In vitro cytotoxicity of dental implant cements on human gingival and mouse preosteoblast cell lines

Abstract

Purpose: This study aims to evaluate the cytotoxicity of implant luting cements and to visualize the morphological changes in the cells.

Methods: Seven experimental groups Cem Implant Cement (CIC), EsTemp Implant Cement (EIC), Harvard Implant Cement (HIC), MIS Crown Set Implant Cement (MCIC), Oxford Cem Implant Cement (OCIC), Premier Implant Cement (PIC), and Adhesor Carbofine (ZPC) were generated including one conventional, and six implant cements (n = 9). Specimens were applied to human fibroblast cell (HGF) and mouse pre-osteoblast cell line (MC3T3-E1) cells by direct contact and extract text methods. The extracts were prepared by sterilizing the discs under ultraviolet light for 24 h in a cell culture medium at 37°C, 5% CO, and 95% humidity. Cell lines were confluent in the cell culture module in 25 cm² and 75 cm² flasks in a carbon dioxide incubator with 5% CO and 95% humidity. Discs and extracts were placed in a 96-well plate. Cell viability was evaluated after 24 h by means of a cell proliferation assay with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT assay).

Results: Dual-cured OCIC and HIC cements comprising methacrylate and zinc oxide elicited relatively lower cytotoxicity than self-cure cements with various compositions. The OCIC revealed the highest cell viability (89%) in the extract method on the HGF cells. Immortalized MC3T3 cells showed more sensitivity to cement exposure than the primary HGF cells.

Conclusion: All tested cements elicited a cytotoxic effect with differences depending on cell type and cement material in extract and direct contact methods. Dual polymerized semi-permanent cement (OCIC) showed higher cell viability in the extract method.

Introduction

In modern dentistry, implant-supported restorations are a better treatment option for partially or totally edentulous patients [1]. The prosthodontic rehabilitation of implants can be performed as a cemented or screw-retained restoration based on the clinician’s choice, according to the case [2,3]. The advantages of cement-retained prostheses, including simplicity, esthetics, passivity, and cost-effectiveness, have been reported [3,4]. In cement-retained implant restorations, unlike natural teeth, two metal or ceramic structures are connected [5,6]. Thus, cementation is an essential process in implant-supported fixed prostheses. Implant cements are clinically classified as temporary, semi-permanent, and permanent cements [4,7,8] and have been developed for effective cementation of implant restorations.

Besides all the advantages, cementation of restoration on implants involves the risk of excess cement remaining in the peri-implant sulcus, and the complete removal of dental cement is nearly impossible, especially in areas deeper than 0.5 mm [1,2,8]. Peri-implant soft tissue health can impact implant success and survival. Although multiple factors may play a role in the etiology of peri-implant diseases, authors have noted the risk of residual excess cement [4,7]. Cement-associated peri-implantitis is explained by bacterial colonization around the cement and damage to the surrounding tissue by chemicals released from the cement composition [9,10,11]. Types of cement can cause toxic effects on tissues due to their chemical content and polymerization reactions. Some of these cement compositions contain potent reagents such as phosphoric acid, eugenol, and acrylics that may impair cell viability, proliferation, and differentiation [12,13]. Resin cements contain monomers, and unbounded monomers can be released after the polymerization reaction. In previous studies, it was reported that the released monomers, such as triethyleneglycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA), induced cytotoxicity [14,15]. The release of monomers from resin cement causes temporary peri-implantitis and bone resorption in the tissues surrounding the implant [12,16]. Currently, numerous implant cement types with different compositions are available on the market, and there is no official guidance for dental cement selection; thus, the wide variety of types makes the decision process complex for clinicians [17].

Cytotoxicity of dental materials refers to their potential to cause harmful effects on living cells, impacting cell viability, growth, or function [18]. Ohlsson E et al. [19] reported that the cytotoxic effects of materials should be considered in developing materials concerning clinical implications. A dental material used in such a compound environment might cause unnecessary disturbance. Many different methods can be used to evaluate the cytotoxicity of dental materials [20,21]. In vitro cytotoxicity tests are less expensive to survey newly developed materials, reducing the probability of surprises when animal usage tests or clinical trials are performed [21,22]. The cell proliferation assay with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT assay) is fast, sensitive, and easy to use. It has high sensitivity and accuracy. Cell growth and death rates can be measured since a linear relationship exists between cell activity and absorbance [20,23]. Despite good mechanical properties, only a few studies demonstrated the cytotoxic effects of some implant-luting cement [24,25]. This study aims to evaluate the cytotoxicity of seven different types of luting cements (one conventional and six implant luting cements) used for bonding implant-based restorations in vitro and to visualize the morphological changes in the cells by scanning electron microscope (SEM). The null hypothesis is that cements used for cementing the implant-supported fixed prosthesis have no cytotoxic effect on HGF and MC3T3-E1 subclone 4 preosteoblast cells in direct contact and extract method.

