Hard chairside reline resins are acrylic-based prosthetic biomaterials used to restore temporary, or even permanently, the fit of removable dentures when there is a change of the underlying oral tissues. They are bonded to the fitting surface of dentures reestablishing their support, stability, and retention [
During the free radical polymerization reaction, the monomer-polymer conversion is never complete [
The curing process of these kinds of autopolymerizing resins is achieved in direct contact with the oral mucosa, leading to high levels of RM content once the material had set
In recent years, different postpolymerization treatments have been proposed to reduce the oral exposure to the RM and the degradation products of reline acrylic resins, including immersion in hot water [
To date, in the mentioned studies, water has been used as the postpolymerization immersion medium. Generally, apart from water, ethanol aqueous solutions have been used in order to increase and accelerate compounds solubility, indicating the importance of this solvent in leaching processes [
Since water immersion postpolymerization treatments are dependent on temperature [
The hypothesis of this work was that postpolymerization treatment based on immersion in ethanol solutions would improve the biocompatibility of two different hard chairside reline resins. Biocompatibility can be defined in terms of the ability of a material to perform a specific function with an appropriate host response [
The materials evaluated in this study are presented in Table
Materials under evaluation in the study.
Product | Manufacturer | Batch number | P/L ratio (g/mL) | Composition | Curing cycle |
---|---|---|---|---|---|
Kooliner (K) | GC America Inc., Alsip, IL, USA | 0701222(P); 0704052(P); 0708151(P); 0610041(L). | 1.4/1 | P: PEMA |
10 minutes |
| |||||
Ufi Gel Hard (U) | Voco GmbH, Cuxhaven, Germany | 0905422(P); 771715(P); 0905421(L); 760494 (L). | 1.77/1 | P: PEMA |
7 minutes |
P: powder; L: liquid; PEMA: poly(ethylmethacrylate); IBMA: isobutylmethacrylate; 1,6-HDMA: 1,6-hexanedioldimethacrylate.
Specimens of each material were prepared from stainless steel molds as ISO 20795-1 recommends [
Specimens of each material with a diameter of
After the postpolymerization treatments, all specimens were milled into small pieces in order to prepare three samples of each specimen. To each sample, of approximately 300 mg, 5 mL of acetone was added (extraction solvent) [
Total quantity of RM (
The mechanical tests were carried out only on the groups submitted to
Specimens of each material (64 × 10 × 3.3 mm) obtained as described above (see Section
Each specimen was tested for both microhardness and flexural strength values. Microhardness of the specimens was tested prior to flexural strength, since the load applied for fracture could create superficial tension forces that can be propagated and interfere with the microscopic measures of superficial microhardness.
The microhardness of the specimens was obtained using a Vickers diamond indenter attached to a microhardness indenter machine (Duramin, Struers DK 2750 Ballerup, Denmark) using a 25 gf (245 mN) load for 30 seconds, as described elsewhere [
After microhardness testing, all specimens were submitted to flexural strength test in a servo-hydraulic universal machine (Instron Model 4502) using 3-point loading. A crosshead speed of 5 mm per minute was used and the distance between the supports was 50 mm, as described elsewhere [
Load was applied until failure and the fracture load was recorded in Newton (N). The flexural strength was expressed in megapascal (MPa) and calculated using the following formula:
Evaluation of cytotoxicity was assessed only on the groups of the specimens submitted to water and ethanol solutions (
Eluates of each material were prepared by placing each disk into a sterile glass vial with 25 mL of Dulbecco’s Modified Eagle’s medium (Sigma-Aldrich), supplemented with penicillin-streptomycin and fetal bovine serum (Sigma-Aldrich). Disks were incubated at 37°C for 24 h. A medium without disks was also incubated as above to serve as the control medium [
The
Cell viability was determined by the ability of the cells to metabolically reduce the tetrazolium salt (MTT) to a purple formazan dye [
Cytotoxicity based on quantifying the release of LDH from membrane-damaged cells was determined using the assay kit TOX7 provided by Sigma-Aldrich, which measures the conversion of a tetrazolium salt into a red formazon product (absorbance was recorded at 490 nm). The percentage release of LDH from the treated cells was calculated by comparing it to the maximum release of LDH achieved by incubating the cells with a DMSO solution (20%).
