Effects of Different Self-Adhesive Resin Cements and Curing Through Zirconia on Gingival Fibroblasts

Kittipat Termteerapornpimol; Karn Tongchairati; Narin Intarak; Sasiprapa Prommanee; Soranun Chantarangsu; Dusit Nantanapiboon; Junji Tagami; Thantrira Porntaveetus

doi:10.1016/j.identj.2024.11.004

. 2024 Dec 12;75(2):1047–1057. doi: 10.1016/j.identj.2024.11.004

Effects of Different Self-Adhesive Resin Cements and Curing Through Zirconia on Gingival Fibroblasts

Kittipat Termteerapornpimol ^a,^b, Karn Tongchairati ^a, Narin Intarak ^a, Sasiprapa Prommanee ^a, Soranun Chantarangsu ^c, Dusit Nantanapiboon ^d,^e, Junji Tagami ^f,^g, Thantrira Porntaveetus ^a,^h,^⁎

PMCID: PMC11976558 PMID: 39672781

Abstract

Introduction

Self-adhesive resin cements (SARCs) are widely used for fixed prostheses. These cements and their eluted products may affect periodontal tissues. This study aimed to investigate the response of human gingival fibroblasts (HGFs) to eluates from SARCs in vitro, simulating clinical conditions after prosthesis fixation, to gain insights into their potential effects on gingival health.

Methods

Two SARCs, RelyX U200 (RX) and Maxcem Elite Chroma (MC), were polymerised according to the manufacturer's protocols using various curing methods (light-cured, light-cured through 1- or 2 mm zirconia, and self-cured). HGFs were exposed to cement eluates at different concentrations. Cell viability, vitality, wound healing, and gene expression were assessed at different time points.

Results

Self-cured MC and MC cured through 2-mm zirconia (both undiluted and 1:5 dilution) significantly decreased HGFs’ viability. Lower cell viability and vitality were detected in MC compared with RX. Wound healing was delayed in cells treated with MC cured through zirconia compared to those cured with direct light, whereas zirconia had no effect on cells treated with RX. The expression of NRF2, a key regulator of cellular defence against oxidative and toxic insults, showed an increasing trend in cells treated with MC compared to RX. This finding suggests that MC may induce more oxidative stress than RX, leading to a more pronounced inflammatory response in HGFs and aligning with the observed delay in wound healing.

Conclusion

The use of MC, especially when cured through zirconia, may negatively impact gingival tissue health, highlighting the importance of carefully selecting cement types and curing methods in clinical practice.

Clinical relevance

This study highlights the potential risks of using MC, especially when cured through zirconia, which may impair gingival tissue health and delay wound healing. Proper choice of cement and curing methods is essential for optimal patient outcomes.

Key words: Cytotoxicity, Fibroblasts, Health care, Resin cements, Wound healing, Zirconia

Introduction

In current dental practices, self-adhesive resin cement (SARC) is widely used in prosthodontic dentistry for fixed prostheses. Cement or their eluted products stand a great chance to interfere with periodontal tissues surrounding natural teeth or dental implants, and possibly dental pulp. In 2017, a systematic review showed that excess dental cement is a risk factor causing periimplant mucositis and periimplantitis.¹

Molecular and cellular cytotoxicity of SARCs has been studied and showed that the cements reduced viability of gingival fibroblasts2, 3, 4 and compromised proliferation of periodontal ligament cells.⁵^,⁶ Gallegos et al demonstrated that SARC significantly reduced viability of human gingival fibroblasts (HGFs) at day 3 after direct contact with cement. SARC obtained the lowest cell viability when contacted with preosteoblasts, compared with bioceramic cement, zinc phosphate cement, and resin-modified glass ionomer cement.² These indicate that the inadequate removal of excess SARC can lead to periodontal inflammation. To date, studies on the effects of SARCs on gene expression are limited. Alvarez et al studied the effects of the cements on expression of genes involved in osteogenic and odontoblastic differentiation and inflammatory and antioxidant responses of odontoblast-like cells. The results showed that expression of those genes varied among different cements.⁷ However, to date, there is no study that describes the effects of SARCs on such responses in HGFs.

