2.1

Materials

A commercially available Ni–Cr dental casting alloy (d.Sign ^® 10) was employed. The main constituents of the alloy (in mass%) as reported by the manufacturers were 75.4% Ni, 12.6% Cr and 8% Mo and the alloy indications included the production of crowns, short and long spans bridges, posts and telescope crowns . Disc-shaped specimens (15 mm diameter and 1.0 mm thickness) were prepared from wax patterns (Blue inlay casting wax, Kerr Italia SpA, Salerno, Italy) connected by a 3 mm diameter sprue (Dentaurum, Turnstrase 31, Ispringen, Germany) to the sprue former (Whip Mix™, Kentucky, USA) and positioned in the center of the casting ring (Whip Mix 4088, Whip Mix™, Kentucky, USA) resulting in a 5 mm sprue length. A carbon-free phosphate-bonded casting investment (GC Fujivest II, GC Europe NV, Leuven, Belgium) and expansion liquid (GC Fujivest II) were selected and using low vibration, the investment material was poured into the casting ring and allowed to set for 20 min in accordance with the manufacturers instructions. The ring was placed in the center of a pre-heated furnace at 820 °C for 40 min to burn out the wax pattern and thermally expand the mould. Additionally, to control the expansion throughout the investing process a 1 mm thick dry casting liner (GC New Casting Liner) was used. To cast each ring, a 9 g ingot of alloy was pre-heated for 60 s in a carbon-free ceramic crucible (Heraeus Kulzer GmbH, Hanau, Germany) using a vacuum pressure casting machine (Heracast, Heraeus Kulzer). After pre-heating the casting ring was removed from the furnace, positioned in the casting machine and melted until the thin oxide glaze broke and the molten alloy was forced into the casting mould using a combination of pressure and vacuum. Divesting was carried out on the next day to ensure that all the castings had cooled sufficiently and the investment material was broken carefully under water using a plaster knife.

To remove the residual investment material from each casting was alumina particle air abraded (Renfert Basic Master, Buckinghamshire, UK) with 50 μm aluminum oxide abrasive using 2 bar pressure for 20 s from a distance of 5 cm. The disc-shaped specimens were separated from the sprues using a cutting disc. The discs were used in the alumina particle air abraded finishing condition, and a further series of disc-shaped specimens with a finishing condition equivalent to that employed clinically was achieved by polishing with rubber polishing wheels suitable for non-precious alloys .

2.2

Profilometry

Three d.Sign ^® 10 discs from each finishing condition examined (polished and alumina particle air abraded) were examined using a contact stylus profilometer (Talysurf CLI 2000 Taylor-Hobson Precision, Leicester, UK) to determine the surface texture of the finishing condition. Three discs were selected at random and traces were performed with a 90° conisphere stylus tip of 2 μm radius, across a 100 mm ² area coincident with the center of the specimen. Profilometry was performed at a stylus velocity of 0.5 mm/s, recording data points every 5 μm ( x -direction) with a 8.6 nm resolution ( z -direction) resulting in 2001 traces with a 5 μm step-size ( y -direction). The roughness parameter estimated for the current study was the Ra value which represented ‘the arithmic mean of the absolute departures of the roughness profile from the mean line’. The Ra value was quantified from the ‘raw data’ of the profilometric profiles generated across the 100 mm ² area (composed of 2001 traces) at the operating conditions of stylus velocity, applied force and step-size outlined above. The mean Ra value was determined following the employment of a 0.25 mm cut-off Gaussian roughness filter in accordance with ISO 4287 .

2.3

Cell culture

The human oral epithelium cell line used throughout the current study was TR146 (SkinEthic Laboratories, Nice, France) first described in 1985 by Rupniak et al. . The histological origin of the cell line was a squamous cell carcinoma of the buccal epithelium. The chemicals and antibiotics used in the current study were of analytical-grade, molecular biology-grade or cell culture-grade and were purchased from Sigma–Aldrich Ltd., Dublin, Ireland, unless otherwise specified. The cell line was maintained in Complete Medium (CM) which consisted of Dulbeccos’ Modified Eagle’s medium (DMEM, pH 7.0) containing 4500 mg/L glucose, l -glutamine, sodium bicarbonate and phenol red without sodium pyruvate, and was supplemented with 10% (v/v) fetal bovine serum (FBS) which was supplied as heat-inactivated (60 °C) and sterile-filtered. CM was also supplemented with penicillin (100 units/mL) and streptomycin (100 units/mL).