Materials and Methods

Specimen preparation

Seven commercially available dental luting cements used for implant prostheses were investigated in this study (Table 1). All specimens were prepared in a disk-shaped Teflon mold with 5 mm diameter and 2 mm height to ensure standardization (n = 9 for each group). Self-cured cement specimens were prepared in a dark environment at room temperature and left to set for 24 h. Dual-cure cement specimens were prepared using a curing unit (BA Optima; BA International, Northampton, UK). After light curing, the specimens were left in the dark for 24 h. They were sterilized by keeping all surfaces of the specimens under ultraviolet light for 15 min.

Table 1 Luting cements used in the study

Cement/ Abbreviation	Content	Cement type	Manufacturer
Cem Implant Cement (CIC)	base and catalyst: urethane dimethacrylate, triethyleneglycol dimethacrylate, polymerization activator, fumed silica	auto-polymerized eugenol-free acrylic urethane polymer-based temporary cement	BJM Laboratories Silmet Ltd, Or-Yehuda, Israel Lot no. 4320CIHRXGTR
EsTemp Implant Cement (EIC)	zinc oxide, white mineral oil, petrolatum	dual polymerized eugenol-free resin-based temporary cement	Spident Co. Ltd., Incheon, Republic of Korea Lot no. EI20004
Harvard Implant Cement (HIC)	methacrylate zinc oxide	dual polymerized semi-permanent cement	Harvard Dental International GmbH, Hoppegarten, Germany Lot no. 91909494
MIS Crown Set Implant Cement (MCS)	urethane diacrylate resilient oligomer	auto-polymerized permanent cement	MIS Implant Technologies Ltd., Shlomi, Israel Lot no. 4317CI
Oxford Cem Implant Cement (OCIC)	methacrylate esters zinc oxide	dual polymerized semi-permanent cement	Oxford Cem Scientific, Elmshorn, Germany Lot no. 71910124
Premier Implant Cement (PIC)	base: methacrylate monomer, 2-hydroxymethyl methacrylate, triethyleneglycol dimethacrylate, aliphatic urethane diacrylate resilient oligomer, pigment, stabilizer catalyst: methacrylate monomer, benzoyl peroxide, triethyleneglycol dimethacrylate, aliphatic urethane diacrylate resilient oligomer, pigment, stabilizer	auto-polymerized eugenol-free urethane dimethacrylate-based temporary cement	Premier Dental Products, Plymouth Meeting, PA, USA Lot no. 4309CI
Adhesor Carbofine (ZPC)	powder: 60-95% zinc oxide, magnesium oxide, fillers liquid: 30-50% polyacrylic acid	zinc polycarboxylate cement	Spofa Dental, Jicin, Czech Republic Lot no. 7012157

Cell culture

Human gingival fibroblast (HGF, ATCC #: PCS-201-018, Passage 16) and mouse preosteoblast (MC3T3 E1 subclone 4, ATCC #: CRL-2593, Passage 18) cell lines associated with the peri-implant soft and hard tissues were used for the cytotoxicity studies. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Serana Europe GmbH, Brandenburg, Germany) containing 10% fetal bovine serum (FBS) (EURx, Gdańsk, Poland) and 1% antibiotic/antimycotic solution (100 IU/ mL penicillin-streptomycin-gentamicin) (BioShop Canada Inc., Burlington, Canada) at 37°C in a fully humidified atmosphere of 5% CO₂. Media was routinely changed every 2 days, and cell lines were passaged upon reaching 80% confluency by trypsinization.

Cytotoxicity evaluation via extracts

Extracts were prepared as described in the ISO 10993-12: Sample Preparation and Reference Materials standard [26]. Briefly, prepared as described above, the dental luting cement specimens were incubated in DMEM with a 0.5 cm²/mL specimen surface area to media ratio for 24 h. Then, the extracts were filter sterilized using a 0.22 µm syringe filter, and the stock extract solutions (1:1) were serially diluted to 1:2, 1:4, and 1:8 in DMEM. The extracts were used immediately upon preparation.