Resin monomers found in these cement products have been shown to affect healthy cells surrounding teeth or implants differently. For example, among Bis-GMA, hydroxylethyl methacrylate (HEMA), triethylene glycol methacrylate (TEGDMA), and urethane dimethacrylate (UDMA), Bis-GMA is considered the most cytotoxic resin monomer.⁸ Mendonca et al demonstrated that HEMA-based resin cement and TEGDMA/Bis-GMA–based resin cement had a significantly different impact on MDPC-23 odontoblast cell line.⁹ Besides their components, handling techniques of SARCs also contribute to their biocompatibility. Schmid-Schwap et al reported that light-curing application in RelyX Unicem led to a significant increase in fibroblasts compared to autopolymerised specimens.⁸ Many studies attempted to simulate clinical scenarios in which the restoration was placed between the light-curing unit and the cement. While there is evidence of light attenuation caused by ceramic restorations, the impact of this phenomenon at the cellular level has not yet been well understood.¹⁰^,¹¹

The aims of this study were to investigate and compare the response of HGFs to two types of SARCs, namely RelyX U200 (RX) and Maxcem Elite Chroma (MC), subjected to different polymerisation methods (light-cure, light-cure through zirconia or self-cure). The goal was to enhance our comprehension of how SARCs influence the cellular and molecular behaviours of HGFs.

Materials and methods

Cements and eluates preparation

Two products of SARCs, RelyX U200 (RX, 3M ESPE) and Maxcem Elite Chroma (MC, Kerr Corporation), were used and prepared according to a manufacturer's instruction. The compositions and handling protocols of selected SARCs¹² are listed in Table 1. Briefly, cement specimens were prepared using transparent cylindrical acrylic moulds (height = 2 mm, diameter = 5 mm) with mylar strips and glass slides in an aseptic environment. The mylar strips were used to separate the cement and zirconia disc during the curing process. Four polymerisation groups were designed as follows: self-cured (left polymerised in the dark for 4-6 minutes), light-cured, light-cured through 1 mm (1 mm-Z), and 2 mm zirconia (2 mm-Z). The 3M Elipa DeepCure-S LED Curing Light device (3M ESPE) with a light intensity of 1470 mW/cm² was used. Prior to the experiment, the device was calibrated with a Demetron LED radiometer (Kerr Corporation) to ensure the claimed light intensity of 1470 mW/cm². The A2 shade 3M Lava Esthetic zirconia was used in the light-curing through zirconia conditions. Immediately after polymerisation, cements were immersed in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco), 50 mg/mL streptomycin, and 100 U/mL penicillin, with a surface area-to-volume ratio of 1.25 cm²/mL. The samples were incubated at 37°C for 24 hours, following ISO 10993-12:2013 guidelines.¹³ Depending on the volume of extracts required for each experiment, we prepared 2 cement discs per group for the MTT assay and 5 cement discs per group for all other experiments, including the wound healing assay, cell vitality assessment, and RNA extraction. Freshly prepared eluates were diluted into 1:5, 1:10, 1:20, 1:40, and 1:80 concentrations using 10% FBS DMEM. The MTT cell viability test utilised eluates at both full and diluted concentrations of cement eluates, whereas the cell vitality test, scratch wound assay, and gene expression analyses employed either full concentration and/or a 1:20 dilution of cement extract.

Table 1.

Details of self-adhesive resin cements (SARCs) used in this study.

Material	Manufacturer	Composition	Curing protocols
RelyX U200 (RX) Lot no. 9953335	3M ESPE	Methacrylate monomers containing phosphoric acid groups, methacrylate monomers, silanated fillers, initiator components, stabilisers, rheological additives, alkaline initiator components, pigments	Self-cure: 6 min Light-cure: 20 s
Maxcem Elite Chroma (MC) Lot no. 8994778	Kerr Corporation	HEMA, GDM, UDMA, 1,1,3,3-tetramethylbutyl hydrogen peroxide, TEGDMA, fluoroaluminosilicate glass, GPDM, barium glass fillers, fumed silica	Self-cure: 4 min Light-cure: 10 s

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Cell culture

HGFs obtained from 3 unrelated healthy donors (age 19-25 years old) were maintained in 10% FBS DMEM and incubated at 37°C with 5% CO₂. Cultures from passages 3 to 6 were used in this study. The protocols were approved by the Human Research Ethics Committee of the Chlalongkorn University (No. 087/2023, Date of approval: 1 September 2023).

Characterisation of gingival mesenchymal cells

The immunophenotype of gingival-derived mesenchymal cells (MSCs) was determined. Briefly, at cell passage three or four, an approximately 1.0 × 10⁵ cell suspension in 2% FBS phosphate buffered saline (PBS) was directly stained for 30 minutes in the dark at room temperature with specific monoclonal immunoglobulin G (IgG) antibodies against human CD44, CD45, CD73, CD90 and CD105 conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or peridinin chlorophyll (PerCP) protein. The stained cells were then fixed with 600 μL of 4% paraformaldehyde solution. Acquisition and analysis were performed with a flow cytometer (BD FACSCalibur). The isotype controls used were monoclonal antibodies FITC-IgG1 and PE-IgG1.