Statistical analysis for RM content consisted of a two-way analysis of variance (ANOVA) followed by Tukey multiple means comparisons (at a
Data of microhardness, flexural strength tests, and cytotoxicity assays were analyzed by Kruskall-Wallis test and individual differences were investigated by Tukey test (both at a
The RM content of K and U showed higher quantities of residual IBMA compared with residual 1,6-HDMA (Table
Mean (×104 µg/g) and SD of RM content of experimental groups from material Kooliner (
Material | Temp. | Dry conditions | Type of solution | |||
---|---|---|---|---|---|---|
Water | Ethanol 20% | Ethanol 50% | Ethanol 70% | |||
Kooliner | 23 ± 2°C | 2.77 (0.29)aA | 2.51 (0.16)aA | 2.56 (0.25)aA | 2.57 (0.31)aA | 2.46 (0.41)aA |
55 ± 2°C | 2.67 (0.22)aA | 2.04 (0.21)bB | 1.88 (0.15)bB | 1.59 (0.14)cB | 0.80 (0.16)dB | |
| ||||||
Ufi Gel Hard | 23 ± 2°C | 1.90 (0.15)aA | 1.62 (0.26)bA | 1.58 (0.17)bA | 1.52 (0.23)bA | 1.45 (0.11)bA |
55 ± 2°C | 1.85 (0.32)aA | 0.95 (0.05)bB | 0.86 (0.42)cB | 0.72 (0.13)dB | 0.39 (0.05)eB |
Horizontally identical superscripted small letters denote no significant differences among groups (
Vertically identical superscripted capital letters denote no significant differences among groups (
For both materials, higher concentrations of ethanol led to lower levels of RM content when submitted to
All specimens of the 70% ethanol group from both materials presented an irregular surface that prevented the determination of Vickers microhardness (Figure
Microscopic images of Vickers indentations produced on specimens from the ethanol 70% groups submitted to
Considering K specimens (Figure
Mean and SD of Vickers microhardness (VHN) of K and U experimental groups (
For K specimens (Figure
Mean and SD of flexural strength (MPa) of K and U experimental groups (
For U specimens (Figure
For both materials, eluates obtained from ethanol-treated groups resulted in higher cell viability compared to without-treatment (dry conditions) and water-treated groups (Figures
Effect of postpolymerization treatment on cytotoxicity assessed by the MTT test. Values represent mean and SD of at least three experiments. W/T = without-treatment group. C(−) = Incubation of cells with culture medium. Identical characters (*) denote no significant differences among groups (
Effect of postpolymerization treatment on cytotoxicity assessed by the LDH release assay. Values represent mean and SD of at least three experiments. W/T = without-treatment group. C(+) = incubation of cells with 20% DMSO. Identical characters (*) denote no significant differences among groups (
Cell viability determined by the ability of cells to metabolically reduce MTT to a formazan dye showed that postpolymerization treatment of the K resin with ethanol increased the cell viability from ~38% to ~56% (compared with water treatment). In what refers to U resin, ethanol treatment increased the viability from ~62% to ~77% (Figure
In the case of LDH release, ethanol-treated groups showed a significant decrease compared to positive control,
In the present work, the effect of ethanol solutions as postpolymerization treatment was evaluated in order to improve the biocompatibility of two distinct hard chairside reline resins (K and U). For this purpose, different parameters were tested and monitored, namely, the RM content, the microhardness, and flexural strength of the material, as well as the cytotoxicity of the corresponding eluates.
