2.1

Specimens preparation

Four materials were tested: two resin-composite blocks (RCB), Grandio Blocs (GR) and Block HC (HC); one polymer-infiltrated ceramic network (PICN), Enamic (EN); and one conventional resin-composite, Grandioso (GND), Table 1 .

Each CAD/CAM block was sectioned into 5 × 5 × 5 mm specimens using a diamond blade (MK 303, MK diamond, CA, USA) mounted on a saw (Isomet 1000 Precision Saw; Buehler Co., IL, USA) under constant water irrigation. Conventional resin-composite samples were prepared using a polytetrafluoroethylene (PTFE) mould with 5 × 5 × 5 mm dimensions, and were cured according to manufacturer recommendations. The composite was applied into increments of 2 mm maximum thickness and light cured for 20 s using a LED light-curing unit with an output irradiance of 1200 mW/cm ² (Elipar™, 3M ESPE, USA) under standard curing mode and afterwards the specimen was cured from all surfaces to ensure full curing.

All specimens (CAD/CAM composite blocks and the conventional composite) were wet ground and polished with a lapping machine (MetaServ 250, Buehler Co., IL, USA) with a series of silicon carbide papers (SiC) P320, P500, P1200, P2400, and P4000 grit (Buehler Co., IL, USA) under water cooling. Specimens were then cleaned in an ultrasonic bath (Ultrasonic cleaning system, L&R Co., NJ, USA) with phosphate-buffered saline solution, rinsed four times with purified cell culture water and then ultrasonically cleaned in 80% ethanol. Finally, they were sterilized by UV exposure for 1 h per top half and then flipped over for UV exposure of the bottom half. Finally, once UV sterilisation process was completed all samples were transferred immediately to 24 well plates to initiate experimentation (prepared as in Section 2.2 ). In total, 96 specimens were prepared comprising 24 of each material divided into 12 for each cell line (HGK and HGF) of each material that were divided into four specimens (n = 4) for each of three replicas. All experiments were performed using appropriate controls with biological and instrumental triplicates.

2.2

Cell culture preparation

Commercially available cells were purchased and cultured accordingly to manufacturer’s recommendations and standard protocol for cell culture, maintenance, freezing and thawing (see below). Two cell lines, primary human gingival fibroblast (Lot-201018) and primary human gingival keratinocytes (Lot-201014) (ATCC, VA, USA), were grown in their relevant growth media.

Human gingival fibroblasts (HGF) were cultured with fibroblast basal medium supplemented with fibroblast growth kit (Lot-804014). The final concentration of each component was as follows (HGF complete growth media): l -glutamine: 7.5 mM; rh FGF beta: 5 ng/mL; rh insulin: 5 μg/mL; hydrocortisone: 1 μg/mL; ascorbic acid: 50 μg/mL; foetal bovine serum: 2%. Also, 1% penicillin/streptomycin was added.

Human gingival keratinocytes (HGK) were grown in their relevant growth media: dermal cell basal medium and Keratinocyte growth kit (Lot-80923177). The final concentration of each component in complete keratinocyte growth medium was as follows: 0.4% bovine pituitary extract (BPE); 0.5 ng/mL rh TGA-alpha; 3 mM l -glutamine; 100 ng/mL hydrocortisone; 5 μ/mL insulin; 1.0 μg/mL epinephrine; 5 μg/mL apo-transferrin with 1% penicillin/streptomycin.

The cells were expanded and passaged at regular periods based on their growth characteristics and manufacturer’s protocol. Incubation was performed at 37 °C, CO ₂ 5%. Once confluent cells were detached using 0.25% trypsin (Life Technologies, Inc.), cells were counted using Millipore Scepter counter (Merck Millipore, UK) and 5 × 10 ⁴ cells seeded into a 24-well culture plate (15.6 mm diameter) in 500 μL of complete growth medium. Once the cells were attached (24 h later), composite specimens (conventional and CAD/CAM) were placed in the centre of each well signifying the start of the experiment according to ISO standard 10993-5 [ ]. The ratio of the surface area of the sample to medium volume was 3 cm ² /mL, which is within the ISO standard ratio of 0.5−6 cm ² /mL, ISO 10933, 12.

2.3

Cell viability

Cellular viability of 100% was attributed to control wells, where cells were cultured with no composite blocks (LC or positive growth control). Cellular viability was quantified via a colorimetric assay using AlamarBlue™ cell viability reagent, DAL1100 (Thermo Fisher Scientific, IL, USA). At least one biological replica (24-well plate) was used for each assay at each time point (1, 3, 5 and 10 days).

HGF and HGK at each time point were exposed to AlamarBlue™ (1:10) for 1 h at 37 °C. Then 100 μL of supernatant was transferred into a 96-well plate for analysis at each time point. Cell viability was measured at the four-time points 1, 3, 5 and 10 days of cell growth. The 96-well plate was read with a UVM 340-microplate reader at 570 nm and 600 nm (ASYS, Scientific laboratory supplies). Cell viability was calculated according to Eq. (1) [ ]:

where A ₅₇₀ and A ₆₀₀ are absorbance at 570 and 600 nm respectively and R _O is the correction factor calculated from (A ₅₇₀ /A ₆₀₀ ) of the positive growth control.

2.4

Cytotoxicity

The cytotoxic potential of the tested materials was investigated using a Pierce™ LDH cytotoxicity assay kit, 88954 (Thermo Fisher Scientific, IL, USA). At least one biological replica (24-well plate) was used for each assay at each time point.

Cytotoxicity in HGF and HGK at each of the four time points (day 1, 3, 5 and 10) were measured using 50 μL of the supernatant and 50 μL of LDH cell reaction solution incubated for 30 min at room temperature in the dark. The reaction was stopped using the LDH “stop” solution. Appropriate controls were used as per the manufacturers’ protocol; maximum LDH release from the cells was set by adding membranolytic-particles, and was considered the positive (high) control, and the spontaneous LDH release control (water-treated) was considered the low control. The 96-well plate was read with a UVM 340-microplate reader at 490 nm subtracted from 680 nm (ASYS, Scientific laboratory supplies) and cytotoxicity was calculated according to Eq. (2) [ ]:

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