Cell viability

HGFs were seeded in triplicate for each condition at a density of 5000 cells/well in 96-well plates. After 48 hours of incubation, the experimental groups were treated with various dilutions of cement eluates, while the control group was cultured in 10% FBS DMEM. The cells were then incubated for either 30 minutes or 24 hours in CO₂ incubator. At the designated timepoints, cell viability was observed using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assays following previously described protocols.¹⁴^,¹⁵ Briefly, cells were treated with MTT reagent (5 mg/mL) and kept at 37°C with 5% CO₂ for 1 hour. Formazan precipitations were dissolved using dimethyl sulfoxide (DMSO). Absorbance measurement at 570 nm was performed using Microplate reader (BioTek, Winooski, VT). The experiment was carried out for 3 independents. To measure cell viability, the optical density (OD) values obtained from cells treated with either RX or MC were each normalised to the same control OD value as follows¹⁴: % Cell viability = [OD_sample ÷ OD_control] × 100.

Cell vitality

HGFs were seeded at a density of 5000 cells/well in 96-well plates. After 48 hours, the cells were treated with either undiluted and 1:20 diluted eluates for each curing condition (RX and MC) and compared with cells treated with 10% FBS DMEM as the control group. Then, the treated cells were stained using DAPI (4′,6-diamidino-2-phenylindole) and the FITC Annexin V apoptosis detection kit with propidium iodide (PI) (Biolegend) according to manufacturer's instruction. Images were visualised using a ZEISS Axio Imager Apotome 2 microscope (Carl Zeiss Microscopy). The cells were classified according to staining outcomes, which are divided into 4 groups as follows: (1) vital cells: DAPI(+), FITC(–), PI (–); (2) cells in an early stage of death: DAPI(+), FITC(+), PI (–); (3) cells in a middle stage of death: DAPI(+), FITC(+), PI (+); and (4) cells in a late stage of death: DAPI(+), FITC(–), PI (+). Cell numbers were quantified using ImageJ version 8.0.2 software. The percentage of cells in each category was calculated. The experiments were performed in duplicates for three independent experiments.

Wound scratch assay

To evaluate the effect of the cements on the wound healing ability of HGFs, a scratch wound assay was performed. HGFs were seeded in 24-well plates at a density of 30,000 cells/well and allowed to reach 90% confluence growth. After 48 hours, the wound was established in the middle of each well using a sterile 200 μL micropipette tip and subsequently washed with PBS to remove any dislodged cells or debris. The cells were then treated with 1:20 dilutions of the eluates or 10% FBS DMEM (control), and the gaps were observed and captured at the same locations under light microscope (Olympus Primovert) at 0, 24, 48, and 72 hours after treatment. The cell-free area was analysed using ImageJ version 8.0.2 software. The experiments were carried out in duplicates for three independent experiments. Wound healing ability was assessed by calculating the percentage of wound closure using the following formula¹⁶: % Wound closure = [(Initial gap area – Gap area at each timepoint) ÷ Initial gap area] × 100.

Gene expression

HGFs were seeded at a density of 100,000 cells/well in 6-well plates. After 48 hours, the cells were treated with an undiluted concentration of cement eluates for 30 minutes and a 1:20 diluted concentration for 24 hours, alongside 10% FBS DMEM serving as the control. At desirable timepoints, total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) and then subjected to cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). The mRNA expression level was determined through quantitative RT-PCR CFX using the Real-Time PCR Detection Systems (Bio-Rad) with specific primers for the target genes of interest, as listed in Supplementary Table S1, including GAPDH (endogenous control). Relative gene expression was calculated using the 2^-ΔΔCT method.¹⁷ The experiments were repeated at least three times independently.

Statistical analysis

Each experimental condition, including both RX and MC, along with a single shared control group for each experimental condition, was performed in triplicate. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc.). Mann-Whitney U test was applied for two-independent group comparisons and Kruskal-Wallis H and Dunn's post-hoc test for comparisons of 3 or more independent groups. A significance level (P value) less than .05 was considered statistically significant.

Results

MSCs characterisation

HGFs exhibited cell morphology resembling fibroblasts. These cells expressed the mesenchymal stem cell markers including CD44, CD73, CD90, and CD105, without the expression of the hematopoietic stem cell marker (CD45), as shown in Supplementary Figure S1.

