Digital technology in prosthetic practice uses milled PMMA discs.
DMA and Brillouin spectroscopy are innovative techniques in dentistry.
Tested materials exhibit better mechanical properties in frequency ranges used.
Tested materials have less cytotoxicity than traditional ones.
To compare the mechanical and biological features of a polymethylmethacrylate (PMMA) disc for CAD/CAM prostheses (test samples, TG) with a traditional resin (control samples, CG).
Mechanical analysis was performed using Dynamic Mechanical Analysis (DMA) and Brillouin’s micro-spectroscopy. Human keratinocyte morphology and adhesion were analyzed by scanning electronic microscopy (SEM), cytotoxicity by the MTT assay, apoptosis by flow cytometry and p53, p21 and bcl2 gene expression by real time PCR.
TG exhibited a higher elastic modulus than CG (range 5100–5500 ± 114.3 MPa vs 3000–3300 ± 99.97 MPa). The Brillouin frequency was found at ω B = (15.50 ± 0.05) GHz for TG and at ω B_1 = (15.50 ± 0.05) GHz and ω B_2 = (15.0 ± 0.1) GHz for CG where two peaks were always present independently of the sample point. SEM analysis revealed that keratinocytes on TG disks appeared to be flattened with lamellipodia. Keratinocytes on CG disks rose above the substrate with cytoplasmatic filaments. MTT viability data at 3 h and 24 h showed TG was significantly less cytotoxic than CG (p < 0.001). No significant differences emerged in apoptosis on CG and TG. Real-time PCR showed p53 expression increased after 3 h by about 9-fold in keratinocytes on TG (p < 0.001) and about 5-fold in those on CG (p < 0.001). High p53 expression persisted after 24 h on both disks. No significant variations were observed in p21 and bcl2 expression at any time-point.
PMMA resins, as used in CAD/CAM technology, displayed suitable biocompatible and mechanical properties for removable prostheses.
In modern dentistry one of the most interesting advances was CAD/CAM development and application in the design, analysis and manufacture of fixed prostheses such as inlays, onlays, crowns, implant abutments etc. [ ]. Its use in removable prostheses re-awakened the interest of the Italian National Health Service due to their longer life-spans, reduced costs and potential for widespread application [ , ].
In creating removable prostheses CAD/CAM technology includes two patient appointments. In the first, all required clinical data for denture construction are registered and in the second the dentures are inserted. With CAD/CAM anatomical information is digitized, and the denture base and occlusions are designed virtually [ ]. Two processes are then available. Rapid prototyping, also known as the additive process, layers resinous materials on a support structure which is cured by visible light, UV light, heat or laser. Alternatively, the subtractive technique mills the prosthetic bases and teeth from a pre-polymerized commercially-manufactured polymethylmethacrylate (PMMA) disk. The teeth are then either manually fixed into the sockets using methacrylate-based bonding agents [ ] or base and teeth are made in one blank without bonding. Among the advantages of CAD/CAM technology compared with standard denture construction are simpler laboratory process, fewer dental appointments, better clinical outcomes in terms of, for example, patient satisfaction, soft tissue adaptation etc. and more precise tooth alignment [ , ]. Disadvantages include doubts about denture adaptation to soft tissue, costs and the steep learning curve for clinicians and for dental technicians [ ].
Whether produced by standard methods or CAD/CAM technology all removable prosthetics are usually obtained from acrylic resins (PMMA) due to their physical and chemical properties, low costs, ease of processing, aesthetic properties, repairability, low solubility and low water adsorption [ ]. Monomeric PMMA residues may, however, reduce denture mechanical properties and have major repercussions on prosthesis biocompatibility with ensuing allergic reactions, symptoms such as burning stomatitis, edema, oral mucosal ulcers and greater susceptibility to bacterial infections and superinfections [ ]. Disappointing results were achieved in attempts to reduce monomeric PMMA residues by raising pressure, extending processing times and using water bath post-polymerization [ ]. A more promising approach is use of CAD/CAM pre-polymerized PMMA billets. Polymerized under high temperature and press values, residual monomers are reduced in number as assessed by infrared spectroscopy, high performance liquid chromatography and gas chromatography [ ]. The present study compared the mechanical and physical properties of new CAD/CAM and standard resins and assessed their biocompatibility with oral cell populations.
Many studies have focused on the mechanical properties of dental materials [ ]. Static tests [ , ] determined maximum strength, but provided limited information on structure and related physical properties and were not suitable for measuring elastic properties or viscoelastic behavior under load [ ]. Furthermore, sample destruction precluded repeated testing [ ]. Since dental composites are exposed to dynamic, rather than static, loads dynamic tests appeared more useful [ ]. The Dynamic Mechanical Analysis (DMA), for example, determines the elastic and viscous responses of a sample in one experiment, and is particularly well-suited for visco-elastic materials [ ]. Furthermore, since dynamic tests simulate cyclic masticatory loading of dental composites better than static tests [ ], they might be better predictors of clinical performance.
Since substrate mechanical properties are however, known to influence cellular behavior, microscale is the appropriate scale length for cellular interactions [ ]. Brillouin micro-spectroscopy, a valuable tool in tissue engineering and the mechanical characterization of biomaterials [ ] is an innovative technique that tests cellular mechanics [ , ].