The TR146 cells were maintained in 10 mL CM in tissue culture dishes (100 mm height, 200 mm diameter) (Sarstedt Ltd., Wexford, Ireland) at 95% relative humidity in an environment containing 5% carbon dioxide (CO ₂ ) at 37 °C (normal incubation conditions) in a Biotech Galaxy CO ₂ incubator (RS Biotech Laboratory Equipment Ltd., Scotland, UK). A serum-free medium (SFM) at pH 7.0 was also used in this study as several components of CM can interfere with the colorimetric and enzymatic assays. SFM consisted of DMEM containing 4500 mg/L of glucose and sodium bicarbonate without phenol red. This media was supplemented with 4 mM/L l -glutamine, penicillin (100 units/mL) and streptomycin (100 units/mL). Dulbecco’s Phosphate Buffer saline (DPBS; pH 7.4) was purchased as a 10× concentrate. Dilutions were made using Millipore water which had been filtered using a Minisart 0.22 μm pore size filter (Sartorius Mechatronics™, Dublin, Ireland) prior to being sterilized in a LTE Touchclave-LAB autoclave (LTE Scientific Ltd., Oldham, UK) operating at 115 °C for 10 min. Triton X-100 solution was supplied sterile-filtered condition and diluted to a 1% solution using sterile-filtered Millipore water, prior to being autoclaved at 115 °C for 10 min.

2.4

Assay format

To investigate the influence of finishing condition (polished and alumina particle air abraded) on the biocompatibility of the d.Sign ^® 10 alloy, TR146 cells were seeded in multiwell dishes. Confluent TR146 monolayers grown in CM were treated with sterile-filtered 0.25% (w/v) trypsin–edetate disodium (trypsin-EDTA [2.5 g porcine trypsin, 0.2 g EDTA.4Na/L Hank’s Balanced Salt solution with phenol red]) for 10 min at 37 °C. TR146 cells were then detached from the tissue culture dish by forcible pipetteing. The cell suspension was centrifuged in an Eppendorf Centrifuge 5804 (Davidson & Hardy Ltd., Belfast, UK) at 250 × g for 10 min. The supernatant was discarded and the pellet was re-suspended in CM or SFM. An Improved Bright Line Neubauer Haemocytometer (Hausser Scientific Ltd., PA, USA) was used to estimate the cell density by adding a 1:1 (v/v) of the re-suspended TR146 pellet and 0.4% trypan blue dye solution (prepared in 0.81% NaCl and 0.06% K ₂ HPO ₄ ). The cells were visualized on a Nikon™ TMS inverted phase contrast microscope (Nikon Instruments™, Micron Optical Co. Ltd., Dublin, Ireland). Cells that absorbed trypan blue dye were considered non-viable and were therefore not counted. Cell suspensions were adjusted to 1 × 10 ³ cells/mL CM or SFM.

For visual assessments of cell morphology and cell density and ELISA analysis of cytokine production, 2 × 10 ³ cells in 2 mL CM were added to each well of a sterile, DNase/RNase-free, flat-bottomed, 6 well cell culture dish (Greiner Bio-One Cellstar, Cruinn Diagnostics Ltd., Dublin, Ireland) and the cells were incubated for 24 h prior to biocompatibility testing. For analysis of metabolic activity with XTT and cellular toxicity with the Cytotox™ kit, 2 × 10 ² cells in 0.2 mL SFM were added to each well of a 96 well cell culture dish (Cellstar ^® , Greiner Bio-One, Sarstedt Ltd.) and incubated for 24 h prior to testing.

2.5

Direct and indirect alloy exposure

The biocompatibility of d.Sign ^® 10 discs for the finishing conditions investigated were assessed (polished and alumina particle air abraded) by placing the alloy discs directly or indirectly into contact with TR146 cell monolayers. Prior to placement the discs were washed using sterile-filtered Millipore water and were aseptically placed into Defend ^® Self-Sealing Sterilization Pouches (Carl Parker Associates, Mydent Corporation, NY, USA) and sterilized at 115 °C for 15 min to ensure sterility prior to cell culture exposure. Direct exposure involved the placement of the sterilized alloys discs directly onto the surface of confluent cell monolayers with incubation for specific time periods prior to removal. Indirect exposure involved incubation of the alloy discs in 50 mL of SFM at room temperature for 1, 5, 9 and 14 day time periods. At each of the time points the alloy discs were aseptically removed from the SFM and the resulting immersion solutions were either used immediately for biocompatibility analysis or stored at −20 °C for future testing and analysis.

