Cytocompatibility of a self-adhesive gutta-percha root-filling material.

CONTEXT: A novel root-filling material based on the incorporation of ultrafine alkaline bioactive glass particles (bioactive gutta-percha, [BGP]) was developed to work without sealer. AIM: In the present study, the objective was to verify the in vitro biological response to this material by assessing its cytocompatibility.
MATERIALS AND METHODS: Prototypes of BGP were compared to conventional gutta-percha (GP), dense polystyrene beads as a negative control and fragments of latex as a positive control. Extracts of each material were prepared according to ISO 10993-5:2009, and human osteoblast-like cells in primary culture were exposed to all extracts for 24 h. Cell viability was assayed sequentially for three different parameters: mitochondrial activity, membrane integrity, and cell density. STATISTICAL ANALYSIS USED: Nonparametric analysis (using Kruskal-Wallis test combined with post hoc Dunn's test) was performed for comparison among groups, with significance established at 5%.
RESULTS: BGP reduced mitochondrial activity to 62% of control, but presented no toxicity on membrane integrity and proliferation assays. BGP effect on metabolism was dose-dependent and reduced to acceptable levels with dilution.
CONCLUSION: The novel GP material presented slight dose-dependent effects on cell metabolism but did not affect cell survival.

INTRODUCTION

Gutta-percha (GP) remains the most commonly used and universally accepted core root-filling material.[1] One favorable aspect of GP is related to its thermoplastic properties, which allowed the development of filling techniques using heated material.[2] These techniques are, to some extent, able to improve the overall filling quality of the root canal space as compared to cold compaction techniques.[34] However, even when used in the thermoplastic form, GP has well-known shortcomings, specifically regarding its shrinkage on cooling,[5] lack of rigidity and adhesiveness, and usual displacement under pressure,[6] features that frequently characterize the suboptimal standard of current root canal fillings.

Alternative approaches as well as new root-filling materials have been introduced with the clear purpose to circumvent the limitations of GP and try to produce a reliable fluid-tight seal monoblock root filling. Bioactive glass (BG) is being extensively explored to design novel dental materials, even though there is a lack of literature on its complete biologic effects.[7] The expected properties of BGs include the ability of strong interfaces with hard and soft tissues as well as effects on the ionic equilibrium by either the release or uptake of ions from the biological environment (often leading to bioactivity).[8] The capability of forming an hydroxycarbonate apatite layer allows for the strong interaction and binding to bone by interacting with collagen fibrils, protein adsorption, incorporation of collagen molecules osteoblast attachment.[8] In this context, a new self-adhesive root-filling material was developed, and an initial report claimed that it is a potentially interesting material, bioactive GP (BGP).[9] This material consists of radiopaque flame-sprayed glass nanoparticles (45S5) incorporated into polyisoprene, the matrix polymer of GP. ZnO was also incorporated to maintain the gum-like thermoplasticity of GP and to enhance its radiopacity.[10] The recent studies with composites containing 45S5 nanoparticles have confirmed their improved bioactivity.[1112] Moreover, studies on the incorporation of the 45S5 bioglass nanoparticles onto polymers also had shown that those materials also present antimicrobial properties and good immediate sealing properties when heated and applied into simulated root canals in resin blocks.[9] Subsequent studies have shown that this novel GP can induce a high pH environment and is self-adhesive to root dentin.[10] However, as can be expected from any new material, there is a current lack of scientific evidence regarding the cytocompatibility of this composite material. Therefore, the present study was designed to assess, through a multiparametric in vitro assay, the effects of the self-adhesive GP, BGP, on primary human osteoblast (HOB) viability. Conventional GP was employed as the gold-standard filling material for comparison.

MATERIALS AND METHODS

Materials

The following materials were used: Conventional GP (Dentsply, Petrópolis, Rio de Janeiro, Brazil) as a gold-standard material; a novel GP containing 20% of radiopaque nanometric BG (BGP, Smartodont llc, Zurich, Switzerland) as a tested material; dense polystyrene (PS) as a negative control; and fragments of latex (L) as a positive control. The sample preparation was performed as recommended for irregularly shaped solid devices according to ISO 10993-5:2009.[13] Each material was fragmented into small pieces of <0.5 cm measured in the largest dimension, and portions of 0.2 g were separated and subsequently sterilized by gamma radiation (25 kGy). Extracts of 0.2 g of each material/mL of culture medium alpha-minimum essential medium (MEM) without fetal calf serum were prepared after sterilization. After 24 h of incubation at 37°C, the extracts were used immediately in the cytotoxicity assay.