Trypsinized cells were counted using a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA) and inoculated to 96-well tissue culture-treated plates at 1 × 10^⁵ cells/mL concentration in triplicates. The cells were maintained in DMEM with 10% FBS and 1% PSG for 24 h. At the end of 24 h, the media was removed, and serial dilutions of 1:1, 1:2, 1:4, and 1:8 extracts were added. DMEM containing no extract was used as a negative control group, and a 1:1 extract of natural rubber was used as a positive control for internal verification of tests. DMEM added to wells containing no cells were used as blank. The cells were grown for another 24 h. Then, the viability of the cells was evaluated using a cell proliferation kit (Cell Proliferation Kit, XTT based: Biological Industries, Beit-HaEmek, Israel, lot 2031578) following the manufacturer’s instructions. The reaction solution was briefly prepared by mixing the XTT activation and reagent solutions at a 1:50 ratio. The reaction solution was added to each well at a 1:2 solution-to-media ratio. The plates were incubated for 4 h at 37°C. Then, the absorbance of each well was measured using a Varioskan Flash plate reader (Thermo Scientific, Waltham, MA, USA) at 450 nm. Non-specific absorbance for each well was measured at 630 nm and subtracted from the readings obtained at 450 nm. Lastly, the average absorbance of the blank specimens was subtracted from the other specimens. The percent viability of the cells was calculated assuming a 100% viability in the negative control group. Half maximal inhibitory concentration (IC₅₀) values were calculated by plotting the logarithm of the percent concentrations of the extracts against the percent viability values.

Cytotoxicity evaluation via direct contact

Cytotoxicity Evaluation via direct contact was performed as described in the ISO 10993-5: Biological evaluation of medical devices standard [27]. Briefly, the dental luting cement specimens, prepared as described above, were placed in a 48-well plate in triplicates, and DMEM was added on top at a 0.5 cm²/mL specimen surface area to media ratio. The cells were prepared as described above and added on top of the specimens at a final concentration of 1 × 10⁵ cells/mL. Wells containing no cement specimens were used as negative control and DMEM added to wells containing no cells were used as blank. The cells were grown for 24 h, and the viability of the cells was evaluated using an XTT based cell proliferation kit. The percent cell viability was calculated assuming 100% viability in the negative control group.

Scanning electron microscopy imaging (SEM)

For SEM imaging, the cells were grown on the luting cement specimens for 24 h, as described in the direct contact method. At the end of 24 h, the media was removed, and the specimens were washed three times with phosphate-buffered saline. Then, the cells were fixed in a 4% paraformaldehyde solution for 10 min. The specimens were dehydrated in 10%, 30%, 50%, 70% and 100% ethanol for 10 min each. Before imaging, the specimens were dried using a Leica CPD 300 critical point dryer (Leica Microsystems, Wetzlar, Germany) and sputter-coated with a thin layer of gold using a Leica ACE 200 vacuum coater (Leica Microsystems, Wetzlar, Germany). Imaging was performed using a Hitachi SU5000 field emission SEM (Hitachi High-Technologies, Tokyo, Japan) under 5 kV acceleration.

Statistical analyses

The statistical significance of the differences in the mean cell viability data obtained with the XTT assay was evaluated using a one-way analysis of variance (ANOVA) procedure with the Tukey HSD post hoc test. The normality of the data was assessed using the Shapiro-Wilk test and the homogeneity of variance was assessed using the Levene test. The Shapiro-Wilk test indicated that the residuals may slightly deviate, but the normality assumption was satisfied (W = 0.940, P = 0.52). The Levene test for equality of variances was not significant (F [6, 56] = 1.08, P = 0.392), indicating that the variances among the groups were approximately equal. All tests were performed using the statistical analysis software SPSS v.25 (IBM SPSS Statistics for Windows, Version 25.0. IBM Corp., Armonk, NY, USA), and P ≤ 0.05 was considered the condition for a statistically significant difference. IC₅₀ values were calculated using the GraphPad Software 8.0.1. (GraphPad Software Inc., San Diego, CA, USA).

Results

Cytotoxicity evaluation via extracts

The average cell viability obtained from the 1:1 dilution of the positive control natural rubber extract was found to be 0% for the HGF cells and 2.49% for the MC3T3 cells, demonstrating the validity of the test. Cell viability data for the luting cements are shown in Fig. 1. The cytotoxicity of the extracts was evaluated by calculating the IC₅₀ values. The IC₅₀ values of the extracts for both cell lines are presented in Table 2.

MC3T3 cells were found to be more susceptible to the cytotoxic effect compared to the HGF cells. According to these findings, extracts from most of the luting cement specimens demonstrated considerable cytotoxicity, especially on HGF cells. OCIC and HIC showed the least cytotoxicity for both cell lines. For the MC3T3 cells, only OCIC showed an IC₅₀ value above 50% extract. For the HGF cells, only MCIC showed an IC₅₀ value below 50% extract. Although the IC₅₀ values varied between different cell lines, the cytotoxicity trend was mostly parallel for the different luting cements.

