Cytotoxic effect of dental luting cement on human gingival mesenchymal stem cell and evaluation of cytokines and growth factor release – An in vitro study

Abstract

Aim:

In routine dental care, various dental luting cements are utilized to cement the dental prosthesis. Thus, the aim of the current study was to assess the Cytotoxic effect of three different dental luting cements on human gingival mesenchymal stem cell and evaluation of cytokines and growth factors release.

Settings and Design:

Cytotoxicity of glass ionomer cement (GIC), resin modified glass ionomer cement (RMGIC) and resin cement (RC) on the human gingival mesenchymal stem cells (HGMSCs) was evaluated. Amongst the cements tested, least cytotoxic cement was further tested for the release of cytokines and growth factors.

Materials and Methods:

MTT test was used to evaluate the cytotoxicity of the dental luting cements at 1 h, 24 h, and 48 h on HGMSCs. Cytokines such as interleukin (IL) 1α & IL 8 and growth factors such as platelet derived growth factor & transforming growth factor beta release from the least cytotoxic RC was evaluated using flow cytometry analysis.

Statistical Analysis Used:

The mean absorbance values by MTT assay and cell viability at various time intervals between four groups were compared using a one way analysis of variance test and Tukey’s post hoc test. The least cytotoxic RC group and the control group’s mean levels of cytokines and growth factors were compared using the Mann–Whitney test.

Result:

As exposure time increased, the dental luting cement examined in this study were cytotoxic. RC was the least cytotoxic, RMGIC was moderate and glass ionomer cement showed the highest cytotoxic effect. Concomitantly, a significant positive biological response of gingival mesenchymal stem cells with the release of ILs when exposed to the RC was observed.

Conclusion:

For a fixed dental prosthesis to be clinically successful over the long term, it is imperative that the biocompatibility of the luting cement be taken into account in order to maintain a healthy periodontium surrounding the restoration.

INTRODUCTION

A fixed dental prosthesis is becoming more popular, since a discernible rise in the number of partially edentulous individuals. Prosthetic restorations like crowns, bridges, and cement-retained implant prosthesis are cemented with various commercially available dental luting cement such as zinc phosphate, glass ionomer cement (GIC), resin-modified GIC (RMGIC), and resin cement (RC). Hence, the selection of suitable cement is essential for the long-term success of a fixed dental prosthesis and for maintaining a healthy periodontium surrounding the restoration.[1] Thus, while assessing the luting agents, it is pivotal to consider their chemical, physical, and mechanical properties along with biocompatibility, which can be defined as a material’s ability to function in living organisms and evoke the necessary tissue reaction.[2]

Among the various luting agents, GIC is routinely used because of its chemical bonding with the tooth structure;[3,4] however, it has low tensile and flexural strength and high solubility, especially during the initial setting where it sets by an acid–base reaction with lower pH during their initial set, thus contributing to inferior mechanical properties. Thus, RMGIC, a combination of resin and glass ionomer, was developed, with enhanced setting and mechanical characteristics in comparison to GIC.[5]

Resin Cement is currently the most common choice of dental luting cement as it imparts enhanced mechanical and esthetic results due to high translucency and the possibility of shade selection.[6,7] Selecting appropriate dental luting cement is crucial for restorations with subgingival margins and restorations with marginal discrepancy because soluble components which leach out before and/or after their set may be cytotoxic due to incomplete polymerization[8,9] along with pH changes and chemical irritation to the gingival tissue of periodontium[10] which is in restorations vicinity, thereby inducing various biological response of gingival tissue to different cement types.[11] The cytotoxic effect and their impact on host cellular proliferation are used to evaluate the biocompatibility of dental luting cement and offer additional guidance in choosing the appropriate luting agent for the cementation of fixed dental prosthesis.

The human gingival mesenchymal stem cells (HGMSCs) acquired from the gingiva are prolific, easily procured, and amenable to minimally invasive cell isolation procedures. Also, has potential to induce tissue reparation/regeneration. These cells exhibit notable immunomodulatory abilities that modulate cytokines such as interleukin (IL)-1α which regulates the release of other cytokines and IL-8 which promotes the healing of the wound by controlling the release of the other cytokines and stimulates the production of growth factors.[12,13,14]

Early stages of wound healing are significantly influenced by platelet-derived growth factor (PDGF), which is found in gingival epithelium, while transforming growth factor-beta (TGF-β) receptors are expressed differently in periodontal cells and tissues. They are more prevalent in regenerated tissue, indicating that they have a role in the process of periodontal repair and wound healing.[15]

Therefore, the study aimed to evaluate the effect of GIC, RMGIC, and RC on the HGMSC viability at 1 h, 24 h, and 48 h. IL-1α, IL-8, cytokines, PDGF, and TGF-β release after 48 h from the least cytotoxic cement.