Effect of cement eluates on HGFs viability

To investigate the viability of HGFs in response to SARC eluates obtained from different polymerised procedures, the MTT assay was performed on cells treated with various concentrations of cement eluates. Treatment with undiluted cement extract at 30 minutes was established to observe the acute cytotoxic effects of SARCs on HGFs. The result showed that the viability of HGFs treated with RX under all curing conditions (self-cured, light-cured, 1 mm-Z, 2 mm-Z) was comparable to that of control (Figure 1). In contrast, both self-cured and 2-mm-Z MC groups exhibited significantly lower HGF viability compared to the control. Within the MC groups, direct light-cured MC showed significantly higher viable cells compared to 2 mm-Z. When comparing the types of cements, RX demonstrated significantly higher cell viability than MC, whether they were self-cured or light-cured through 2 mm Zirconia (Figure 1). These findings suggest that RX is less toxic or more compatible with HGFs and that zirconia thickness can influence the toxicity or compatibility of MC on HGFs. Additionally, MC appears to be more sensitive to zirconia thickness than RX.

Fig 1 — The percentage of HGFs viability in the presence of cement extracts after 30 minutes. * indicates statistically significant differences compared to the controls (P <.05), # indicates statistically significant differences between groups (P <.05).

After 24 hours of exposure, within the RX groups, both full and diluted concentrations (1:5, 1:10, and 1:20) under all conditions exhibited comparable cell viability to the control, except undiluted self-cured RX, which showed a significant reduction in HGFs viability (Figure 2A). In contrast, MC at full and 1:5 concentrations under all curing conditions, except 1:5 light-cured, displayed significantly reduced HGFs viability across all curing conditions, while 1:10 and 1:20 showed comparable cell viability to the control (Figure 2B). These findings indicate that regardless of the types of cement, both undiluted RX and MC, when self-cured, demonstrated a significant reduction in HGFs viability compared to the controls. However, when light-cured through zirconia (both 1 mm and 2 mm thickness), undiluted and 1:5 concentrations of RX showed significantly higher HGFs viability. Additionally, when directly light cured, RX and MC at 1:5, 1:10, and 1:20 concentrations showed comparable cell viability. These results suggest that the cell compatibility of MC is influenced by its concentration/dilution and the presence of zirconia when light cured.

Fig 2 — The percentage of HGFs viability in the presence of cement extracts at 24 hours. A, RX. B, MC. * indicates statistically significant differences compared to the control (P <.05).

HGFs vitality in response to cement eluates

Cement eluates have previously been shown to induce cell death.18, 19, 20 Therefore we investigated whether cement eluates obtained from different curing procedures would affect the vitality of HGFs (Supplementary Figure S2). In line with the observations regarding cell viability, no viable cells were detected under any conditions with the full concentration of MC after 24 hours of treatment. Only dead cells were detected, with a notable increase in the late stage of cell death following this order: 2 mm-Z > self-cured > 1 mm-Z > direct light-cured MC. In the self-cured RX group, some dead cells were detected; however, the light-cured RX groups showed a high percentage of vital HGFs (direct light-cured: 99.75%, 1 mm-Z: 99.75%, and 2 mm-Z: 98.80%) (Figure 3). Furthermore, we observed that a 1:20 dilution, with a 24-hour treatment duration, consistently yielded nearly 100% viable cells across all conditions in both RX and MC groups (Supplementary Figure S3). The 1:20 concentration was chosen for the wound scratch and 24-hour gene expression assays.

Fig 3 — Vitality of HGFs treated with undiluted cement extract at 24 hours. # indicates statistically significant differences between groups (P <.05).

Delayed wound closure of HGFs in response to cement eluates

We investigated whether persistent exposure of HGFs to cement eluates resulted in delayed wound healing (Supplementary Figure S4). The wound scratch assay showed that the percentage of wound closure in HGFs exposed to RX was not significantly different from the control group throughout the 3-day observation period. In contrast, all groups exposed to MC exhibited a markedly lower percentage of wound closure compared to the controls on day 1. Specifically, on day 2, both the 1 mm-Z and 2 mm-Z MC groups displayed significantly delayed wound closure. By day 3, the wound closure of the 2 mm-Z MC group remained significantly lower than that of the control group (Figure 4). When comparing different types of cements, the 2 mm-Z MC group exhibited significantly inferior wound closure compared to the 2 mm-Z RX group. These findings suggest that while all RX groups demonstrated favourable wound closure, the presence and thickness of zirconia in cement influence the wound-healing capability of HGFs.

Fig 4 — Wound healing rate relative to day 0 of HGFs treated with cement extracts. * indicates statistically significant differences compared to the control group on the same experimental day (P <.05), and # indicates statistically significant differences between groups (P <.05).