Fig. 1 IC₅₀ values for the luting cements (the percent viability of the cells was calculated assuming a 100% viability in the negative control group)

Table 2 Descriptive statistics of the IC₅₀ values

Material n = 9	HGF mean (±SD)	MC3T3-E1 mean (±SD)
MIS Crown Set Implant Cement (MCIC)	40.23 (±6.60)	16.18 (±3.00)b
Adhesor Carbofine (ZPC)	59.42 (±7.55)a	14.10 (±1.96)b
Oxford Cem Implant Cement (OCIC)	86.60 (±7.45)	51.80 (±8.18)
EsTemp Implant Cement (EIC)	50.19 (±5.17)	14.4 (±2.15)b
Premier Implant Cement (PIC)	57.67 (±6.42)a	25.23 (±3.31)
Harvard Implant Cement (HIC)	69.69 (±8.50)	36.79 (±5.74)
Cem Implant Cement (CIC)	63.23 (±9.55)	19.82 (±3.41)
Rubber	15.17 (±1.71)	9.37 (±1.39)

The same lowercase letters indicate a statistically insignificant difference among each cell line (P > 0.05).

 

Cytotoxicity evaluation via direct contact

The percent cell viability of the cells grown directly on the luting cement specimens is shown in Fig. 2. Unlike the observations in the extract method, the viability of HGF and MC3T3 cells did not vary as drastically in the direct contact method. For MC3T3 cells, similar viability was observed in all specimens except for ZPC, which had the lowest value. For HGF cells, HIC and EIC showed the highest viability, while ZPC, again, showed the lowest viability. The cell viability values in the direct contact test are presented in Table 3.

Fig. 2 The percent cell viability of the cells grown directly on the luting cement specimens

Table 3 Descriptive statistics of the cell viability in the direct contact test

Material n = 9	HGF mean (±SD)	MC3T3-E1 mean (±SD)
MIS Crown Set Implant Cement (MCIC)	19.90 (±0.92)a	18.84 (±0.35)c
Adhesor Carbofine (ZPC)	14.00 (±0.24)	12.53 (±0.22)
Oxford Cem Implant Cement (OCIC)	22.65 (±1.97)a	21.93 (±0.36)c,d
EsTemp Implant Cement (EIC)	29.68 (±1.82)b	22.26 (±1.50)c,d
Premier Implant Cement (PIC)	22.57 (±0.51)a	18.29 (±0.90)c
Harvard Implant Cement (HIC)	31.91 (±0.81)b	22.88 (±1.16)d
Cem Implant Cement (CIC)	20.25 (±0.54)a	18.40 (±0.26)c

The same lowercase letters indicate a statistically insignificant difference among each cell line (P > 0.05).

 

SEM imaging

SEM observations were in parallel with the direct contact cytotoxicity analyses. Neither of the two cell lines exhibited good spreading on the specimens. The cells mostly retained a spherical shape on the surfaces with occasional filopodia visible (Figs. 3 and 4).

Fig. 3 SEM images of the resin cements (cytotoxicity evaluation via extracts)

Fig. 4 SEM images of the resin cements (cytotoxicity evaluation via direct contact)

Discussion

In the present study, seven different types of cement (one conventional luting and six implant luting cements) were tested by extract (XTT) and direct contact cytotoxicity test to determine the potentially harmful effects of the cement on the HGF and MC3T3-E1 cells, and the morphological changes in the cells were observed by SEM. The results showed that luting cements exhibited different cytotoxic effects on HGF and MC3T3-E1 cells. Thus, the null hypothesis was partially rejected.

Evaluation of the cytotoxicity of dental materials is necessary for determining the materials’ clinical usability. In-vitro experiments are the primary tests and are frequently used to evaluate the effect of the material on the cell cultures [28]. In cytotoxicity studies, the test medium is defined according to the environment to which the tested biomaterial is exposed. In this cytotoxicity study, different implant luting cements were evaluated on human gingival fibroblast (HGF) and mouse preosteoblast (MC3T3-E1) cell lines that were associated with the peri-implant soft and hard tissues, by using two different cell culture methods. Various methods have been used in the literature to evaluate the cytotoxic effects of dental luting cements [11,29,30]; however, there is limited information on the cytotoxicity of implant luting cements [24,25].

