Cytotoxicity of alloying elements and experimental titanium alloys by WST-1 and agar overlay tests

Abstract

Objective

This study was performed to evaluate the biocompatibility of nine types of pure metals using 36 experimental prosthetic titanium-based alloys containing 5, 10, 15, and 20 wt% of each substituted metal.

Methods

The cell viabilities for pure metals on Ti alloys that contain these elements were compared with that of commercially pure (CP) Ti using the WST-1 test and agar overlay test.

Results

The ranking of pure metal cytotoxicity from most potent to least potent was: Co > Cu > In > Ag > Cr > Sn > Au > Pd > Pt > CP Ti. The cell viability ratios for pure Co, Cu, In, and Ag were 13.9 ± 4.6%, 21.7 ± 10.4%, 24.1 ± 5.7%, and 24.8 ± 6.0%, respectively, which were significantly lower than that for the control group ( p < 0.05). Pure Pd and Pt demonstrated good biocompatibility with cell viabilities of 93.8 ± 9.6% and 97.2 ± 7.1%, respectively. The Ti–5Pd alloy exhibited the highest cell viability (128.4 ± 21.4%), which was greater than that of CP Ti. By alloying pure Co or Cu with Ti, the cell viabilities for the Ti– x Co and Ti– x Cu alloys increased significantly up to 10 wt% of the alloying element followed by a gradual decrease with a further increase in the concentration of the alloying element. Based on the agar overlay test, pure Ag, Co, Cr, Cu, and In were ranked as ‘moderately cytotoxic’, whereas all Ti alloys were ranked as ‘noncytotoxic’.

Significance

The cytotoxicity of pure Ag, Co, Cr, Cu, and In suggests a need for attention in alloy design. The cytotoxicity of alloying elements became more biocompatible when they were alloyed with titanium. However, the cytotoxicity of titanium alloys was observed when the concentration of the alloying element exceeded its respective allowable limit. The results obtained in this study can serve as a guide for the development of new Ti-based alloy systems.

Introduction

Metallic materials are used widely in dentistry for restorative materials, prostheses, orthodontic appliances, surgical fixation plates, wires, and implants . These materials remain in intimate contact with living cells for long periods of time. During their clinical service, these alloys can affect the cell viability, and the released ions can evoke toxic, allergic, inflammatory, and/or mutagenic reactions . Recently, commercially pure titanium (CP Ti) and its alloys have been selectively used for dental prostheses and implant-supported crowns because of their biocompatibility, strength, and corrosion resistance . However, further improvements in CP Ti are required to overcome its shortcomings such as higher elastic modulus than that of the natural bone, low wear resistance, excessive oxide formation, poor castability, and poor grindability . Correspondingly, many experimental titanium alloys have been developed for dental use, and studies of their properties have been focused mainly on improvement of their strength and castability . The biocompatibility of these alloys with human tissues is a prerequisite for their use as biomaterials, and special attention should be paid to their effects on specific cellular functions. For the development of new metallic biomaterials with superior biocompatibility, a systematic and quantitative evaluation of the cytotoxicity of potential alloying elements is required.

Previous reports identified possible biocompatibility problems of dental alloys including those of Ti–Al–V alloys . The cytotoxicity of metallic biomaterials has been examined using extracted solutions of the materials that contained metallic compounds released from the metals. Alternatively, metal salts or solutions of metal cations have been used also for cytotoxicity tests to avoid a multiple evaluation of an element’s cytotoxicity . In some reports, the materials themselves or their particulate form have been used for the cytotoxicity evaluation. Results that were obtained previously by several different methods and the types of cell lines cannot be compared directly with each other. Furthermore, these previous results are contradictory . In the present study, the cytocompatibility of the bulk alloy was evaluated.

It is well known that the cytocompatibility of an element can be altered by the alloying elements. For example, palladium has been shown to reduce the tendency for release of copper from Pd–Cu alloys . Moreover, the mass release of a particular element is generally not related to its atomic concentration in the alloy. Thus, cytocompatibility responses should be evaluated with the bulk alloys. Moreover, assays made with pure metals are useful to study the effect on the cell behavior for each component in an alloy . Because information on the cytotoxicity of Ti alloys is limited, this study was performed to determine whether the alloying elements will be less cytotoxic when combined with Ti by evaluating the biocompatibility of nine types of alloying elements and 36 experimental Ti alloys using WST-1 and agar overlay tests.