MATERIALS AND METHODS

Groups for evaluation of cytotoxic effect of the dental luting cements on human gingival mesenchymal stem cell (GMSC) (performed in triplicate):

• Group 1: Control group without cement pellet

• Group 2: GIC (GC Corporation, Tokyo, Japan)

• Group 3: RMGIC (3M™ RelyX™ Luting 2 Automix)

• Group 4: RC (3M ESPE RelyX™ U200 Self-Adhesive Automix).

Sample collection of healthy gingival tissue

Healthy gingival tissue of 2–4 mm in size was collected from healthy adult patients undergoing crown-lengthening surgical procedures otherwise indicated for fixed restoration with appropriate oral hygiene. To reduce the oral microbe content, the patients received chlorhexidine mouth rinse before the surgical crown-lengthening procedure. Patients on medications for blood thinner, drugs that may lead to gingival enlargement, pregnant/lactating women, tobacco consumption in any form, and with a history of any diseases were excluded. Informed consent was acquired from the patient in accordance with institutional ethics. Immediately after the biopsy, the tissue was collected in a 15 ml centrifuged tube containing Dulbecco’s modified Eagle’s medium (HiMedia) with enriched antibiotic and actinomycotic solution (AA) and was transferred to the laboratory immediately. Ethical Committee number: DYPDCH/EC/648/07/2021.

Isolation of gingival mesenchymal stem cells

Gingival tissue was cut into 1 mm fragments (E0) in a bioseptic cabinet (Generic; Class 100) and placed in 60 mm culture dishes with 10% fetal bovine serum (Gibco). Thereafter, culture dishes were incubated for 24 h at 37°C with 5% CO2 (Thermo Scientific; Forma Study Cycle I 160). 0.25% Trypsin EDTA solution was used to detach the 70%–80% confluent cells, was transferred to a 75 cm² flask, and was continuously passaged up to passage 4–5.

Characterization of gingival mesenchymal stem cells using flow cytometry analysis

Following the harvesting of confluent GMSCs, they were treated with phosphate-buffered saline (PBS) and incubated for 30 min at 4°C with antibodies against CD73, CD90, CD105, CD34, CD45, and HLA-DR (all from eBioscience, USA). Flow cytometry was used to analyze the data after the cells were cleaned with PBS.

Preparation of dental cement pellets

The dental cement were taken out of sealed packing and mixed in a bioseptic environment as per the manufacturer’s instructions. In a 96-well cell culture flask, experiment dental cement pellets were formed using 1 mm × 2 mm polytetrafluoroethylene molds, which accounted for more than 10% of the well’s surface area.

Assessment of cytotoxicity of cement (cell survival)

GMSCs were seeded with 200 μL of cell culture media in a 96-well cell culture plate along with a test specimen and then incubated. The cytotoxicity was assessed for all four groups at 1-, 24-, and 48-h intervals using MTT assay. The yellow MTT dye is converted into insoluble blue formazan crystals by the vital cell’s mitochondrial succinate dehydrogenase. The number of vital cells and the amount of formazan are directly correlated. Following the solvent addition, a microtiter plate reader (TECAN Infinite 200 PRO) was used to measure the optical density (OD) of the resultant solution at 570 nm. The cell survival ratio (CSR%) was computed using the OD comparing the test (OD test) and control specimens (OD control). A greater CSR% value translates into less cytotoxicity and a more viable cell count. To ensure the accuracy of the results, all specimens were tested in triplicate. ELISA reader was used to read the readings.

Cytokines and growth factors release assessment from the least cytotoxic cement

After 48 h of exposure to the RC, the release of growth factors and cytokines was examined using the least cytotoxic RC, as determined by the cell viability MTT test. GMSCs were treated with RC at 37°C for another 48 h in the presence of serum-free culture media. A conditioned medium was collected to determine the release of the cytokine IL-1α, IL-8, PDGF, and TGF-β using flow cytometry analysis.