The results from the XTT method showed significantly different cytotoxic effects of the tested cements on human HGF and MC3T3-E1 cells. Also, there were partially different results between extract and direct contact cell culture tests. The cytotoxicity of the luting cement extracts was assessed using IC₅₀ values. IC₅₀ is a measure of the effectiveness of a substance in inhibiting a specific biological function, in this case, cell viability and proliferation. This quantitative metric indicates the % concentration of the extracts from the luting cements required to inhibit the cell viability by 50% in vitro. Since the dose-dependent effect of the extracts is often not linear and the compounds released from different types of cement may have different effects on the cells, the IC₅₀ values were calculated in the present study. A significant difference was observed in the tested cell lines’ susceptibility to the cement extracts’ cytotoxic effects. Fibroblast cells showed more cell viability than preosteoblast cells after exposure to cement and cement extracts. In contrast to the present study, Rodríguez et al. [31] stated that gingival fibroblast cells were more sensitive than preosteoblast cells in the direct contact method. However, other studies have demonstrated that preosteoblast cells are more sensitive than gingival fibroblast cells, and cell viability is less in preosteoblast cells in cytotoxicity studies [32,33,34]. This is mainly due to the genetic differences between the immortalized MC3T3 and primary HGF cells. Established cell lines may exhibit a more uniform phenotype due to clonal selection during their establishment. On the other hand, primary cell lines often retain the original tissue’s heterogeneity, involving various cell types and states, which can grant greater adaptability and resilience to environmental changes and challenges [35,36]. In addition, primary cell lines can undergo a limited number of passages before reaching replicative senescence. This inherent limitation helps preserve their genetic stability and physiological characteristics over time, whereas immortalized cell lines may accumulate genetic alterations and phenotypic changes [37]. This could contribute to decreased resistance to the cytotoxicity of the cement extracts in MC3T3 cells. Therefore, the cell lines’ physiological relevance should be considered in future studies to prevent misleading results. Moreover, clinicians must be careful when deciding on the cement type to be used and also pay attention to removing the excess cement after cementation.

According to the findings of the present study, all the tested cements showed cytotoxicity with varying degrees in the extract method. OCIC and HIC were the most cytocompatible cements for both cell lines. ZPC showed the highest cytotoxicity for the MC3T3 cell while showing relatively moderate cytotoxicity for the HGF cells. Lastly, EIC and MCIC showed relatively high cytotoxicity for both cell lines (Fig. 1). In the direct contact method, the % cell viability values were well below the 70% threshold determined in the ISO 10993-5 standard [27] and much lower compared to the extract method for all specimens and cell lines (Fig. 2). Fujioka-Kobayashi et al. [38] also reported more pronounced cytotoxicity in the direct contact test compared to the extract test. This is expected since the negative control group in the direct contact method is the tissue culture-treated polystyrene optimized for cell adhesion and growth, which is not the case for the cement specimens. Although the method and the types of the material were different, the findings of the present study regarding the overall cytotoxicity of the implant luting cements are in line with the current literature [11,24,30].

The material contents of luting cement can cause inflammation in the peri-implant tissue. In the present study, resin-containing implant cements were cytotoxic in the direct contact method. In contrast, these types of cement decreased the cell viability at varying rates in the extract method, in agreement with previous studies [11,24]. However, the results of these studies cannot be directly compared due to the differences in the materials and the methods used. Alkurt et. al. [24] have shown a range of cytotoxicity for different types of luting cements. Diemer et al. [11] have also reported a significant cytotoxic effect of different resin-cements on different cell lines. Resin cements often have hydrophobic characteristics due to the nature of the resin matrix, making them suitable for use in oral environments where exposure to saliva and other fluids is inevitable and contributes to lower plaque accumulation rates. However, this also impairs cellular attachment to the cement. Also, due to incomplete polymerization and resin degradation, monomers can be released from the resin matrix and interact with surrounding tissues. About 15-50% of the methacrylate groups remain unreacted. As a result, it can cause adverse biological effects, including local and systemic toxicity, pulp reactions, and allergic and estrogenic effects [12]. It has been reported that cell viability in the peri-implant tissue will decrease if the cement is cytotoxic to cells, and this region will tend to have bacterial colonization [31]. Resin cements that contained Bis-GMA, HEMA, UDMA, and TEDGMA cause cytotoxicity in mouse fibroblasts for 24 to 72 h [39]. Additionally, the surface of composite resins exposed to oxygen during polymerization forms a surface layer containing formaldehyde, which does not polymerize and is toxic to the cell [16]. Diemer et al. [11] evaluated the cytotoxicity of 12 different resin-cements and zinc phosphate cement on different cell lines. They reported that all resin cements reduced cell viability of human cells with significant differences depending on cell type and cement material and they pointed out the importance of the removal of the excess cement residues after prosthetic placement of cement-retained implant restorations.