Materials and methods

Preparation of specimens

Binary Ti– x A alloys varying in concentration of substituting metal A in Ti (where A represents the concentration of 5, 10, 15 or 20 wt% of Ag, Au, Co, Cr, Cu, In, Pd, Pt, and Sn) were fabricated using vacuum arc melting on a water-cooled hearth ( Table 1 ). The alloy specimens were homogenized for 4 h at temperatures 150 °C below to the respective alloy’s solidus temperature followed by cooling at a rate of 10 °C/min to 600 °C, followed by air-cooling to room temperature. This thermal treatment was performed in a tube furnace under a high purity argon atmosphere. The prepared alloy buttons were embedded in epoxy resin and cut into disks with a diameter of 10 mm and a thickness of 1.2 mm. The disks were polished successively through 2000 grit SiC abrasive, and ultrasonically cleaned in acetone, ethanol, and distilled water. Then, the disks were sterilized by exposing them to ultraviolet light for 30 min on a clean bench prior to the test. The CP Ti was used as a control.

Table 1
Pure metals used.
Raw material Specification Lot no. Manufacturer
CP Ti Rod, 10 mm D, ASTM B265 04RB-10 Daito Steel Co, Japan
Titanium Sponge, 3 mm and down 99.9% 129Q001 Alfa Aesar, USA
Chromium Pieces, 2–3 mm thick, 99.995% 38494 Alfa Aesar, USA
Cobalt Pieces, 99.9+% 10454 Alfa Aesar, USA
Copper Shot, 13 mm D, 99.99%, oxygen free 36686 Alfa Aesar, USA
Gold Plate, 99.99% 10041w0067 LS-Nikko, Korea
Indium Plate 99.995% 0907 LS-Nikko, Korea
Palladium Pieces, 99.95% 100413 LS-Nikko, Korea
Platinum Plate 99.8% 100830 LS-Nikko, Korea
Silver Silver shot, 1–5 mm, Premion ® , 99.99% 12186 Alfa Aesar, USA
Tin Granules, ACS, 99.9% 36691 Alfa Aesar, USA

Cell viability

The viability of cells was analyzed using a colorimetric assay for quantification of the cleavage of the tetrazolium salt WST-1 (Roche, Germany) by mitochondrial dehydrogenases. The resulting dye can be quantified by a spectrophotometer and directly correlated with the number of metabolically active cells in the culture. For the test, L-929 mouse fibroblast cells were grown on a 100 mm-diameter Petri dish containing 10 mL RPMI1640 (with 10% FBS). When enough cells were produced, 100 μL of the cell suspension at a cell density of 5 × 10 4 cells/mL were seeded on the test samples and incubated at 37 °C under a 5% CO 2 atmosphere. After 24 h of incubation, the cells were detached with 10 μL of 0.25% Trypsin-EDTA solution (Gibco, Canada) for 3 min, and 100 μL RPMI1640 were added per well. Then, 100 μL of the mixture were transferred into a 96-well culture plate, 10 μL WST-1 solutions were added to each well, and the cells were incubated at 37 °C in 5% CO 2 for 4 h. After the reaction period, the specimens were shaken for 1 min and absorbance was measured at 450 nm by a microplate reader (Bio-RAD, USA). Seven separate analyses were performed.

Agar overlay test

Cell suspensions in 10 mL L-929 at a cell density of 3 × 10 5 cells/mL were seeded in 100 mm-diameter cell culture dishes, and incubated to confluence at 37 °C in 5% CO 2 . After a 24-h incubation process, the medium was replaced with 10 mL of freshly prepared agar/nutrient medium containing RPMI1640, 5% FBS, and 3% agarose mixture. A 10 mL neutral red solution (0.01% in phosphate-buffered saline, Sigma, USA) was added and the cells were incubated for 15 min at room temperature. Excess dye was then removed, and the test specimens were placed on the agar surface. A 0.25% ZDBC polyurethane film was used as a positive control and a polyethylene sheet as a negative control. The dishes were incubated for 24 h at 37 °C in 5% CO 2 . Thereafter, the cultures were examined under a microscope.