Working at high frequency (GHz), it measures the real and imaginary parts of the longitudinal elastic modulus on the cellular length scale [ ], assessing viscoelastic properties and determining tensile micro-stresses [ ]. Moreover, since it is a non-contact method it was used to evaluate elastic heterogeneity in cells and tissues and to study dentin micromechanics [ ]. The present study characterized the mechanical properties of an innovative dental resin polymer by means of Dynamic Mechanical Analysis and Brillouin microscopy, flanked by biological viability assays. After polymer exposure, scanning electronic microscopy (SEM) assessed cell morphology and substrate adhesion while the MTT assay, which was reported to be more sensitive than the sulforhodamine B (SRB) assay [ ] and the lactate dehydrogenase assay (LDH) [ ], investigated cell viability and cytotoxicity in accordance with ISO-10993-5 recommendations [ ].
Since the MTT assay is not informative on the mechanism of cell damage or death, apoptosis and expression of two key apoptosis-related genes (p53 and BCL2) were analyzed [ ]. Cell cycle analysis and expression of cell cycle regulatory proteins (p16 and p21) provided data on cell proliferation patterns [ ].
The first null hypothesis was that the two resins did not present any mechanical differences in terms of the elastic modulus, rigidity and morphology. The second null hypothesis was that no differences could be detected in in vitro biological behavior of oral mucosa cells grown on resin.
Materials and methods
Control materials (CG) were custom made samples of a cold-curing, preheated PMMA resin (Palapress Vario, Haraeus Kulzer, Germany) that were manufactured in a conventional dental laboratory by a master technician. They were made up of PMMA powder and liquid which contained methylmethacrylate (MMA), 1,4-BDMA Dimetacrylate and tertiary amine in a mixing ratio of 10 g/7 m1, in accordance with the manufacturer’s instructions. The Palamat practice at 2 bar pressure was inserted 13 min after the start of polymerization for 15 min, at a temperature of 55 °C. After polymerization, specimens were trimmed for excess, finished and polished using pumice powder (Polyglass Ultra ponce, KALADENT, Urdorf, Switzerland) and felt-tip polishing buffs. Final finishing was achieved with a universal high-gloss polishing paste for PMMA resins (KMG, CANDULOR, Wangen, Switzerland) and a high lustre polishing buff for 1 min (High luster buff Acryl, Bredent, Senden, Germany). Specimens were stored in sterile plastic containers in a cool dry place for 24 h before testing.
PMMA cylinders of test samples (TG) (Ivobase CAD, lvoclar Vivadent, Lichtenstein) were milled from a pre-polymerized PMMA pod that was manufactured under high pressure. Made up of polymethylmethacrylate, co-polymers and pigments, specimens were cut by a rotary table saw (Inca, Injecta, Teufenthal, Switzerland) equipped with a 3 mm thick stainless circular blade (Oertli Werkzeuge, Hori, Switzerland). Specimens were finished, polished and stored, as described above for the CG. Specimen number and dimension:
3 CG and 3 TG specimens, all 60 × 8 × 2.5 mm in size for dynamic mechanical analysis (DMA)
5 CG disks and 5 TG disks of 1.9 cm 2 surface area for Brillouin spectroscopy
60 CG disks and 60 TG disks of 1.9 cm 2 surface area for biocompatibility assays
30 CG disks and 30 TG disks of 1.9 cm 2 surface area for morphological analysis (SEM).
All samples were measured using a 150 mm caliper (Mitutoyo,Kawasaky, Japan) which is accurate to 1/20 mm.
Dynamic Mechanical Analysis (DMA)
To determine the elastic modulus, 3 CG and 3 TG samples underwent isotherm mode at 37 °C. Frequencies ranged from 0.1 to 100 Hz, maintaining an 1 N amplitude force. Specimens were analyzed in the linear elastic field. Tests were replicated 6 times for each sample. Mechanical properties were assessed by the Mettler Toledo DMA/SDTA861 instrument (Mettler Toledo Inc. USA), equipped with “Three Points Bending” ( Fig. 1 a and b).
The Young modulus formula
quantified deformation of the applied load, where:
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stress as expressed in Mega Pascals (MPa) in the International System
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strain, as a dimensionless quantity, which is often expressed as a percentage. It is defined as length variation over initial length.
In viscoelastic materials force reactions are both tangential and normal. Thus, the Young modulus becomes
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= storage module indicates stored material elasticity linked to reversible elastic energy.
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E ‘ ‘
= loss module indicates capacity for irreversible energy loss transformed into heat
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= imaginary unit <SPAN role=presentation tabIndex=0 id=MathJax-Element-8-Frame class=MathJax style="POSITION: relative" data-mathml='i=’>𝑖=i=
Elasticity: Prism deformation derives from the <SPAN role=presentation tabIndex=0 id=MathJax-Element-9-Frame class=MathJax style="POSITION: relative" data-mathml='δ’>𝛿δ
angle between applied and load forces. The <SPAN role=presentation tabIndex=0 id=MathJax-Element-10-Frame class=MathJax style="POSITION: relative" data-mathml='δ’>𝛿δ
phase angle quantifies delay in deformation with respect to load force. In solid elastic materials <SPAN role=presentation tabIndex=0 id=MathJax-Element-11-Frame class=MathJax style="POSITION: relative" data-mathml='δ’>𝛿δ
= 0, in viscous fluids <SPAN role=presentation tabIndex=0 id=MathJax-Element-12-Frame class=MathJax style="POSITION: relative" data-mathml='δ’>𝛿δ
= 90, and in visco-elastic systems 0 ≤ <SPAN role=presentation tabIndex=0 id=MathJax-Element-13-Frame class=MathJax style="POSITION: relative" data-mathml='δ’>𝛿δ
The loss factor, tan( <SPAN role=presentation tabIndex=0 id=MathJax-Element-14-Frame class=MathJax style="POSITION: relative" data-mathml='δ’>𝛿δ