The implant cements used in the present study, except EIC and ZPC, are resin-based luting materials. The fact that the cements found to be most cytocompatible had a similar composition (OCIC: methacrylate esters, zinc oxide, HIC: methacrylate, zinc oxide) indicates the significance of composition regarding cytotoxicity and should be considered besides the mechanical properties in clinical applications. The differences in cytotoxicity may also be attributed to the polymerization methods. The most cytotoxic effect was observed in MCIC, which is a self-cure cement. Studies also reported that chemically polymerized cements were more cytotoxic than dual-cure and light-cure cements [29,30]. Schmid-Schwap et al. [30] have reported that adhesive and self-adhesive resin cements showed significantly less cytotoxicity when dual-cured. This finding may be due to the higher probability of residual monomers remaining in chemically polymerized cements, which generate a cytotoxic effect on the cell. Further analyses of the individual components of the cements and the polymerization methods need to be investigated to identify the underlying mechanisms of the observed cytotoxicity. Among 7 different types of cement, polycarboxylate cement also reduced cell viability in both extract and the direct contact method for 2 cell lines compared to the control group. Öztürk et al. [40] reported that polycarboxylate cement was the most cytotoxic cement for human oral buccal epithelial cells. The cytotoxic potential of polycarboxylate cement may be explained by its content of zinc and residual acids [25].

According to SEM results, cements affected cell morphology and viable cell count. In the SEM images (Figs. 3 and 4), the cells mostly retained a spherical shape with no spreading on the surface. Cellular attachment (or the lack of it) has two significant consequences regarding the success of the implant. If the cement surface is not populated by the host cells, healing of the gingival tissues in the immediate proximity of the cement may be impaired [38], and the bacterial population of the surface may lead to the eventual loss of the implant [33].

In the present study, data were obtained using two different in-vitro test methods; however, for clinical relevance, in vitro studies should be supported by animal and clinical studies. In addition, research work can focus on improving the cellular response of these implant cement materials. Within the limitations of this in vitro study, the following conclusions were drawn:

Immortalized MC3T3 cells showed more sensitivity to cement exposure compared to primary HGF cells. All tested types of cement elicited a cytotoxic effect with differences depending on cell type and cement material in extract and direct contact methods. Dual-cured OCIC and HIC types of cement comprising methacrylate and zinc oxide elicited relatively lower cytotoxicity compared to self-cure types of cement with various compositions. The OCIC revealed the highest cell viability (89%) in the extract method on the HGF cells.

Abbreviations

ANOVA: analysis of variance; Bis-GMA: Bisphenol a glycidyl methacrylate; CIC: Cem Implant Cement; CO: carbon monoxide; DMEM: Dulbecco’s modified eagle medium; ET: Estemp Implant Cement; FBS: fetal bovine serum; HEMA: 2-hydroxyethyl methacrylate; HC: Harvard Implant Cement; HGF: human fibroblast cell; ISO: International Organization for Standardization; MCS: MIS Crown Cet Implant Cement; MC3T3-E1: mouse preosteoblast cell; OCIC: Oxford Cem Implant Cement; PIC: Premier Implant Cement; SD: standard deviation; SEM: scanning electron microscopy; TEGDMA: triethylene glycol dimethacrylate; UDMA: urethane dimethacrylate; XTT assay: cell proliferation assay with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide; ZPC: Adhesor Carbofine

Ethical Statements

Not applicable

Conflicts of Interest

The authors have no conflict of interest.

Funding

This study was supported by the Scientific Research Projects Coordination Unit of Gazi University with project number 03/2020-13.

Author Contributions

KA: methodology, investigation, original draft writing; BTB: conceptualization, resources, formal analysis, original draft writing, review, editing, supervision; MB: methodology, investigation; writing, MBG: writing, review, editing, supervision, SKN: writing, review, editing, supervision

ORCID iD

¹⁾KA: [e-mail], https://orcid.org/0000-0003-1043-936X

²⁾BTB*: [e-mail]; [e-mail], https://orcid.org/0000-0001-7825-712X

³⁾MG: [e-mail], https://orcid.org/0000-0001-8894-0467

²⁾MBG: [e-mail]; [e-mail], https://orcid.org/0000-0002-4002-6390

²⁾SKN: [e-mail], https://orcid.org/0000-0001-8836-0673

Data Availability Statements

Data related to this article are available from the corresponding author upon request.