Neutral red is a weak cationic dye that readily diffuses across plasma and organelle membranes, accumulating in the lysosomes. Any loss of membrane integrity induced by a toxic substance will result in decreased retention of neutral red dye. Damaged or dead cells are decolorized compared with healthy control cells . The decolorized zones and cell lysis around and/or under the specimens were evaluated according to ISO 7405 . Each test was repeated five times. The decolorized zones were scored as follows: 0, no decolorization detectable; 1, decolorization only under the specimen; 2, decolorization zone not greater than 5 mm from the specimen; 3, decolorization zone not greater than 10 mm from the specimen; 4, decolorization zone greater than 10 mm from the specimen; and 5, total culture is decolorized. Cell lysis was defined as loss of cell membrane integrity. Cell lysis was scored as follows: 0, no cell lysis detectable; 1, less than 20% cell lysis; 2, 20–40% cell lysis; 3, >40% to <60% cell lysis; 4, 60–80% cell lysis; and 5, more than 80% cell lysis. For each specimen, the median score from each specimen was calculated for both the decolorization zone index and the lysis index. The cytotoxicity was classified as follows: 0–0.5, noncytotoxic; 0.6–1.9, mildly cytotoxic; 2.0–3.9, moderately cytotoxic; and 4.0–5.0, markedly cytotoxic. The median values were calculated to describe the central tendency of the scores because the results were expressed as an index in the ranking scale .

Statistical analysis

Data were expressed as a mean ± standard deviation (SD) for each of the seven tests. Version 19.0 of the statistical software, SPSS (SPSS, Inc., an IBM Company, Chicago, Illinois, USA), now called “IBM SPSS Statistics”, was used to analyze the data from the WST-1 test by means of the Kruskal–Wallis one-way analysis of variance and Duncan’s multiple range test with α = 0.05.

Materials and methods

Preparation of specimens

Binary Ti– x A alloys varying in concentration of substituting metal A in Ti (where A represents the concentration of 5, 10, 15 or 20 wt% of Ag, Au, Co, Cr, Cu, In, Pd, Pt, and Sn) were fabricated using vacuum arc melting on a water-cooled hearth ( Table 1 ). The alloy specimens were homogenized for 4 h at temperatures 150 °C below to the respective alloy’s solidus temperature followed by cooling at a rate of 10 °C/min to 600 °C, followed by air-cooling to room temperature. This thermal treatment was performed in a tube furnace under a high purity argon atmosphere. The prepared alloy buttons were embedded in epoxy resin and cut into disks with a diameter of 10 mm and a thickness of 1.2 mm. The disks were polished successively through 2000 grit SiC abrasive, and ultrasonically cleaned in acetone, ethanol, and distilled water. Then, the disks were sterilized by exposing them to ultraviolet light for 30 min on a clean bench prior to the test. The CP Ti was used as a control.

Table 1
Pure metals used.
Raw material Specification Lot no. Manufacturer
CP Ti Rod, 10 mm D, ASTM B265 04RB-10 Daito Steel Co, Japan
Titanium Sponge, 3 mm and down 99.9% 129Q001 Alfa Aesar, USA
Chromium Pieces, 2–3 mm thick, 99.995% 38494 Alfa Aesar, USA
Cobalt Pieces, 99.9+% 10454 Alfa Aesar, USA
Copper Shot, 13 mm D, 99.99%, oxygen free 36686 Alfa Aesar, USA
Gold Plate, 99.99% 10041w0067 LS-Nikko, Korea
Indium Plate 99.995% 0907 LS-Nikko, Korea
Palladium Pieces, 99.95% 100413 LS-Nikko, Korea
Platinum Plate 99.8% 100830 LS-Nikko, Korea
Silver Silver shot, 1–5 mm, Premion ® , 99.99% 12186 Alfa Aesar, USA
Tin Granules, ACS, 99.9% 36691 Alfa Aesar, USA

Cell viability

The viability of cells was analyzed using a colorimetric assay for quantification of the cleavage of the tetrazolium salt WST-1 (Roche, Germany) by mitochondrial dehydrogenases. The resulting dye can be quantified by a spectrophotometer and directly correlated with the number of metabolically active cells in the culture. For the test, L-929 mouse fibroblast cells were grown on a 100 mm-diameter Petri dish containing 10 mL RPMI1640 (with 10% FBS). When enough cells were produced, 100 μL of the cell suspension at a cell density of 5 × 10 4 cells/mL were seeded on the test samples and incubated at 37 °C under a 5% CO 2 atmosphere. After 24 h of incubation, the cells were detached with 10 μL of 0.25% Trypsin-EDTA solution (Gibco, Canada) for 3 min, and 100 μL RPMI1640 were added per well. Then, 100 μL of the mixture were transferred into a 96-well culture plate, 10 μL WST-1 solutions were added to each well, and the cells were incubated at 37 °C in 5% CO 2 for 4 h. After the reaction period, the specimens were shaken for 1 min and absorbance was measured at 450 nm by a microplate reader (Bio-RAD, USA). Seven separate analyses were performed.

