically asymptomatic dental implants in a consensus.
Many in vivo and in vitro studies have been performed to understand the mechanisms of bone cells behavior on implant materials. The cell proliferation, ALP activity, cell attachment, collagen synthesis, DNA content, mineralization, and extracellular matrix protein synthesis are some of the utilized parameters to determine cell responses to biomaterials. ,,,,,
The limitation of this study was using only cell counting for evaluation of cell proliferation and ALP activity for differentiation of osteoblast cells. However, as stated above, DNA content evaluation and DNA quantification which should be done for further studies.
Hamada  stated that limited information of radiation effect on osseointegration. Dare et al.  discouraged the diagnostic radiation for immediate postsurgical assessment of osseointegrated dental implants, due to the possibility of detrimental effects of ionizing radiation on healing and remodeling of bone. They exposed the cells with a single dose of X-rays at 40, 100, 400, or 4000 mGy, respectively, from a linear accelerator radiotherapeutic machine (Linac) or a 40 mGy dose from a diagnostic chest X-ray machine. After the irradiation, they investigated the proliferation and ALP activity of the cells. These results indicated that ionizing radiation at a single dose of up to 400 mGy induced no significant changes in cell growth and differentiation of osteoblast-like cells. Some researchers investigated the effects of gamma irradiation and Cesium 137 on osteoblast cell activities. , They found that high doses of gamma radiation decreased the cell proliferation and increased ALP activity of the osteoblast-like cells. Ahmad et al.  signalized high expression of ALP following high dose of irradiation with a probably a defense mechanism.
In many radiation-osteoblast studies, gamma irradiation was used. All the radiation doses were high doses compared to X-ray doses produced by dental imaging devices and different radiation types have different effects on osteoblast activities as seen in the literature. ,,
In the present study, we found that X-ray doses produced by dental imaging devices (except dental X-ray tube and CBCT) increased the cell proliferation compared with nonirradiated control cells but this increment was not statistically significant. The radiation doses of dental X-ray tube or CBCT decreased the cell growth, but this decrement was statistically insignificant. The X-ray radiation doses of dental imaging devices have no any effect on osteoblast cell proliferation at cellular stage.
ALP activity is a marker for osteoblast differentiation and the cells produces this enzyme for the early osteogenesis. Our results showed that X-ray doses produced by dental imaging devices (except digital dental X-ray tube and CBCT) decreased the ALP activity compared with nonirradiated control cells but this increment was not statistically significant. Therefore exposure with these devices does not affect osteoblast differentiation statistically. Radiation doses of CBCT exposures decreased the ALP activity compared with nonirradiated cells but it is not also significant statistically. When the radiation doses increased, the ALP activity of the osteoblast cells reached nonirradiated cells' ALP activity levels. This situation disclosed with a possible defense mechanism of the cells against high dose radiation as Ahmad et al.  reported. Radiation doses, produced by digital dental X-ray tube, were increased the ALP activity of the osteoblast cells. Irradiation with 0.05 and 0.1 s exposure times insignificantly increased the ALP activity. These radiation doses are insufficient causing any meaningful changes in osteoblast cells. Exposure time settings at 0.2 s was found statistically significant (P < 0.05). Digital dental X-ray radiation dose values are very low, and very low doses (required sufficient radiation doses) of X-ray radiation change the behavior of cells.
The increment of cells' ALP activity and cell proliferation enhancement, especially using digital dental X-ray device, indicates that low dose X-rays have a possible stimulating effects. The radiation doses of dental X-ray devices are reliable for imaging postoperative control of dental implants based on this information.
Only a few studies report on the effects of radiation on osteoblasts in the literature. Radiation-osteoblast studies have used several osteoblast lineages. MC3T3-E1 cells are of murine origin, and thus may not demonstrate all the reactions of human osteoblasts (HOB). HOB cell lines can be suitable for radiation studies.  The handicaps of primary cells are their heterogeneity, lack of reproducibility and the limited harvesting from a particular patient. In addition, primary osteoblasts retain their phenotype for a relatively short period of time compared to osteoblast cell lines.  Therefore transformed hFOB was a better choice for the current study. In vitro cell culture studies have some advantages, including relatively well-controlled variables or to allow parameters that are difficult to study in vivo. The primary bone cells or immortal cell lines have been used for in vitro investigations.  This culture system is a suitable model for the investigation of osteoblast differentiation or proliferation.
Cell proliferation is usually measured by clonogenic assay in radiation biology. Counting colony formation is the preferred method of evaluating radiation effects. Mesenchymal stem cells that do not form colonies require other assays to determine cell growth. , Other radiation-osteoblast studies used cell counting method,  colorimetric assay,  or MTT assay.  To conduct a clonogenic assay, the culture surface must be transparent. Clonogenic assay is not suitable on opaque implant metals. Therefore, we used cell counting to determine proliferation with hemocytometer.
The surface and topography of dental implants affect cell growth, attachment or mineralization of the cells. This purpose proved in many studies. Cell growth is higher on roughed Ti surfaces than normal surfaces. ,,, Hakki et al.  showed that osseointegration is higher on smooth sand-blasted surfaces than rough sand-blasted surfaces. Therefore we made smooth sand-blasted surfaces on Ti disks in our study. Park et al.  emphasized the effect of Ti implants' sterilization and cleaning. Their results indicated that recleaned and resterilized Ti implant surfaces cannot be considered the same as the first surfaces. The refuse of Ti implants after resterilization may not result in the same tissue responses as found with never-before-implanted specimens. Therefore we used the disks only 1 time in the present study. UV irradiation on Ti surfaces enhanced the bioactivity of the osteoblast cells. Aita et al.  and Li et al.  showed the positive effect of UV on bioactivity of the osteoblast cells. They expressed this positive influence with the effect of UV on hydrocarbons. The UV decomposed the hydrocarbons on Ti surfaces by photo catalysis. The UV promoted osteoblast cell attachment, proliferation and differentiation. In addition, UV irradiation was recognized as a trustworthy method for surface cleaning without any change of topography. We preferred cleaning the disks with UV after autoclave sterilization in our study.
To the best of our knowledge, this is the first in vitro study on the interaction of irradiated (using dental X-ray devices) cells to Ti surface. Primary focus of this study was to determine the radiation doses of dental imaging methods and culture period that affects cell-implant interaction at cell stage. Although in vitro studies are important and should be conducted prior to in vivo or clinical studies, in vitro investigations do not necessarily reflect a clinical situation.
| Conclusion|| |
Our in vitro study suggests that the ionizing radiation produced by digital dental imaging devices may result to certain increment of the cell number and activities. After all, the most importing situation is the stochastic effects. Hence, the clinicians must be careful using the recurrent exposures for routine postoperative control. More controlled study should be made with cell cultures to verify the exact activity of digital dental imaging.
Ankara University Scientific Research Projects Coordination Agency (Project No: 11B3334002) supported this study. Cell cultures were made in Cell Culture Laboratory at Ankara University Institute of Biotechnology. This study was awarded as the best oral presentation and study on VI. Turkish Oral Diagnosis and Dentomaxillofacial Radiology Symposium, 15-17 April 2015, İzmir, Turkey.
Financial support and sponsorship
Ankara University Scientific Research Projects Coordination Agency (Project No: 11B3334002) supported this study.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]