References

• 1.  Pjetursson BE, Thoma D, Jung R, Zwahlen M, Zembic A (2012) A systematic review of the survival and complication rates of implant‐supported fixed dental prostheses (FDPs) after a mean observation period of at least 5 years. Clin Oral Implants Res 23, 22-38. doi: 10.1111/j.1600-0501.2012.02546.x
• 2.  Wittneben JG, Joda T, Weber HP, Brägger U (2017) Screw retained vs. cement retained implant‐supported fixed dental prosthesis. Periodontol 2000 73, 141-151. doi: 10.1111/prd.12168
• 3.  Vigolo P, Givani A, Majzoub Z, Cordioli G (2004) Cemented versus screw-retained implant-supported single-tooth crowns: a 4-year prospective clinical study. Int J Oral Maxillofac Implants 19, 260-265.
• 4.  Bhoyar A, Jain A, Parlani S, Pandey H (2016) Selection of luting agent in implant retained prosthesis: a review. Int J Oral Health Dent 2, 19-25. doi: 10.5958/2395-499X.2016.00003.4
• 5.  Hill EE (2007) Dental cements for definitive luting: a review and practical clinical considerations. Dent Clin North Am 51, 643-658. doi: 10.1016/j.cden.2007.04.002
• 6.  Schiessl C, Schaefer L, Winter C, Fuerst J, Rosentritt M, Zeman F, Behr M (2013) Factors determining the retentiveness of luting agents used with metal-and ceramic-based implant components. Clin Oral Investig 17, 1179-1190. doi: 10.1007/s00784-012-0798-x
• 7.  Gultekin P, Gultekin BA, Aydin M, Yalcin S (2013) Cement selection for implant‐supported crowns fabricated with different luting space settings. J Prosthodont 22, 112-119. doi: 10.1111/j.1532-849X.2012.00912.x
• 8.  Hamed MT, Abdullah Mously H, Khalid Alamoudi S, Hossam Hashem AB, Hussein Naguib G (2020) A systematic review of screw versus cement-retained fixed implant supported reconstructions. Clin Cosmet Investig Dent 12, 9-16. doi: 10.2147/CCIDE.S231070
• 9.  Bouillaguet S, Wataha JC, Hanks CT, Ciucchi B, Holz J (1996) In vitro cytotoxicity and dentin permeability of HEMA. J Endod 22, 244-248. doi: 10.1016/s0099-2399(06)80141-x
• 10.  Pauletto N, Lahiffe BJ, Walton JN (1999) Complications associated with excess cement around crowns on osseointegrated implants: a clinical report. Int J Oral Maxillofac Implants 14, 865-868.
• 11.  Diemer F, Stark H, Helfgen EH, Enkling N, Probstmeier R, Winter J et al (2021) In vitro cytotoxicity of different dental resin-cements on human cell lines. J Mater Sci Mater Med 32, 4. doi: 10.1007/s10856-020-06471-w
• 12.  Kraus D, Wolfgarten M, Enkling N, Helfgen EH, Frentzen M, Probstmeier R et al (2017) In-vitro cytocompatibility of dental resin monomers on osteoblast-like cells. J Dent 65,76-82. doi: 10.1016/j.jdent.2017.07.008
• 13.  Goldberg M (2008) In vitro and in vivo studies on the toxicity of dental resin components: a review. Clin Oral Investig 12, 1-8. doi: 10.1007/s00784-007-0162-8
• 14.  Kurt A, Altintas SH, Kiziltas MV, Tekkeli SE, Guler EM, Kocyigit A et al (2018) Evaluation of residual monomer release and toxicity of self-adhesive resin cements. Dent Mater J 37, 40-48. doi: 10.4012/dmj.2016-380
• 15.  Geurtsen W, Lehmann F, Spahl W, Leyhausen G (1998) Cytotoxicity of 35 dental resin composite monomers/additives in permanent 3T3 and three human primary fibroblast cultures. J Biomed Mater Res 41, 474-480. doi: 10.1002/(sici)1097-4636(19980905)41:3<474::aid-jbm18>3.0.co;2-i
• 16.  Schmalz G (1998) The biocompatibility of non-amalgam dental filling materials. Eur J Oral Sci 106, 696-706. doi: 10.1046/j.0909-8836.1998.eos10602ii05.x
• 17.  Weber HP, Sukotjo C (2007) Does the type of implant prosthesis affect outcomes in the partially edentulous patient? Int J Oral Maxillofac Implants 22 Suppl, 140-172.
• 18.  Williams DF (2008) On the mechanisms of biocompatibility. Biomaterials 29, 2941-2953. doi: 10.1016/j.biomaterials.2008.04.023
• 19.  Ohlsson E, Bolay C, Arabulan S, Galler KM, Buchalla W, Schmalz G et al (2024) In-vitro-cytotoxicity of self-adhesive dental restorative materials. Dent Mater 40, 739-746. doi: 10.1016/j.dental.2024.02.015