Agar overlay test

Cell suspensions in 10 mL L-929 at a cell density of 3 × 10 5 cells/mL were seeded in 100 mm-diameter cell culture dishes, and incubated to confluence at 37 °C in 5% CO 2 . After a 24-h incubation process, the medium was replaced with 10 mL of freshly prepared agar/nutrient medium containing RPMI1640, 5% FBS, and 3% agarose mixture. A 10 mL neutral red solution (0.01% in phosphate-buffered saline, Sigma, USA) was added and the cells were incubated for 15 min at room temperature. Excess dye was then removed, and the test specimens were placed on the agar surface. A 0.25% ZDBC polyurethane film was used as a positive control and a polyethylene sheet as a negative control. The dishes were incubated for 24 h at 37 °C in 5% CO 2 . Thereafter, the cultures were examined under a microscope.

Neutral red is a weak cationic dye that readily diffuses across plasma and organelle membranes, accumulating in the lysosomes. Any loss of membrane integrity induced by a toxic substance will result in decreased retention of neutral red dye. Damaged or dead cells are decolorized compared with healthy control cells . The decolorized zones and cell lysis around and/or under the specimens were evaluated according to ISO 7405 . Each test was repeated five times. The decolorized zones were scored as follows: 0, no decolorization detectable; 1, decolorization only under the specimen; 2, decolorization zone not greater than 5 mm from the specimen; 3, decolorization zone not greater than 10 mm from the specimen; 4, decolorization zone greater than 10 mm from the specimen; and 5, total culture is decolorized. Cell lysis was defined as loss of cell membrane integrity. Cell lysis was scored as follows: 0, no cell lysis detectable; 1, less than 20% cell lysis; 2, 20–40% cell lysis; 3, >40% to <60% cell lysis; 4, 60–80% cell lysis; and 5, more than 80% cell lysis. For each specimen, the median score from each specimen was calculated for both the decolorization zone index and the lysis index. The cytotoxicity was classified as follows: 0–0.5, noncytotoxic; 0.6–1.9, mildly cytotoxic; 2.0–3.9, moderately cytotoxic; and 4.0–5.0, markedly cytotoxic. The median values were calculated to describe the central tendency of the scores because the results were expressed as an index in the ranking scale .

Statistical analysis

Data were expressed as a mean ± standard deviation (SD) for each of the seven tests. Version 19.0 of the statistical software, SPSS (SPSS, Inc., an IBM Company, Chicago, Illinois, USA), now called “IBM SPSS Statistics”, was used to analyze the data from the WST-1 test by means of the Kruskal–Wallis one-way analysis of variance and Duncan’s multiple range test with α = 0.05.

Results

Cell viability

The cell viability for the control group (cells seeded on CP Ti) was 89.9 ± 18.8% compared with that of the cells seeded into culture wells containing only media. The samples could be considered biocompatible if the cell viability on the metal was equivalent to or greater than that of the control group. Pure metals showed different degrees of cell viability with the greatest viability for Pt followed by Pd, Au, Sn, Cr, Ag, In, Cu, and Co in cell culture for 24 h ( Fig. 1 ). The cell viabilities for pure Ag, In, Cu, and Co, 24.8 ± 6.0%, 24.1 ± 5.7%, 21.7 ± 10.4%, and 13.9 ± 4.6%, were significantly lower than that for the control group ( p < 0.05, Fig. 1 ). Pure Ag, In, Cu, and Co were moderately cytotoxic. The L-929 cells were inhibited in the extract of each metal. The cell viability for pure Au, Sn, and Cr displayed moderate viabilities of 82.1 ± 6.7%, 78 ± 12.9%, and 61.2 ± 20.0%, respectively. Pd and Pt demonstrated good biocompatibility as indicated by cell viabilities of 93.8 ± 9.6% and 97.2 ± 7.1%, respectively, compared with that of the control group.

Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Cytotoxicity of alloying elements and experimental titanium alloys by WST-1 and agar overlay tests
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