Gene expression of HGFs in response to cement eluates

We investigated the expression of genes related to cell proliferation and wound healing, MMP2 and CCR3. The 1 mm-Z MC displayed a notably elevated MMP2 with 2-fold increase in expression at 30 minutes compared to the control (Figure 5A). After 24 hours, both 1 mm-Z MC and 2 mm-Z MC exhibited a significant upregulation in MMP2 expression with 2.3- and 2.6-fold increases, respectively, compared to the control. Additionally, 2 mm-Z MC displayed a higher expression than 2 mm-Z RX at 24 hours (Figure 5B). Conversely, the expressions of CCR3 in both RX and MC under all conditions at 30 minutes and 24 hours were comparable to the control, except for self-cured RX, which showed a significantly higher level (5.5-fold) than the control at 30 minutes (Figure 5C).

Fig 5 — Gene expression profiling of HGFs following exposure to undiluted cement extracts at 30 minutes and 1:20 diluted extracts at 24 hours. A and B, *MMP2*. C and D, *CCR3*. E and F, *MMP2*. G and H, *CCR3*. (A, C, E, and G) 30 minutes, (B, D, F, and H) 24 hours. * indicates statistically significant differences compared to the controls (P <.05), # indicates statistically significant differences between groups (P <.05).

To further explore the molecular alterations–associated stress response, NRF2 and HO-1. At 30 minutes, all RX and MC groups displayed similar NRF2 expression compared to the control, except for the 1 mm-Z MC group, which showed a significant increase in expression (1.7-fold) (Figure 5E). When comparing different types of cements, the 1 mm-Z and self-cured MC groups demonstrated significantly higher NRF2 expression compared to the RX under the same conditions (Figure 5E). At 24 hours, both RX and MC did not show any significant difference to the control (Figure 5F). In contrast, HO-1 did not exhibit any significant differences between RX or MC and control at 30 minutes (Figure 5G). However, at 24 hours, light-cured and 1 mm-Z MC showed significantly reduced HO-1 expression compared to the control (0.4-fold in both groups), and 1 mm-Z MC also showed a significantly reduced expression compared to 1 mm-Z RX (Figure 5H).

Furthermore, we examined the expression of inflammatory cytokine (TNF-α, IL-6, IL-8) and apoptotic markers (BAX and BCL-2). No significant change in the expression levels of the observed genes was found among MC, RX, and control groups (Supplementary Figures S5 and S6).

Discussions

Optimal periodontal health significantly contributes to the success of fixed prostheses. As SARCs become more prevalent in routine dental practice, it becomes imperative to thoroughly assess the biocompatibility of these materials. This is crucial because the cements or their leached components may interface with and potentially interact with the surrounding periodontium, including the gingiva.

In this study, we demonstrated that the viability, vitality, wound healing ability, and gene expression of HGFs were influenced by the thickness of zirconia, cement manufacturers, and concentration of cement extract. We employed the cement extract dilution method to mimic clinical scenarios, as cement products washed out over time by gingival crevicular fluid and saliva after prosthesis cementation. Our findings align with previous studies conducted on MDPC-23 immortalised rat odontoblast cells, indicating that MC was more toxic than RX.⁷^,⁸^,²¹^,²² The observed variations in cytotoxicity between RX and MC may be attributed, at least in part, to differences in the quantity and combinations of resin monomers in their formulations, such as HEMA, TEGDMA, and UDMA. These monomers have been demonstrated to interfere with normal cellular functions, affecting both cellular and systemic levels with varying degrees of toxicity, as confirmed by dose-response studies.23, 24, 25, 26, 27 Additionally, previous research highlighted significant differences in the effects of HEMA-based resin cement compared to TEGDMA/Bis-GMA–based resin cement on cell lines.⁹ A previous study has shown that TEGDMA was detected in extracts from light-cured RX, exhibiting a steady release profile after 24 hours of incubation.²⁸ Another study identified significant TEGDMA levels specifically when RX was cured through zirconia.²⁹ In contrast, UDMA and BisGMA were not detected in RX extracts using HPLC analysis.²⁸^,²⁹ However, HEMA and GDMA were present in the extracted DMEM from MC samples. The levels of these residual monomers in MC were elevated, when the polymerisation process was hindered by zirconia or the absence of light activation in self-cured groups.²⁹ HEMA was exclusively present in the MC formulation. Despite its relatively low toxicity towards HGFs,²³ it exhibited the highest release in eluates among the resin monomers due to its hydrophilicity.³⁰ This characteristic could potentially contribute to differing levels of cytotoxicity between the two brands of SARCs examined in our study. Additionally, the presence of functional monomers in SARCs, which are not typically found in most conventional resin cements, could lead to greater toxicity compared to other cement types.³^,⁴^,³¹ These functional monomers, such as 10-MDP and 4-META, have low pH levels that can potentially disrupt cell functions. Furthermore, their acidity interferes with polymerisation by reducing the activity of the free-radical initiator,³² resulting in a lower degree of conversion and the release of toxic substances into the oral environment.³³