2.6

Analysis of metal ion release by ICP-MS

The d.Sign ^® 10 alloy discs in the finishing conditions under investigation (polished and alumina particle air abraded) were aseptically placed into 50 mL of SFM and statically incubated at room temperature for 1, 7 and 14 days. After the specified time periods, the discs were aseptically removed from the solution and prepared for ICP-MS analysis. The samples were diluted 1:10 (v/v) in deionized water and were acidified to pH 2.0 with nitric acid prior to analysis. ICP-MS analysis was performed using an Agilent 7500a Series ^® ICP-MS (Agilent Technologies, Dublin, Ireland) within the detection limits of the apparatus (ng/L).

2.7

Cellular morphology

Directly exposed cells to the alloy finishing condition were incubated under normal conditions for a further 48 h and images were taken at 24 and 48 h with a Nikon™ COOLPIX CP990 camera. The effects of indirect exposure to the alloy finishing condition was examined by the addition of 2 mL of the 1, 5, 9 and 14 day immersion solutions to TR146 cells in 6 well cell culture dishes. Indirectly exposed cells were incubated under normal incubation conditions for 48 h and visualized at 24 and 48 h time points. A control of TR146 cell monolayers maintained with CM with no alloy exposure was set up for each experiment (direct and indirect). An additional control of TR146 cells exposed to 1% Triton-X 100 solution was also included. The morphology of the control and alloy exposed cells were examined for loss of cellular symmetry, presence of blebbing, cell detachment and cell death.

2.8

Analysis of cell density by trypan blue exclusion

The density of the control and alloy exposed cells (for the polished and alumina particle air abraded discs) were examined at 2, 24, 48 and 72 h with the treated cells counted with trypsinized cells and additionally in situ with trypan blue dye. The surface area (of the measured area) was determined and calculated for the entire surface area of the well. Cells that received no alloy exposure were used as the control and the experiment was performed in triplicate.

2.9

Analysis of metabolic activity with XTT

In addition to the trypan blue cell counts, a quantitative method of assessing cellular metabolic activity was employed using XTT sodium salt. A solution containing 0.5 mg/mL XTT plus 0.4 mg/mL Coenzyme Q ₀ (2, 3-dimethoxy-5-methyl- p -benzoquinone) was prepared in DPBS (pH 7.4). Following 24 h incubation in 96 well dishes, TR146 cells were exposed to 100 μL of 1, 5, 9 or 14 day alloy immersion solutions generated for each finishing condition. Cells were then reincubated under normal conditions. The XTT reduction assay was performed (in triplicate) on these cells at 2, 24, 48 and 72 h intervals. After the specified time periods, the SFM in each of the wells was discarded. A 200 μL aliquot of XTT solution was added to each well and incubated for a further 24 h. A 100 μL aliquot was placed into a fresh 96 well plate and the absorbance was measured at 480 nm with a spectrophotometer (Tecan Genios Spectrophotometer, Unitech Ltd., Dublin, Ireland). TR146 cells treated with SFM only were used as controls. All samples were processed in triplicate on at least three separate occasions.

2.10

Analysis of cellular toxicity by LDH

Cellular toxicity was determined by measuring LDH release from TR146 cells with the CytoTox 96 ^® Non-Radioactive Cytotoxicity Assay (Promega, Medical Supply Company Ltd., Dublin, Ireland). Following 24 h incubation in 96 well dishes, TR146 cells in SFM were exposed to 100 μL of 1, 5, 9 or 14 day alloy immersion solutions generated for each finishing condition and the cells were then reincubated under normal conditions. At 2, 24, 48 and 72 h time intervals, the growth medium was removed and assayed for LDH content (in triplicate). For the specified time periods (2, 24, 48 and 72 h), the SFM in each of the wells was analyzed to eliminate background absorbance interference and TR146 cells treated with a 1% solution of Triton X-100 in DPBS were used as the control.