The RM content of both K and U was quantified by HPLC showing higher quantities of residual IBMA compared with residual 1,6-HDMA, respectively, in all conditions evaluated. These results are in accordance with previous studies [
Moreover, an important finding of our study was the reduction of the RM content of both specimens, at 55°C, due to ethanol solutions treatment when compared with water. Indeed, it has been previously suggested that the chemistry of different solvents is a key element in postpolymerization treatments [
The synergic effect of high temperature and ethanol solutions on reducing the RM content was evaluated as well. High temperature (~55°C) has already been considered a crucial element in the postpolymerization treatment of acrylic resins, since it seems to be responsible for a further consumption of RM during polymerization [
Another important consideration is the impact of these novel proposed postpolymerization treatments on the mechanical properties of the resins. Replacement of RM molecules with solvent molecules has been associated with a plasticizing effect observed in postpolymerization treatments [
In the present study, immersion in water at 55°C of K specimens produced a significant increase of their microhardness and flexural strength compared with the controls. This can be explained by a stronger effect of the temperature compared with the water plasticizing effect. Nevertheless, U specimens did not show any differences on microhardness and flexural strength after hot water bath (at 55°C), possibly because of differences in the polymeric structure between the two resins. Material U undergoes rapid polymerization reaction and solidifies quickly. It is likely that air voids are entrapped during mixing of the powder and liquid components, which result in a porous structure [
When determining the most effective postpolymerization treatment, the one that reduces the RM content more effectively should be chosen. However, in this choice, professionals must also consider if the mechanical properties of the resin are not negatively affected. Since a water bath at 55°C has already proven to be an effective postpolymerization treatment to reduce the RM content of materials K and U [
The clinical success of acrylic resins depends not only on the chemical and mechanical properties of the materials but also on their biological safety. As such, the postpolymerization treatment that proved to promote a higher reduction in RM content while maintaining the mechanical properties (i.e., 50% or 20% ethanol solution at 55°C, for K and U, resp.) was further evaluated in what concerns cytotoxicity.
In this study,
Two different endpoints commonly used as a measure of dental materials cytotoxicity, mitochondrial enzyme activity (MTT assay), and plasmatic membrane damage (LDH assay) were used to assess the
In the present study, the results from both cytotoxicity assays were consistent and showed that the use of ethanol aqueous solutions at 55°C as postpolymerization treatment significantly decreased the cytotoxicity of both materials. In contrast, immersion in water at 55°C had no significant effect on materials cytotoxicity. Similar results were reported by others who found that postpolymerization heat-water treatments did not markedly influence
At this point, we may conclude that the hypothesis of our study was found to be partially accepted, since postpolymerization treatments based on ethanol solutions did improve the biocompatibility of the acrylic resins in the groups submitted to a combination approach of ethanol-water solutions and temperature.
Autopolymerized hard reline resins are commonly used for direct relining of dentures. Advantages of time, cost, and logistics of these acrylic resins compared to the laboratory-processed reline systems became very relevant in the oral rehabilitation of a growing geriatric and frail population. However, the biocompatibility of these materials is still a problem [
Under our experimental conditions, a postpolymerization treatment based on a combination approach of ethanol-water solutions and temperature enabled the reduction of the monomer content and the cytotoxicity of acrylic reline resins, while keeping their mechanical properties. Specifically, for Kooliner, the immersion on 50% ethanol solution at 55°C during 10 min showed to be the best condition. In Ufi Gel Hard, the most effective postpolymerization treatment was the 20% ethanol solution at 55°C during 10 minutes.
The authors alone are responsible for the content and writing of the paper.
The authors are grateful to Drs. Sara Oliveira, Teresa Eliseu, Elysse Filipe, and Sílvia Coelho for their contribution on the laboratory experiments. The authors would like to thank Voco GmbH (Cuxhaven, Germany) for the donation of the Ufi Gel Hard material evaluated in this study and Fundação para a Ciência e Tecnologia (Portugal) (PEst-OE/SAU/UI4062/2011; PEst-OE/SAU/UI4013/2011; Ciência 2008 for J. P. M. and EXCL/CTMNAN/0166/2012) for providing financial support to this project.