Various curing techniques in cementing fixed prostheses represent distinct clinical scenarios. Limited light transmission, as seen in metal crowns, tends to result in self-curing, while light-transmissible materials allow light activation. In our study, the use of dual-cure SARCs exhibited a significant difference in viable gingival cells between light-cure and self-cure groups, possibly indicating varying amounts of residual unreacted monomers.³⁴^,³⁵ A previous study revealed that the cytotoxicity of SARCs in NIH/3T3 fibroblast cells was independent of interposing ceramic or resin nanoceramic materials (such as Lava Ultimate) with a thickness not exceeding 2 mm³. However, the efficacy of curing these cements is notably compromised by light-transmissible restorative materials. Specifically, a statistically significant reduction in the degree of conversion was observed in RelyX U100 when light-cured under 2 mm-thick IPS Empress II ceramic discs.³⁶ In our study, we utilised 3M Lava Esthetic zirconia, a light-transmissible material, and found that a 2 mm thickness of zirconia, when in contact with MC, influenced the viability, vitality, and wound healing capabilities of HGFs. Consequently, our findings emphasise the importance of considering the thickness of zirconia when employing SARCs, as it can impact the well-being of surrounding gingival cells.

Beyond cell viability, the scratch wound assay assesses the impact of SARCs on HGF migration, a crucial aspect of wound healing. Previous research has indicated that cements can impede wound healing.³⁷ In our study, MC exhibited a slower wound closure rate compared to RX under identical curing conditions, aligning with the observed cell viability. The impact of SARCs on gene expression, particularly in HGFs, remains poorly understood. In 2018, live cell imaging of HGFs treated with RX showed increased membrane permeability. Notably, cell death primarily occurred through the necrotic pathway, as evidenced by the absence of caspase-3 and caspase-7 activities.³⁸ In a study by Şişmanoğlu et al, the impact of different SARCs on apoptosis induction in NIH/3T3 mouse fibroblasts was examined, showing minimal effect on the apoptotic pathway.¹⁹ In our study, immunofluorescence staining revealed no significant increase in apoptosis for either MC or RX in HGFs. While some restorative materials can activate the intrinsic pathway of apoptosis in gingival cells, characterised by BAX upregulation and BCL-2 downregulation,³⁹ our findings did not detect any impact of RX and MC on the expression levels of these genes. This confirms that SARCs do not strongly induce apoptosis in gingival cells, suggesting the involvement of alternative pathways in cell response.

Concerning wound healing capability, the interaction between CCR3/CCL5 and CCL28 has been known to stimulate gingival fibroblast proliferation and migration. Specifically, the interaction between CCR3 and CCL28 can induce IL-6 secretion, activating the inflammatory process.⁴⁰ These ligand-receptor interactions may be linked to the pathogenesis of SARCs toxicity. However, our findings did not identify any significant difference in CCR3 expression under all conditions, suggesting that further investigation is needed to elucidate the potential associations between these interactions and SARCs-induced pathogenic effects. Additionally, maintaining a balanced expression between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is crucial in different stages of wound healing, including extracellular matrix organisation.⁴¹^,⁴² In our study, the expression of MMP2 was increased in both 1 mm-Z and 2 mm-Z MC at 24 hours, aligning with the observed delayed wound closure in these groups. This suggests that MC cured through zirconia might contain residual monomers, potentially inducing an overexpression of MMP2 and consequently leading to delayed wound healing.

There has been a growing concern surrounding the impact of resin monomers and eluted products from SARCs on cellular metabolism and stress. Our study demonstrated changes in stress response markers within the NRF2-KEAP1 pathway, crucial for maintaining antioxidant balance during the stress. Previous research indicates that oxidative stress and the presence of HEMA activate this pathway, affecting NRF2 and HO-1 expression.43, 44, 45 In our study, RX conditions showed no significant difference in NRF2 and HO-1 expression, while dysregulation of NRF2 was observed in 1 mm-Z MC after 30 mins, and that of HO-1 in 1 mm-Z MC and 2 mm-MC after 24 hours. Additionally, an increased trend in NRF2 expression was observed in MC compared to RX. These findings suggest that RX induces lower oxidative stress compared to MC, potentially indicating a defensive response by gingival cells against oxidative stress from uncured resin during the curing process. Figure 6 illustrates the overall findings of this study.