2.11

Analysis of inflammatory cytokine release by ELISA

TR146 cells were seeded at 2 × 10 ³ cells in 2 mL CM in 6 well plates and incubated under normal incubation conditions for 24 h. For direct exposure, alloy discs (polished and alumina particle air abraded) were placed onto the confluent cell monolayers and incubated for 24 h. Aliquots of 1 mL were then removed and used immediately or stored at −20 °C until required for further testing and analysis. For the indirect exposure analysis, 1, 5, 9 or 14 day alloy immersion solutions were generated for polished and alumina particle air abraded discs and 1 mL of each immersion solution was added to the TR146 cells and incubated for a further 24 h. An aliquot of 1 mL was then removed and used immediately or stored at −20 °C until required for testing and analysis. IL-1α, IL-8, PGE ₂ and TNF-α ELISAs were performed using the Quantikine ^® Immunoassay Systems (RnD Systems Ltd., Abingdon, UK) according to the manufacturer’s instructions. All samples were processed in triplicate on at least three separate occasions.

2.12

Statistical analysis

One- and two-way analyses of variance (ANOVAs) were made in GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA) using a critical significance level of P = 0.05. A one-way ANOVA was performed on the mean Ra values for the surface finishing conditions (polished or alumina particle air abraded) investigated using profilometry. The cell densities analyses using trypan blue dye exclusion assay, were independently reduced to two-way ANOVAs (surface finishing condition × time) for the direct and the indirect exposure to the surface of confluent cell monolayers. Cellular metabolic activity (XTT) and cell toxicity (LDH) were also reduced to two, two-way ANOVAs (surface finishing condition × time) for indirect exposure of the immersion solutions to the surface of confluent cell monolayers. Four two-way ANOVAs (surface finishing condition × exposure method) were used to determine the expression of the inflammatory cytokines (IL-1α, IL-8, PGE ₂ and TNF-α, respectively) for the untreated control or following direct exposure to the d.Sign ^® 10 discs (in the surface finishing condition investigated) onto the 2D TR146 cell monolayer structures at 24 h (exposure time).

2.1

Materials

A commercially available Ni–Cr dental casting alloy (d.Sign ^® 10) was employed. The main constituents of the alloy (in mass%) as reported by the manufacturers were 75.4% Ni, 12.6% Cr and 8% Mo and the alloy indications included the production of crowns, short and long spans bridges, posts and telescope crowns . Disc-shaped specimens (15 mm diameter and 1.0 mm thickness) were prepared from wax patterns (Blue inlay casting wax, Kerr Italia SpA, Salerno, Italy) connected by a 3 mm diameter sprue (Dentaurum, Turnstrase 31, Ispringen, Germany) to the sprue former (Whip Mix™, Kentucky, USA) and positioned in the center of the casting ring (Whip Mix 4088, Whip Mix™, Kentucky, USA) resulting in a 5 mm sprue length. A carbon-free phosphate-bonded casting investment (GC Fujivest II, GC Europe NV, Leuven, Belgium) and expansion liquid (GC Fujivest II) were selected and using low vibration, the investment material was poured into the casting ring and allowed to set for 20 min in accordance with the manufacturers instructions. The ring was placed in the center of a pre-heated furnace at 820 °C for 40 min to burn out the wax pattern and thermally expand the mould. Additionally, to control the expansion throughout the investing process a 1 mm thick dry casting liner (GC New Casting Liner) was used. To cast each ring, a 9 g ingot of alloy was pre-heated for 60 s in a carbon-free ceramic crucible (Heraeus Kulzer GmbH, Hanau, Germany) using a vacuum pressure casting machine (Heracast, Heraeus Kulzer). After pre-heating the casting ring was removed from the furnace, positioned in the casting machine and melted until the thin oxide glaze broke and the molten alloy was forced into the casting mould using a combination of pressure and vacuum. Divesting was carried out on the next day to ensure that all the castings had cooled sufficiently and the investment material was broken carefully under water using a plaster knife.

