This study aimed to investigate the influence of CAD/CAM composite materials on human gingival fibroblasts (HGF) and gingival keratinocytes (HGK).
Four materials were investigated: 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). HGF and HGK were cultured as per the supplier’s protocol (ATCC, UK). Cell proliferation and cytotoxicity were evaluated at 1, 3, 5 and 10 days using LDH and Alamar Blue assays. Indirect immunostaining was used to assess the Caspase-3 activity. Data were analysed via two-way ANOVA, one-way ANOVA and Tukey’s post hoc test ( α = 0.05 for all tests).
There was significant difference in cell proliferation of the HGK and HGF cells in contact with different composite materials but no significant differences in their cytotoxicity. There was a significant effect on cell proliferation and cytotoxicity with different exposure times, for each type of resin-composite. HGF cell proliferation was higher than HGK with almost all investigated materials and at all time points. No Caspase-3 activity was detected in either cell lines.
HGK proliferation and cytotoxicity appeared to be more influenced by composite materials compared to HGF, demonstrating EN cytotoxic effects in HGK. Different manufacturing techniques of resin-composites (photo curing versus heat/pressure curing) had no significant effect on their biocompatibility.
Biocompatibility of dental materials is crucial, especially for materials that are in direct contact with oral tissues such as dentine, the dental pulp and adjacent gingiva [ ]. Ceramic is more biocompatible compared to resin-composite [ ] as resin-composite components can be released due to incomplete polymerisation or degradation of components over time [ ].
Resin-composites designed for use with CAD/CAM systems are polymerised under high temperature and/or high pressure with high filler content and sometimes with innovative compositions [ ]. In theory, these formulations of CAD/CAM composites should have superior biocompatibility compared to conventional resin-composites due to their higher degree of conversion [ , ] and lower levels of residual monomer [ ]. Additionally, fewer potentially toxic monomers and no photoinitiators are used in the manufacture of CAD/CAM composite blocks [ , ]. In addition, UDMA is the main monomer used in CAD/CAM composite rather than Bis-GMA. UDMA is considered less toxic than Bis-GMA as it is not synthesised from bisphenol A (BPA), and has been found to induce less cytotoxicity on human gingival and pulp fibroblasts in vitro [ ]. Finally, CAD/CAM blocks exhibit high breakdown-resistance, therefore the release of toxic components release is less likely [ , , ].
CAD/CAM composite blocks can be classified into two main categories based on their microstructural geometry and fabrication method: resin-composite blocks (RCB), which are manufactured by incorporation of filler particles into a monomer mixture; and polymer-infiltrated ceramic networks (PICN) [ , ]. Production of PICNs is achieved in two stages: fabrication of a porous pre-sintered ceramic network followed by coupling agent conditioning, then polymer infiltration into this ceramic network takes place [ , ].
Human gingival keratinocytes (HGK) are considered the main cell population of the marginal keratinised gingiva (epithelial layer) of the oral mucosa, which is supported by the subepithelial connective tissues in which human gingival fibroblasts (HGF) reside. Both cell types play an essential role in the maintenance of soft tissue integrity and the oral wound healing and regeneration process [ ]. CAD/CAM composite blocks used for a coronal restoration, whether on natural teeth or implants, will come into close contact with oral soft tissues including the keratinised marginal gingiva. Hence, adverse side effects such as inflammation, allergic reactions or tissue cytotoxicity could occur [ ].
There is little research regarding the biocompatibility of the newly introduced CAD/CAM composite materials. Therefore, this study aimed to evaluate the biocompatibility of three manufacturing techniques (conventional resin-composite, resin-composite blocks and PICN) and their influences on HGF and HGK proliferation and cytotoxicity.
The null hypotheses are –
There is no difference in the proliferation of the HGK and HGF cells in contact with the composite blocks.
There is no difference in the cytotoxicity levels of the HGK and HGF cells in contact with the composite blocks.
There is no effect on either cell proliferation or cytotoxicity with different exposure times, for each type of block.
There are no different outcomes, with each specific cell-line, between any of the investigated CAD/CAM blocks.
There are no different outcomes, with each specific cell-line, for resin-composite formed by different processing techniques (photo curing versus heat/pressure curing).
Materials and methods
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 .
|Materials (Code)||Composition by weight||Manufacturer|
|Polymer-infiltrated ceramic network (PICN)||VitaEnamic (EN)||86% fine structure feldspar ceramic||14% UDMA + TEGDMA||Vita Zahnfabrik, Germany|
|Resin-composite blocks (RCB)||Grandio Blocs (GR)||86% nanohybrid fillers||14% UDMA + DMA||VOCO GmbH, Germany|
|Block HC (HC)||61% silica powder, microfumed silica and zirconium silicate||UDMA + TEGDMA||Shofu, Japan|
|Conventional resin-composite||GrandioSO (GND)||89% glass ceramic and silica-nanoparticles||Bis-GMA, Bis-EMA, TEGDMA||VOCO GmbH, Germany|
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.
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.
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) [ ]:
Cellviability%=A570– (A600x RO) for test wellA570- (A600x RO) positive growth control×100%
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.
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) [ ]:
Cytotoxicity%=Specimen-treated LDH activity-Spontaneous LDH activityMaximum LDH activity-Spontaneous LDH activity×100%