Cells

HOB from the HUAP Cell Culture Bank were cultured in alpha-MEM (Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (FBS) and three antibiotics (penicillin 10.000 UI/mL, streptomycin 10 mg/mL, and sulfate gentamicin 50 mg/mL) under humidified atmosphere containing 5% CO2 at 37°C on 25 cm culture flakes. All cells were used in the second passage and still presented osteoblast phenotype as indicated by routine biochemical and morphological assessments.

pH

pH-indicator strips (Merck, Darmstadt, Germany) were used to measure pH either when the extract was placed on the cells or 24 h after exposure. pH values were approximated to the next 0.5 unit.

Cytotoxicity assay

To assess the acute effects of indirect exposure to the materials, a multiparametric assay was performed as described previously.[14] In brief, cells were analyzed using a kit (In-Cytotox, Xenometrix Inc., Allschwill, Switzerland) which allows the sequential assessment on the same cells, of three different cell viability parameters, i.e., the mitochondrial activity XTT (2,3-bis [2-methoxy-4-nitro-5-sulfopheny]-2H-tetrazolium-5-carboxyanilide, XTT), membrane integrity (neutral red [NR] test), and total cell density (crystal violet dye exclusion [CVDE]) on the same cells.

The HOBs were seeded into 96-well plates at a density of 1 × 104 cells/well. The cells were incubated in alpha-MEM and maintained at 37°C under humidified atmosphere containing 5% CO2. After 24 h, the culture medium was removed and replaced by 180 μl of each extract (BGP, GP, PS, and L) or by unconditioned culture medium (alpha-MEM, control), followed by the addition of 20 μl (10%) FBS to each well.

After the 24-h exposure of HOB to each conditioned medium, the cells were submitted to XTT assay. This test is based on the ability of mitochondrial enzymes from metabolically active cells to reduce 2,3-bis (XTT) molecules to a soluble salt of formazan, detectable by its absorbance at 480 nm, as measured by a ultraviolet-visible microplate reader (Synergy II; Biotek Inst., Winooski, VT, USA). For this assay, the extracts were removed from each well by inversion, and wells were filled with 200 μl of serum-free alpha-MEM. Fifty microliters of XTT reagent were added, followed by incubation at 37°C/5% CO2 for 2 h. Optical density (OD) was measured at 480 nm. The same cells submitted to the XTT test were washed with phosphate-buffered saline (PBS) and assayed with the NR test. The vital dye NR is incorporated through endocytosis and accumulates preferentially on the lysosomes of membrane-intact viable cells. After 3 h of exposition to the dye, cells were fixed and the NR was extracted on an acetic acid: Ethanol solution, and the OD of the supernatant was measured at 540 nm.

After the NR test, cells were washed again with PBS and assessed by the CVDE test. Cells were treated with concentrated crystal violet (CV) dye for 10 min, and after four sequential washing steps with PBS, the dye was extracted with a solution containing acetic acid and ethanol. As CV preferentially binds to DNA, the OD at 540 nm of the dye extracted after washing was directly related to the total amount of cells in each well.

To determine if dilution was able to diminish any observed level of cytotoxicity presented by the materials, the extracts of each material (BGP, PS, and L) were diluted on alpha-MEM at the concentrations of 100%, 50%, 10%, or 5% (v/v) on a flow hood, and osteoblasts were exposed to each diluted extract by 24 h as described above. Cell viability was assessed by the XTT assay. For this assessment, cells were transferred to 96-well plates at densities of 1 × 104 cells/well. After 24 h, the culture medium was changed according to each dilution of the extracts. Each experiment was conducted in quintuplicate (n = 5).

Statistical analysis

Statistical analysis was carried out using the InStat® 3.01 software (GraphPad Software, San Diego, CA, USA). The mean values and standard deviations were calculated for each group, and normal distribution of variables was verified with D'Agostino test. Nonparametric analysis of variance test (Kruskal–Wallis), with post hoc Dunn's test, was performed for comparison among groups. The significance of all tests was established at 5%.

RESULTS

Cytotoxicity was evaluated after 24-h exposure to extracts of the tested materials. For each material, data were expressed as a percentage of the control value. There was no difference among the experimental material (BGP), conventional GP, and the negative control (PS) (P > 0.05) for all three methods employed while the positive control (L) induced significant cytotoxic effects with survival rates of <20% of the control group [Figure 1a–c]. BGP induced a mild effect on mitochondrial activity (62% of control), but presented no statistical difference from GP and PS on the NR and CVDE assays (P > 0.05).