To remove the residual investment material from each casting was alumina particle air abraded (Renfert Basic Master, Buckinghamshire, UK) with 50 μm aluminum oxide abrasive using 2 bar pressure for 20 s from a distance of 5 cm. The disc-shaped specimens were separated from the sprues using a cutting disc. The discs were used in the alumina particle air abraded finishing condition, and a further series of disc-shaped specimens with a finishing condition equivalent to that employed clinically was achieved by polishing with rubber polishing wheels suitable for non-precious alloys .

2.2

Profilometry

Three d.Sign ^® 10 discs from each finishing condition examined (polished and alumina particle air abraded) were examined using a contact stylus profilometer (Talysurf CLI 2000 Taylor-Hobson Precision, Leicester, UK) to determine the surface texture of the finishing condition. Three discs were selected at random and traces were performed with a 90° conisphere stylus tip of 2 μm radius, across a 100 mm ² area coincident with the center of the specimen. Profilometry was performed at a stylus velocity of 0.5 mm/s, recording data points every 5 μm ( x -direction) with a 8.6 nm resolution ( z -direction) resulting in 2001 traces with a 5 μm step-size ( y -direction). The roughness parameter estimated for the current study was the Ra value which represented ‘the arithmic mean of the absolute departures of the roughness profile from the mean line’. The Ra value was quantified from the ‘raw data’ of the profilometric profiles generated across the 100 mm ² area (composed of 2001 traces) at the operating conditions of stylus velocity, applied force and step-size outlined above. The mean Ra value was determined following the employment of a 0.25 mm cut-off Gaussian roughness filter in accordance with ISO 4287 .

2.3

Cell culture

The human oral epithelium cell line used throughout the current study was TR146 (SkinEthic Laboratories, Nice, France) first described in 1985 by Rupniak et al. . The histological origin of the cell line was a squamous cell carcinoma of the buccal epithelium. The chemicals and antibiotics used in the current study were of analytical-grade, molecular biology-grade or cell culture-grade and were purchased from Sigma–Aldrich Ltd., Dublin, Ireland, unless otherwise specified. The cell line was maintained in Complete Medium (CM) which consisted of Dulbeccos’ Modified Eagle’s medium (DMEM, pH 7.0) containing 4500 mg/L glucose, l -glutamine, sodium bicarbonate and phenol red without sodium pyruvate, and was supplemented with 10% (v/v) fetal bovine serum (FBS) which was supplied as heat-inactivated (60 °C) and sterile-filtered. CM was also supplemented with penicillin (100 units/mL) and streptomycin (100 units/mL).

The TR146 cells were maintained in 10 mL CM in tissue culture dishes (100 mm height, 200 mm diameter) (Sarstedt Ltd., Wexford, Ireland) at 95% relative humidity in an environment containing 5% carbon dioxide (CO ₂ ) at 37 °C (normal incubation conditions) in a Biotech Galaxy CO ₂ incubator (RS Biotech Laboratory Equipment Ltd., Scotland, UK). A serum-free medium (SFM) at pH 7.0 was also used in this study as several components of CM can interfere with the colorimetric and enzymatic assays. SFM consisted of DMEM containing 4500 mg/L of glucose and sodium bicarbonate without phenol red. This media was supplemented with 4 mM/L l -glutamine, penicillin (100 units/mL) and streptomycin (100 units/mL). Dulbecco’s Phosphate Buffer saline (DPBS; pH 7.4) was purchased as a 10× concentrate. Dilutions were made using Millipore water which had been filtered using a Minisart 0.22 μm pore size filter (Sartorius Mechatronics™, Dublin, Ireland) prior to being sterilized in a LTE Touchclave-LAB autoclave (LTE Scientific Ltd., Oldham, UK) operating at 115 °C for 10 min. Triton X-100 solution was supplied sterile-filtered condition and diluted to a 1% solution using sterile-filtered Millipore water, prior to being autoclaved at 115 °C for 10 min.