• 20.  Khalef L, Lydia R, Filicia K, Moussa B (2024) Cell viability and cytotoxicity assays: Biochemical elements and cellular compartments. Cell Biochem Funct 42, e4007. doi: 10.1002/cbf.4007
• 21.  Wataha JC (2001) Principles of biocompatibility for dental practitioners. J Prosthet Dent 86, 203-209. doi: 10.1067/mpr.2001.117056
• 22.  Wataha JC (2012) Predicting clinical biological responses to dental materials. Dent Mater 28, 23-40. doi: 10.1016/j.dental.2011.08.595
• 23.  Kamiloglu S, Sari G, Ozdal T, Capanoglu E (2020) Guidelines for cell viability assays. Food Frontiers 1, 332-349. doi: 10.1002/fft2.44
• 24.  Alkurt M, Duymus ZY, Sisci T (2019) Comparison of the effects of cytotoxicity and antimicrobial activities of self-adhesive, eugenol and noneugenol temporary and traditional cements on gingiva and pulp living cells. J Adv Oral Res 10, 40-48. doi:10.1177/2320206819850960
• 25.  Winkler C, Schäfer L, Felthaus O, Allerdings J, Hahnel S, Behr M et al (2014) The bacterial adhesion on and the cytotoxicity of various dental cements used for implant-supported fixed restorations. Acta Odontol Scand 72, 241-250. doi: 10.3109/00016357.2013.828320
• 26.  International Organization for Standardization (2021) Biological evaluation of medical devices - Part 12: Sample preparation and reference materials. ISO 10993-12:2021, Geneve.
• 27.  International Organization for Standardization (2009) Biological evaluation of medical devices - part 5: Tests for in vitro cytotoxicity. ISO 10993-5:2009, Geneve.
• 28.  Hanks CT, Wataha JC, Sun Z (1996) In vitro models of biocompatibility: a review. Dent Mater 12, 186-193. doi: 10.1016/s0109-5641(96)80020-0
• 29.  D’Alpino PHP, de Dantas Moura GED, de Arruda Barbosa SC, de Azevedo Marques L, Eberlin MN, Nascimento FD et al (2017) Differential cytotoxic effects on odontoblastic cells induced by self-adhesive resin cements as a function of the activation protocol. Dent Mater 33, 1402-1415. doi: 10.1016/j.dental.2017.09.011
• 30.  Schmid-Schwap M, Franz A, König F, Bristela M, Lucas T, Piehslinger E et al (2009) Cytotoxicity of four categories of dental cements. Dent Mater 25, 360-368. doi: 10.1016/j.dental.2008.08.002
• 31.  Rodriguez LC, Saba JN, Chung KH, Wadhwani C, Rodrigues DC (2017) In vitro effects of dental cements on hard and soft tissues associated with dental implants. J Prosthet Dent 118, 31-35. doi: 10.1016/j.prosdent.2016.10.002
• 32.  de Souza Costa CA, Hebling J, Garcia-Godoy F, Hanks C (2003) In vitro cytotoxicity of five glass-ionomer cements. Biomaterials 24, 3853-3858. doi: 10.1016/s0142-9612(03)00253-9
• 33.  Gallegos SI, Parsaei S, Siddiqui DA, Biguetti CC, Palmer KL, Rodrigues DC (2019) Can dental cement composition affect dental implant success? ACS Biomater Sci Eng 5, 5116-5127. doi: 10.1021/acsbiomaterials.9b00956
• 34.  Marvin JC, Gallegos SI, Parsaei S, Rodrigues DC (2019) In vitro evaluation of cell compatibility of dental cements used with titanium implant components. J Prosthodont 28, e705-e712. doi: 10.1111/jopr.12784
• 35.  Evan G, Littlewood T (1998) A matter of life and cell death. Science 281, 1317-1322. doi: 10.1126/science.281.5381.1317
• 36.  Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144, 646-674. doi: 10.1016/j.cell.2011.02.013
• 37.  Rubin H (1997) Cell aging in vivo and in vitro. Mech Ageing Dev 98, 1-35. doi: 10.1016/s0047-6374(97)00067-5
• 38.  Fujioka-Kobayashi M, Miron RJ, Lussi A, Gruber R, Ilie N, Price RB et al (2019) Effect of the degree of conversion of resin-based composites on cytotoxicity, cell attachment, and gene expression. Dent Mater 35, 1173-1193. doi: 10.1016/j.dental.2019.05.015
• 39.  Korsch M, Walther W (2015) Peri‐implantitis associated with type of cement: a retrospective analysis of different types of cement and their clinical correlation to the peri‐implant tissue. Clin Implant Dent Relat Res 17, e434-e443. doi: 10.1111/cid.12265
• 40.  Öztürk F, Yüksel Ş, Toy E, Kurtoğlu EL, Küçük EB (2012) Genotoxic effects of banding procedure with different orthodontic cements on human oral mucosa cells. Turk J Med Sci 42, 1157-1165. doi:10.3906/sag-1201-60