RESULTS

It was observed that all three tested luting cement presented substantial decreases in cell survival at 1-h, 24-h, and 48-h time intervals. At P < 0.001, there was a statistically significant difference in the mean absorbance values across the four groups. This suggests that all three tested cement showed cytotoxic effect at all the time intervals.

Table 1 compares the mean absorbance values, indicating the cytotoxicity in relation to the cement type and the duration of cement exposure [Graph 1].

Table 1.

Statistics for mean absorbance values for cell viability for four groups at 1-h, 24-h, and 48-h time intervals

Comparison of mean absorbance value for cell viability at 1 h between four groups
Groups		n		1 h		24 h		48 h		P
Control		3		0.44610		0.45295		0.32710		<0.001*
GIC		3		0.34040		0.30705		0.12850
RMGIC		3		0.35560		0.31860		0.14197
RC		3		0.41107		0.37860		0.24705

*Statistically significant. GIC: Glass ionomer, RMGIC: Resin-modified GIC, RC: Resin cement

Graph 1.

Mean absorbance values for cell viability at 1 hr, 24 hr, 48 hr between 4 groups. GIC: Glass ionomer, RMGIC: Resin modified GIC, RC: Resin Cement

Second, the second trend showed that cytotoxicity varied with the kind of cement investigated; at all time intervals, cytotoxicity was lowest for RC, higher for RMGIC, and greatest for GIC [Table 2].

Table 2.

Statistics for comparison of mean difference for cell viability for four groups at 1-h, 24-h, and 48-h time intervals

Multiple comparisons of mean difference in absorbance value for cell viability at 1 h, 24 h, and 48 h between groups
Groups		C versus G		C versus RMG		C versus RC		G versus RMG		G versus RC		RMG versus RC
Mean difference at 1 h		0.10570		0.09050		0.03503		−0.01520		−0.07067		−0.05547
P		<0.001*		<0.001*		0.03*		0.47		<0.001*		0.002*
Mean difference (24 h)		0.14590		0.13435		0.07435		−0.01155		−0.07155		−0.06000
P		<0.001*		<0.001*		0.002*		0.82		0.003*		0.008*
Mean difference (48 h)		0.19860		0.18513		0.08005		−0.01347		−0.11855		−0.10508
P		<0.001*		<0.001*		<0.001*		0.14		<0.001*		<0.001*

*Statistically significant. RC: Resin cement, RMG: Resin modified GIC, G: GIC, C: Control group

Cell survival quantitative analyses

HGMSCs were exposed to luting cement at various times, and the percentage of cells that survived was utilized to objectively evaluate the cells’ survival. The cell survival declined with an increase in the time interval from 1 h to 24 h to 48 h consecutively for all three cement [Table 3 and Graph 2]. Initially, at 1-h time interval observation, GIC showed the maximum decline in cell survival followed by RMGIC and RC. After, the elapse of 48 h, RC showed the highest cell survival followed by RMGIC and the least with the GIC. Thus, it was interpreted that RC was the least cytotoxic while GIC showed the highest cytotoxicity at 1-h, 24-h, and 48-h time intervals.

Table 3.

Distribution of % cell viability between four groups at different time intervals

Cell viability at 1 h, 24 h, and 48 h between four groups
Groups		1 h		24 h		48 h
Control		97.00		94.00		93.00
GIC		76.31		67.79		39.28
RMGIC		79.71		70.34		43.40
RC		92.14		83.59		75.53

GIC: Glass ionomer, RMGIC: Resin-modified GIC, RC: Resin cement

Graph 2.

Distribution of % cell viability between 4 groups at different time intervals. GIC: Glass ionomer, MGIC: Resin modified GIC, RC: Resin cement

RC which was the least cytotoxic among the tested cement was used further to evaluate the release of growth factors and ILs and was compared to the control group without any cement specimen.

Cytokine and growth factor assay

The differences in mean levels of several growth factors and ILs between the least cytotoxic RC and control groups were compared using the Mann–Whitney test. A significance threshold of P < 0.05 was applied. The mean PDGF levels in the control group were relatively higher (378.394 ± 18.248) as compared to the RC group (312.701 ± 47.445). However, the mean difference between the two groups did not show statistical significance (P = 0.08). The mean TGF-β levels in the control group were relatively higher (360.146 ± 7.299) as compared to the RC group (327.299 ± 62.044). However, the mean difference between the two groups did not show statistical significance (P = 0.51) [Table 4 and Graph 3].

Table 4.

