Material and methods

The orthodontic mini-implants selected were Orthodontic Mini Implant (Leone, Florence, Italy), MTN (MTN, Istanbul, Turkey), AbsoAnchor (Dentos, Daegu, South Korea), IMTEC Ortho (3M Unitek, IMTEC, Ardmore, Okla), VectorTAS (Ormco, Glendora, Calif). The microstructure of the orthodontic mini-implants and temporary anchorage devices was investigated by using scanning electron microscopy and phase identification with energy dispersive x-ray analysis. A scanning electron microscope (Leo Evo-40 VP; Leo Electron Microscopy Ltd, Cambridge, UK) operated at 20 kV and an energy dispersive x-ray spectroscope (RÖNTEC, GmbH, Berlin, Germany) were used. X-ray images of the orthodontic mini-implants are shown in Figure 1 , and the details related to these products and their components are listed in Table I .

Scanning electron microscope energy-dispersive x-ray spectroscopy images of mini-implants used in this study.

Six samples were used for each group. The specimens were sterilized by using ultraviolet light and immersed in 7 mL of culture medium for 24 hours at 37°C to extract metal ions or cytotoxic substances. The culture medium containing the material extracts was filtered in a sterile environment for use on the cell cultures. The test procedures for this investigation were regulated by ISO-standard 10993-5.

The xCELLigence system (Roche Applied Science, Mannheim, Germany; ACEA Biosciences, San Diego, Calif) consists of 4 main components: the impedance-based real-time cell analyzer analyzer, the real-time cell analyzer single-plate station, the real-time cell analyzer analyzer computer with integrated software, and the disposable E-plate 96. The real-time cell analyzer analyzer single-plate station fits inside a standard tissue-culture incubator. The electronic impedance of the sensor electrodes is measured to allow monitoring and detection of physiologic changes of the cells on the electrodes. The voltage applied to the electrodes during real-time cell analyzer analyzer measurement was about 20 mV root mean square. The impedance measured between electrodes in a well depends on electrode geometry, ion concentration in the well, and whether cells are attached to the electrodes. In the absence of cells, electrode impedance is mainly determined by the ion environment both at the electrode-solution interface and in the bulk solution. In the presence of cells, cells attached to the electrode sensor surfaces act as insulators and thereby alter the local ion environment at the electrode-solution interface, leading to increased impedance. Thus, the more cells that are growing on the electrodes, the larger the value of electrode impedance. The data expressed in cell index units can be exported to Excel (Microsoft, Redmond, Wash) for any mathematical analysis.

Human gingival fibroblasts were isolated from human gingival connective tissue of systemically healthy subjects. The human gingival fibroblasts were obtained from samples of gingiva removed for crown lengthening purposes. All patients gave informed consent before providing the samples. This protocol was approved by Ethics Committee of the Faculty of Dentistry of Selçuk University, Konya, Turkey. Briefly, gingival tissues were cut into small pieces, rinsed with biopsy media, placed in tissue culture dishes, and incubated in the biopsy medium in a humidified atmosphere of 95% air and 5% carbon dioxide at 37°C overnight. The next day, the biopsy medium was replaced with culture medium (Dulbecco’s modified eagle’s medium [Biological Industries, Beit Haemek, Israel] with 10% fetal bovine serum, 100 units/mL of penicillin, 100 μg/mL of streptomycin). After reaching confluency, the cells were passaged with 0.25% trypsin and 0.1% ethylene diaminotetraacetic acid. Gingival fibroblasts were used between the fourth and sixth passages for all experiments.

MC3T3-E1 (subclone 4), an immortalized cell line provided by Dr Martha J. Somerman from the University of Washington and Renny T. Franceschi from the University of Michigan that was derived from newborn mouse calvaria, was used for the evaluation. The cells were incubated in α-MEM with 10% fetal bovine serum, 1% penicillin-streptomycin, and L-glutamine at 37°C in a humidified atmosphere of 5% carbon dioxide in air.

The materials were incubated in the culture medium for 72 hours according to ISO 10993-5 standards (surface area-to-volume ratio of the specimen to cell-culture medium, 3 cm ² /mL). Human gingival fibroblasts and MC3T3-E1 were maintained with Dulbecco’s modified eagle’s medium containing 10% fetal bovine serum. The real-time cell analyzer was used to evaluate cell survival, according to the supplier’s instructions. After seeding 200 μL of the cell suspensions into the wells (10,000 cells/well) of the E-plate 96, human gingival fibroblasts and MC3T3-E1 were treated with bioactive components released by the metallic alloy material and monitored every 15 minutes for 190 hours.

Results

First, we determined the optimal concentrations for cell proliferation and viability measurements. To this end, 40,000, 20,000, 10,000, and 5000 cells per well were seeded in the E-Plate 96, and the impedance was determined.

According to ANOVA, when left to elute for 25, 34, and 60 hours, there were no significant differences between the human gingival fibroblast cell indexes of the control and study groups ( P >0.05). But all testing groups were significantly different from the untreated control group at 190 hours ( P <0.01) ( Table II and Fig 2 ).

Dynamic monitoring of human gingival fibroblast cell adhesion and cell proliferation.

When evaluated at 27 and 96 hours, only the VectorTAS mini-implants showed statistically significant decreases in MC3T3-E1 cell index ( P <0.001) compared with the control group. There were no significant differences between the other study groups ( P >0.05). No significant differences were found among the control and all study groups ( P >0.05) ( Table III and Fig 3 ).

Table III

MC3T3-E1 cell index by real-time cell analysis and comparison of 27, 60, 96 and 190 hours with ANOVA and Tukey-Kramer multiple comparison tests

Group (n = 6)	27 h Mean ± SD	60 h Mean ± SD	96 h Mean ± SD	190 h Mean ± SD
Control	9.07 ± 0.45	11.66 ± 0.51	12.48 ± 0.24	11.35 ± 0.37
Leone	8.59 ± 0.61	11.83 ± 0.71	11.96 ± 0.46	4.45 ± 2.07 ∗
MTN	9.19 ± 0.19	12.46 ± 0.51	12.04 ± 0.21	6.95 ± 2.13 ∗
AbsoAnchor	9.15 ± 0.36	11.71 ± 0.45	12.08 ± 0.49	9.30 ± 1.85
IMTEC Ortho	8.46 ± 0.65	11.50 ± 0.54	11.91 ± 0.49	9.67 ± 1.15
VectorTAS	7.76 ± 0.44 ∗	10.83 ± 0.40	11.10 ± 0.62 †	8.50 ± 1.37 †

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

1. The best continuing education
2. Residents’ journal review
3. Cone-beam computed tomography is not the imaging technique of choice for comprehensive orthodontic assessment
4. Bone damage associated with orthodontic placement of miniscrew implants in an animal model
5. En-masse retraction dependent on a temporary skeletal anchorage device without posterior bonding or banding in an adult with severe bidentoalveolar protrusion: Seven years posttreatment
6. Modified bonding technique for a standardized and effective indirect bonding procedure

Stay updated, free dental videos. Join our Telegram channel

Join