Fig 6 — Schematic diagram of cellular and molecular responses of HGFs exposed to cement extracts. Reduced HGF proliferation, vitality, and wound healing rate were observed when treated with extracts from MC cement cured through zirconia, as well as altered expressions of genes involved in stress response pathways.

Although the SARCs examined in this study appeared toxic to gingival tissue surrounding human teeth or implants in vitro, their clinical influence or acceptable biocompatibility in vivo could be attributed to various factors. For instance, the dilution effect of saliva and gingival crevicular fluid in the oral cavity may significantly reduce the concentration of potentially harmful substances leached from these materials, mitigating their toxic effects.⁴⁶^,⁴⁷ Moreover, although SARCs undergo self-curing in the dark, they undergo significant polymerisation over time, becoming more stable and less reactive.⁴⁸ The patient's oral care could also contribute to reducing the inflammatory effect on the periodontium. It is noteworthy that, apart from SARCs, recent core build-up materials are predominantly dual-cured, and no clinically apparent harmful effects on periodontal tissues have been reported, despite their contact with these tissues. However, our study raises the possibility that certain commercially available dual-cure dental materials might have a biological impact on surrounding gingival and periodontal cells.

Conclusion

The study demonstrated the impact of the brand and concentration of SARCs, along with the thickness of zirconia, on the viability, vitality, wound healing ability, and gene expressions of HGFs. These insights offer valuable information on SARCs’ biocompatibility and underscore the significance of optimising polymerisation procedures to enhance cellular and molecular responses in dental applications involving gingival cells.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgments

Acknowledgement

This research was supported by the National Research Council of Thailand (N42A650229), Health Systems Research Institute, Thailand Science Research and Innovation Fund Chulalongkorn University (HEA_FF_68_008_3200_001), and Faculty of Dentistry (DRF68_007), Chulalongkorn University. KiT was supported by the 72nd Anniversary of H.M. King Bhumibol Adulyadej Scholarship and the 90th Anniversary Chulalongkorn University Fund (Ratchadapiseksomphot Endowment Fund). NI was supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. JT was supported by the Chulalongkorn University, Second Century High Potential Professoriate Fund at the Faculty of Dentistry. ChatGPT was used to check the grammar of the manuscript.

Author contributions

KiT, KaT, NI, and TP contributed to the conceptualisation; DN assisted with specimen preparation; KiT, KaT, NI, and SP performed the experiments and collected and analysed the data; SC provided assistance with statistical analyses; KiT and KaT prepared the original draft; JT provided critical comments on the manuscript; and TP critically revised the manuscript.

Data statement

The data supporting the findings of this study are available within the article and its supplementary materials.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.identj.2024.11.004.

Appendix. Supplementary materials

Appendix A. Supplementary data.

Supplementary Table S1. Primer sequences of selected markers for qRT-PCR.

Supplementary Figure S1. Characteristics of human gingival fibroblasts (HGFs).

Supplementary Figure S2. Cement extract treated HGFs stained with DAPI/PI/Annexin V-FITC.

Supplementary Figure S3. Vitality of 1:20 cement extract treated HGFs at 24 hours.

Supplementary Figure S4. Representative images of wound scratch assay over 3-day observation.

Supplementary Figure S5. Gene expression profiling of HGFs following exposure to cement eluates at 30 minutes.

Supplementary Figure S6. Gene expression profiling of HGFs following exposure to cement eluates at 24 hours.

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4.Bandarra S, Neves J, Paraíso A, Mascarenhas P, Ribeiro AC, Barahona I. Biocompatibility of self-adhesive resin cement with fibroblast cells. J Prosthet Dent. 2021;125(4):705. doi: 10.1016/j.prosdent.2021.01.002. e1–e7. [DOI] [PubMed] [Google Scholar]
5.Frasheri I, Grimm A, Ern C, Hickel R, Folwaczny M. In-vitro cytocompatibility of self-adhesive dual-curing resin cements on human mesenchymal stem cells (hMSC) and periodontal ligament cells (PDL-hTERT) Dent Mater. 2022;38(2):376–383. doi: 10.1016/j.dental.2021.12.020. [DOI] [PubMed] [Google Scholar]
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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix A. Supplementary data.

Supplementary Table S1. Primer sequences of selected markers for qRT-PCR.

Supplementary Figure S1. Characteristics of human gingival fibroblasts (HGFs).

Supplementary Figure S2. Cement extract treated HGFs stained with DAPI/PI/Annexin V-FITC.

