Preparation and Manufacturing of samples
Preparation and samples manufacturing were done at Digital laboratory at Prosthodontics Department, Faculty of Dentistry Alexandria University, Egypt. By using CAD software (ExocadGMBH,Germany), the test group disc samples were 3D designed with a diameter of 30 mm and a thickness of 4 mm to cover the majority of the culture area of a six well plate, then were translated to a Standard Tessellation Language file " STL file" to tessellate the 3D shape and slice it into digital layers (Fig. 1) [17]. 3D printing was performed via Stereolithography using the Phrozen shuffle (Phrozen Technology, Hsinchu City, Taiwan) used for printing the NextDent Base material (NextDent B.V., Soesterberg, Netherlands) (Fig. 2). The 3D denture base's precise composition is confidential, however the material safety data sheet shows that it is a "monomer based on methacrylate ester" and contains methacrylate oligomers and phosphine oxides (photoinitiator), or more specifically, the bisacylphosphine oxide (BAPO) phenylbis (2, 4, 6-trimethylbenzoyl)-phosphine oxide (Omnirad 819) [10]. While FormLabs Form 2 machine (Formlabs, Somerville, MA, USA.) with the Dental LT Clear resin (FLDLCL01) (Formlabs, Somerville, MA, USA) (Fig. 3) which is composed of trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate, 2-hydroxyethyl methacrylate, bis(1,2,2,6,6- pentamethyl-4-piperidyl) sebacate and methyl 1,2,2,6,6-pentamethyl-4-piperidyl sebacate, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, acrylic acid, monoester with propane-1,2-diol ethylene dimethacrylate, 2-hydroxyethyl acrylate, and mequinol, 4-methoxyphenol, hydroquinone monomethyl ether [30].
Both the TG1 and TG2 samples were removed from the platform by scraping tool, then placed in an ultrasonic bath (CD-4820 Codyson, Misr Sinai, Egypt) filled with pure isopropyl alcohol (IPA) (El Nasr Pharmaceutical Chemicals Co., Egypt) for 15 minutes, then removed and soaked again in fresh IPA for an additional 5 minutes to clean the samples and remove liquid resin before post curing. The samples were allowed to dry completely in the air and the support structures were then removed with a cutter. The printed samples were placed into a UV light curing box as a post cure for 20 minutes for each side, for final polymerization to ensure that materials obtained full polymer conversion and the residual monomer was reduced to a minimal amount with the highest mechanical properties were obtained. This procedure was a necessary step to produce a biocompatible end product as recommended by the manufacturer [10, 11, 28]. For the Heat polymerized acrylic resin samples (RG), (Meliodent, BayerUK, Berkshire, UK.), were fabricated with the same used 3D resins dimensions, by making a print space in stone molds within a dental flask using finished 3D printed sample in the mold, then packing and processing were carried out in accordance with the manufacturer’s instructions (100°C, 30 min). Finally, the acrylic resin samples were finished and polished as the usually done with an actual acrylic resin denture base.
All samples were disinfected by 70% ethanol (October Pharma S.A.E, Egypt) for 5 minutes then washed by phosphate buffer saline (PBS) (Biowest, Business Park Lane, USA). All samples were put separately in sealed sterilization pouches to be sterilized under UV light for 60 minutes in biosafety cabinet (EscoMicroPte.Ltd,Singapore) prior to each experiment to prevent any bacterial contamination. Experiments were designed to assess the in direct effects of each resin after 24 hours, 48 hours, 72 hours, and 168 hours (7 days) intervals in growth media (GM) through their cellular viability among the corresponding time intervals. To assess the in direct cellular response to the chemical leachate from each resin, samples were placed in six well plates of culture area that were acellular, containing only medium (MEDIA ONLY). Each well was used for the transfer of chemical leachate medium to its corresponding experimental well during each repeat, ensuring that transferred medium is directly representative of either cumulative resin degradation or control at each specific time point (Fig. 4) [6]. The conditioned media were stored at -20°C till the commencement of the cytotoxicity study.
Isolation and Culture of Gingival tissue [31]
A sample of attached keratinized gingival tissue was taken under local anesthesia from donor undergoing crown lengthening procedure at Department of Periodontology, Faculty of Dentistry, Alexandria University, Egypt. The gingival samples were then transported to the laboratory (Center of Excellence for Research in Regenerative Medicine and its Applications (CERRMA, Alexandria University, Faculty of Medicine)) in 15 ml falcon tube containing PBS + 3% penicillin/streptomycin/amphotericin (containing 10,000 IU/ mL penicillin; 10,000 µg/mL streptomycin; and 25 µg/mL amphotericin B, Lonza). The sample was de-epithelialized with a scalpel #11, leaving only the connective tissue. The gingival samples then washed three times in PBS then divided into small fragments 1x1 mm. Tissue fragments were cultured in tissue culture dishes in low glucose Dulbecco's modified Eagle's medium (LG-DMEM) supplemented with 10% fetal bovine serum, 2 mm L-glutamine, 1% penicillin/streptomycin (Biowest, Business Park Lane, USA) and left in humidified incubator with 5% CO2 at 37 0C (Fig. 5).
Over a 14-day period, the GM were changed every 2 to 3 days to permit the growth of the tissue explanted fibroblasts. After reaching 80–85% confluence, the cells were detached from the monolayer by treatment with trypsin-EDTA (0.25% trypsin, 1 mM EDTA) (Biowest, Business Park Lane, USA). After that the cells were sub-cultured in tissue flasks in same conditions till reached passage 4.
Once confluent, Fibroblasts were trypsinized and characterized using fluorescent-labeled monoclonal antibodies (mAb) for CD90, CD105, CD 11b, CD 45, and CD 73 markers and then was analyzed using Becton Dickinson, FACS caliber flow cytometer operated with Cell Quest software (Becton Dickinson, New Jersey, USA) [32].
Cell viability assessment [31]
Cells at passage 4 (Fig. 6) were seeded in 96 well plates at a seeding density of 7 X 103 / well and cultured for 24 hours to get adherent and about 70% confluent. Plates were then divided into 4 groups; Control group (CG) where cells were cultured in Complete growth media, RG where cells were cultured in conditioned media of Heat polymerized acrylic, TG1 cells were cultured in conditioned media of NextDent Base and TG2 cells were cultured in Dental LT Clear conditioned media. Growth complete media or conditioned media were transferred to the cells and incubated for 24 hours, 48 hours, 72 hours, and 7 days, according on the time intervals collected from all resins (Fig. 7). The cell viability of HGFs was measured by an MTT [(3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test [31]. After incubation the cells were washed with PBS, and the medium was changed to one containing 0.5 mg/mL MTT (Trevigen, Helgerman CT,Gaithersburg, MD,USA) in DMEM and left for 4 hours at 37 oC (Fig. 8). All readings were carried out using an UV reader at 570 nm (Tecan Trading AG, Switzerland). The MTT assay was performed in four independent experiments, eight replicate wells for each experimental point [31]. All the steps were done in the biosafety cabinet class II to prevent contamination and to protect the operator and samples.