2.4

Assay format

To investigate the influence of finishing condition (polished and alumina particle air abraded) on the biocompatibility of the d.Sign ^® 10 alloy, TR146 cells were seeded in multiwell dishes. Confluent TR146 monolayers grown in CM were treated with sterile-filtered 0.25% (w/v) trypsin–edetate disodium (trypsin-EDTA [2.5 g porcine trypsin, 0.2 g EDTA.4Na/L Hank’s Balanced Salt solution with phenol red]) for 10 min at 37 °C. TR146 cells were then detached from the tissue culture dish by forcible pipetteing. The cell suspension was centrifuged in an Eppendorf Centrifuge 5804 (Davidson & Hardy Ltd., Belfast, UK) at 250 × g for 10 min. The supernatant was discarded and the pellet was re-suspended in CM or SFM. An Improved Bright Line Neubauer Haemocytometer (Hausser Scientific Ltd., PA, USA) was used to estimate the cell density by adding a 1:1 (v/v) of the re-suspended TR146 pellet and 0.4% trypan blue dye solution (prepared in 0.81% NaCl and 0.06% K ₂ HPO ₄ ). The cells were visualized on a Nikon™ TMS inverted phase contrast microscope (Nikon Instruments™, Micron Optical Co. Ltd., Dublin, Ireland). Cells that absorbed trypan blue dye were considered non-viable and were therefore not counted. Cell suspensions were adjusted to 1 × 10 ³ cells/mL CM or SFM.

For visual assessments of cell morphology and cell density and ELISA analysis of cytokine production, 2 × 10 ³ cells in 2 mL CM were added to each well of a sterile, DNase/RNase-free, flat-bottomed, 6 well cell culture dish (Greiner Bio-One Cellstar, Cruinn Diagnostics Ltd., Dublin, Ireland) and the cells were incubated for 24 h prior to biocompatibility testing. For analysis of metabolic activity with XTT and cellular toxicity with the Cytotox™ kit, 2 × 10 ² cells in 0.2 mL SFM were added to each well of a 96 well cell culture dish (Cellstar ^® , Greiner Bio-One, Sarstedt Ltd.) and incubated for 24 h prior to testing.

2.5

Direct and indirect alloy exposure

The biocompatibility of d.Sign ^® 10 discs for the finishing conditions investigated were assessed (polished and alumina particle air abraded) by placing the alloy discs directly or indirectly into contact with TR146 cell monolayers. Prior to placement the discs were washed using sterile-filtered Millipore water and were aseptically placed into Defend ^® Self-Sealing Sterilization Pouches (Carl Parker Associates, Mydent Corporation, NY, USA) and sterilized at 115 °C for 15 min to ensure sterility prior to cell culture exposure. Direct exposure involved the placement of the sterilized alloys discs directly onto the surface of confluent cell monolayers with incubation for specific time periods prior to removal. Indirect exposure involved incubation of the alloy discs in 50 mL of SFM at room temperature for 1, 5, 9 and 14 day time periods. At each of the time points the alloy discs were aseptically removed from the SFM and the resulting immersion solutions were either used immediately for biocompatibility analysis or stored at −20 °C for future testing and analysis.

2.6

Analysis of metal ion release by ICP-MS

The d.Sign ^® 10 alloy discs in the finishing conditions under investigation (polished and alumina particle air abraded) were aseptically placed into 50 mL of SFM and statically incubated at room temperature for 1, 7 and 14 days. After the specified time periods, the discs were aseptically removed from the solution and prepared for ICP-MS analysis. The samples were diluted 1:10 (v/v) in deionized water and were acidified to pH 2.0 with nitric acid prior to analysis. ICP-MS analysis was performed using an Agilent 7500a Series ^® ICP-MS (Agilent Technologies, Dublin, Ireland) within the detection limits of the apparatus (ng/L).

2.7

Cellular morphology

Directly exposed cells to the alloy finishing condition were incubated under normal conditions for a further 48 h and images were taken at 24 and 48 h with a Nikon™ COOLPIX CP990 camera. The effects of indirect exposure to the alloy finishing condition was examined by the addition of 2 mL of the 1, 5, 9 and 14 day immersion solutions to TR146 cells in 6 well cell culture dishes. Indirectly exposed cells were incubated under normal incubation conditions for 48 h and visualized at 24 and 48 h time points. A control of TR146 cell monolayers maintained with CM with no alloy exposure was set up for each experiment (direct and indirect). An additional control of TR146 cells exposed to 1% Triton-X 100 solution was also included. The morphology of the control and alloy exposed cells were examined for loss of cellular symmetry, presence of blebbing, cell detachment and cell death.