Figure 1

Multiparametric cytotoxicity assay. (a) XTT; (b) NR and (c) CVDE. Bars represent mean ± standard deviation (n = 5), normalized as a percentage of the control (untreated) group. PS – Polystyrene: Negative control; Latex: Positive control; BGP: Bioglass gutta-percha; GP: Gutta-percha. An asterisk indicates significant difference from control group(P < 0.05)

As observed in Figure 2, GP extracts showed cell viability data similar to the control in all dilutions used, while the positive control, L, remained cytotoxic even when diluted as 10% of the original extract. BGP, however, presented a dose-response on its effect on mitochondrial activity, decreasing to acceptable levels of toxicity in the first dilution, indicating that dilution has greater effects on the reduction of XTT as compared to the positive control.

Figure 2

Determination of dependence of dose on the XTT assay. Extracts were diluted on culture media in the indicated percentage before cell exposure. Results of XTT reduction presented as mean ± standard deviation (n = 5), normalized as a percentage of the control (untreated) group. Latex: Positive control; BGP: Bioglass gutta-percha; GP: Gutta-percha

GP and L induced stable pH values of 7.5 in all measurements (diluted extracts, 5%, 10%, 50%, and 100%) while BGP increased from pH 7.5 to pH 9 after 24 h of extraction. Dilution of BGP extracts decreased the pH values: 100% pH 9, 50% pH 8, 10% pH 7.5, and 5% pH 7.5.

DISCUSSION

Recently published results have demonstrated through an animal graft model that BGP presents good biocompatibility, even though with an expected initial inflammatory response.[15] However, several limitations on the prediction of efficacy and potential adverse effects in humans from animal models become evident with the high rates of failure during clinical trials of drugs and medical materials. Therefore, the corroboration of those previous findings with human primary cells contributes to a safer animal-to-human extrapolation of biocompatibility.[16] It is important to note HOBs were chosen for this study, as they also presented stable and predictable results on the previous studies on endodontic materials.[1417] While the periapical environment is composed of diverse cell types, such as fibroblasts and DPSCs, the pulp is also attached to the surrounding mineralized tissue which is maintained by osteoblasts, which are vital to system teeth-periodontal ligament-bone. Primary cells are also considered as providing a better simulation of in vivo events and resembling more closely the behavior of cells in their tissue environment than transformed cell lines.[1819]

Another important methodological feature of the present study is the multiparametric assessment of cytocompatibility. This multiple-endpoint assay allows the evaluation of three different parameters from the same cellular sample: (a) Mitochondrial metabolism and respiratory toxicity, (b) lysosome integrity and membrane permeability, and (c) the presence of DNA and cell proliferation.[14] It was observed that the current formulation of BGP showed a significant effect on mitochondrial activity, with 62% of the control XTT reduction, against a proposed cutoff of 75% proposed for nontoxic materials (ISO 10993-5:2009).[13] However, in the multiparametric assay, performed sequentially on the same cells, no toxicity was observed regarding membrane integrity or proliferation. This result may indicate that while BGP is not able to cause cell death, it may induce changes in the culture media, which are sufficient to decrease metabolic activity on the restrained conditions of the in vitro assay. In fact, it is often common to find different results between diverse cell viability parameters on multiparametric tests,[20] usually related to alterations on calcium and phosphate content, or pH of culture media.[2122] It is possible that the effects of the extraction procedure on medium pH could have affected the cell metabolism but not cell survival, when potentiated on the confined conditions and proportions of in vitro tests based on ISO 10993-5:2009.[13]

In the present study, the serial dilutions of the extract revealed that the effects on mitochondrial activity present a dose-dependent response, suggesting the possibility of a reduction of the effect with time or washing, depending on the dilution of the material in body fluids.[23] In this regard, the novel GP material performed much better than the positive control, with a consistent decrease of toxicity with dilution.

The higher pH observed in BGP buffered solutions suggested a material profile with antimicrobial activity.[2425] This issue seems to be important because no endodontic procedure can eliminate the entire intracanal bacterial load. Therefore, further studies are needed to verify the antimicrobial features of this novel GP material.

CONCLUSION

The experimental self-adhesive GP material showed mild dose-dependent effects on cell metabolism at in vitro conditions; however, it did not affect the overall cell survival.

Financial support and sponsorship

The work was supported by the Department of Cell and Molecular Biology of Fluminense Federal University in Niterói, Brazil.

Conflicts of interest

There are no conflicts of interest.