Supplementary Figure S3. Vitality of 1:20 cement extract treated HGFs at 24 hours.

Supplementary Figure S4. Representative images of wound scratch assay over 3-day observation.

Supplementary Figure S5. Gene expression profiling of HGFs following exposure to cement eluates at 30 minutes.

Supplementary Figure S6. Gene expression profiling of HGFs following exposure to cement eluates at 24 hours.

mmc1.pdf^{(461.5KB, pdf)}

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[bib0006] 6.Sun F, Liu Y, Pan Y, Chen M, Meng X. Cytotoxicity of self-adhesive resin cements on human periodontal ligament fibroblasts. Biomed Res Int. 2018;2018 doi: 10.1155/2018/7823467. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib0009] 9.de Mendonca AAM, Souza PPC, Hebling J, de Souza Costa CA. Cytotoxic effects of hard-setting cements applied on the odontoblast cell line MDPC-23. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;104(4) doi: 10.1016/j.tripleo.2007.05.017. e102–e8. [DOI] [PubMed] [Google Scholar]

[bib0010] 10.Kim M-J, Kim K-H, Kim Y-K, Kwon T-Y. Degree of conversion of two dual-cured resin cements light-irradiated through zirconia ceramic disks. J Adv Prosthodont. 2013;5(4):464–470. doi: 10.4047/jap.2013.5.4.464. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib0015] 15.Martínez-Cortés M, Tinajero-Morales C, Rosales C, Uribe-Quero E. Cytotoxicity assessment of three endodontic sealing cements used in periapical surgery. In vitro study. Rev Odontol Mex. 2017;21(1):40–48. [Google Scholar]

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[bib0017] 17.Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ (–delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3(3):71. [PMC free article] [PubMed] [Google Scholar]

[bib0018] 18.Trumpaitė-Vanagienė R, Čebatariūnienė A, Tunaitis V, Pūrienė A, Pivoriūnas A. Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements. Arch Oral Biol. 2018;86:108–115. doi: 10.1016/j.archoralbio.2017.11.011. [DOI] [PubMed] [Google Scholar]

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[bib0034] 34.Vrochari AD, Eliades G, Hellwig E, Wrbas KT. Curing efficiency of four self-etching, self-adhesive resin cements. Dent Mater. 2009;25(9):1104–1108. doi: 10.1016/j.dental.2009.02.015. [DOI] [PubMed] [Google Scholar]

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[bib0037] 37.Guerrero-Gironés J, López-García S, Pecci-Lloret MR, Pecci-Lloret MP, García-Bernal D. Influence of dual-cure and self-cure abutment cements for crown implants on human gingival fibroblasts biological properties. Ann Anat. 2022;239 doi: 10.1016/j.aanat.2021.151829. [DOI] [PubMed] [Google Scholar]

[bib0038] 38.Trumpaite-Vanagiene R, Cebatariuniene A, Tunaitis V, Puriene A, Pivoriunas A. Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements. Arch Oral Biol. 2018;86:108–115. doi: 10.1016/j.archoralbio.2017.11.011. [DOI] [PubMed] [Google Scholar]

[bib0039] 39.Wei Y, Sun J, Men Q, Tian X. Toxic effects of four kinds of dental restorative materials on fibroblast HGF‑1 and impacts on expression of Bcl‑2 and Bax genes. Exp Thera Med. 2018;16(5):4155–4161. doi: 10.3892/etm.2018.6705. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Effects of Different Self-Adhesive Resin Cements and Curing Through Zirconia on Gingival Fibroblasts

Kittipat Termteerapornpimol

Karn Tongchairati

Narin Intarak

Sasiprapa Prommanee

Soranun Chantarangsu

Dusit Nantanapiboon

Junji Tagami

Thantrira Porntaveetus

Abstract

Introduction

Methods

Results

Conclusion

Clinical relevance

Introduction

Materials and methods

Cements and eluates preparation

Table 1.

Cell culture

Characterisation of gingival mesenchymal cells

Cell viability

Cell vitality

Wound scratch assay

Gene expression

Statistical analysis

Results

MSCs characterisation

Effect of cement eluates on HGFs viability

Fig. 1.

Fig. 2.

HGFs vitality in response to cement eluates

Fig. 3.

Delayed wound closure of HGFs in response to cement eluates

Fig. 4.

Gene expression of HGFs in response to cement eluates

Fig. 5.

Discussions

Fig. 6.

Conclusion

Declaration of competing interest

Acknowledgments

Acknowledgement

Author contributions

Data statement

Footnotes

Appendix. Supplementary materials

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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