2.8

Analysis of cell density by trypan blue exclusion

The density of the control and alloy exposed cells (for the polished and alumina particle air abraded discs) were examined at 2, 24, 48 and 72 h with the treated cells counted with trypsinized cells and additionally in situ with trypan blue dye. The surface area (of the measured area) was determined and calculated for the entire surface area of the well. Cells that received no alloy exposure were used as the control and the experiment was performed in triplicate.

2.9

Analysis of metabolic activity with XTT

In addition to the trypan blue cell counts, a quantitative method of assessing cellular metabolic activity was employed using XTT sodium salt. A solution containing 0.5 mg/mL XTT plus 0.4 mg/mL Coenzyme Q ₀ (2, 3-dimethoxy-5-methyl- p -benzoquinone) was prepared in DPBS (pH 7.4). Following 24 h incubation in 96 well dishes, TR146 cells were exposed to 100 μL of 1, 5, 9 or 14 day alloy immersion solutions generated for each finishing condition. Cells were then reincubated under normal conditions. The XTT reduction assay was performed (in triplicate) on these cells at 2, 24, 48 and 72 h intervals. After the specified time periods, the SFM in each of the wells was discarded. A 200 μL aliquot of XTT solution was added to each well and incubated for a further 24 h. A 100 μL aliquot was placed into a fresh 96 well plate and the absorbance was measured at 480 nm with a spectrophotometer (Tecan Genios Spectrophotometer, Unitech Ltd., Dublin, Ireland). TR146 cells treated with SFM only were used as controls. All samples were processed in triplicate on at least three separate occasions.

2.10

Analysis of cellular toxicity by LDH

Cellular toxicity was determined by measuring LDH release from TR146 cells with the CytoTox 96 ^® Non-Radioactive Cytotoxicity Assay (Promega, Medical Supply Company Ltd., Dublin, Ireland). Following 24 h incubation in 96 well dishes, TR146 cells in SFM were exposed to 100 μL of 1, 5, 9 or 14 day alloy immersion solutions generated for each finishing condition and the cells were then reincubated under normal conditions. At 2, 24, 48 and 72 h time intervals, the growth medium was removed and assayed for LDH content (in triplicate). For the specified time periods (2, 24, 48 and 72 h), the SFM in each of the wells was analyzed to eliminate background absorbance interference and TR146 cells treated with a 1% solution of Triton X-100 in DPBS were used as the control.

2.11

Analysis of inflammatory cytokine release by ELISA

TR146 cells were seeded at 2 × 10 ³ cells in 2 mL CM in 6 well plates and incubated under normal incubation conditions for 24 h. For direct exposure, alloy discs (polished and alumina particle air abraded) were placed onto the confluent cell monolayers and incubated for 24 h. Aliquots of 1 mL were then removed and used immediately or stored at −20 °C until required for further testing and analysis. For the indirect exposure analysis, 1, 5, 9 or 14 day alloy immersion solutions were generated for polished and alumina particle air abraded discs and 1 mL of each immersion solution was added to the TR146 cells and incubated for a further 24 h. An aliquot of 1 mL was then removed and used immediately or stored at −20 °C until required for testing and analysis. IL-1α, IL-8, PGE ₂ and TNF-α ELISAs were performed using the Quantikine ^® Immunoassay Systems (RnD Systems Ltd., Abingdon, UK) according to the manufacturer’s instructions. All samples were processed in triplicate on at least three separate occasions.

2.12

Statistical analysis

One- and two-way analyses of variance (ANOVAs) were made in GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA) using a critical significance level of P = 0.05. A one-way ANOVA was performed on the mean Ra values for the surface finishing conditions (polished or alumina particle air abraded) investigated using profilometry. The cell densities analyses using trypan blue dye exclusion assay, were independently reduced to two-way ANOVAs (surface finishing condition × time) for the direct and the indirect exposure to the surface of confluent cell monolayers. Cellular metabolic activity (XTT) and cell toxicity (LDH) were also reduced to two, two-way ANOVAs (surface finishing condition × time) for indirect exposure of the immersion solutions to the surface of confluent cell monolayers. Four two-way ANOVAs (surface finishing condition × exposure method) were used to determine the expression of the inflammatory cytokines (IL-1α, IL-8, PGE ₂ and TNF-α, respectively) for the untreated control or following direct exposure to the d.Sign ^® 10 discs (in the surface finishing condition investigated) onto the 2D TR146 cell monolayer structures at 24 h (exposure time).