Comparison of mean interleukin-1 α and interleukin-8 level release at 48 h between control and resin cement groups

Comparison of mean IL-1α and IL-8 levels and growth factors (pg/mL) release at 48 h between control and RC group
Parameters		Groups		n		Mean±SD		Mean difference		P
IL-1α		Control		3		350.646±25.201		64.019		0.04*
		RC		3		286.627±9.500
IL-8		Control		3		363.796±3.650		164.352		0.04*
		RC		3		199.444±13.717
PDGF		Control		3		378.394±18.248		65.694		0.08
		RC		3		312.701±47.445
TGF-β		Control		3		360.146±7.299		32.847		0.51
		RC		3		327.299±62.044

*Statistically significant. SD: Standard deviation, PDGF: Platelet-derived growth factor, TGF-β: Transforming growth factor-beta, IL-8: Interleukin-8, IL-1α: Interleukin-1 α, RC: Resin cement

Graph 3.

Mean IL and growth factor levels between Control and Resin Cement at 48 hrs. IL: Interleukin, Human PDGF: Platelet derived growth factor, Human TGF: Transforming growth factor beta

The mean IL-1α levels in the control group were significantly higher (350.646 ± 25.201) as compared to RC (286.627 ± 9.500), and the mean difference was statistically significant at P = 0.04. The mean IL-8 levels in the control group were significantly higher (363.796 ± 3.650) as compared to RC (199.444 ± 13.717), and the mean difference was statistically significant at P = 0.04 [Table 4].

DISCUSSION

For the longevity and clinical success of a fixed dental prosthesis, the health of the periodontium is critical since it is in juxtaposition with the marginal interfaces of the restoration. Thus, along with the physical, mechanical properties, the biocompatibility of commercially available dental luting cement should be critically assessed. The current investigation has shown that all of the luting cement tested had significant cytotoxicity as the survival of GMSCs was significantly reduced in all specimens containing cement than in the control specimen devoid of luting cement. The cytotoxicity of various luting cement was assessed in relation to cement type, time, and whether the least cytotoxic cement would trigger the release of cytokines and growth factors to aid in the gingival tissue wound healing.

The cytotoxic effect of tested cement was believed to be due to leached out components from the cement, incomplete polymerization, and pH change during their setting which is in accordance with other in vitro studies.[16,17,18,19,20,21,22,23,24,25] Hence, the null hypothesis, which proposed that HGMSC’s biological response to different test cement would not differ, was rejected. This result is consistent with a study by Rodriguez et al.,[25] which discovered that human gingival fibroblasts and osteoblasts were susceptible to cement exposure, which resulted in decreased cellular viability. According to Brauer et al.’s 2011 description, the main source of cytotoxicity in the GIC liquid is thought to be polyacrylic acid.[24,26] Apart from the leaching of metal ions from the luting cement, the research has suggested that one factor influencing RMGIC’s cytotoxicity is the brief release of free monomers during monomer-to-polymer conversion.[24,25,27,28] Stanislawski et al. showed that unpolymerized resin monomers are the main chemicals responsible for the cytotoxicity of RMGIC.[29,30] 2-hydroxyethyl methacrylate (HEMA) has been shown to drastically affect the morphology of human gingival fibroblasts at even low doses. Despite the decreased proportion of unbound monomers in newly developed RMGIC, the polymerization process still does not result in a complete conversion. Linkevicous et al. work showed that the complete removal of excess residual cement from the subgingival margin of the restoration is nearly impossible due to its radiolucent nature.[31] Thus, in restorations with subgingival margins, it becomes more critical to use a dental luting agent that minimally affects the gingival tissue.

The current study also revealed that the luting cement cytotoxicity peaks up in the first few hours after setting. Long-term acidic pH conditions and significant ion (fluoride ion) leaching from GIC in the first few hours[17,26] may be responsible for the early cytotoxic effects of luting cement. The cytotoxicity of the cement under test decreased with time, as reported in investigations by Smith and Ruse (1986)[32] and Hume and Mount (1988).[33] In contrast, Caughman et al concluded that GIC’s cytotoxic effects were not only due to initial low pH levels.[23] Instead, even after the cement has hardened for 48 h, cytotoxic components can still be leached out. According to Goldberg, the processes of cytotoxicity also pertain to the immediate release of free monomers that take place during the monomer-to-polymer conversion and the delayed release of leachable compounds due to erosion and degradation over time which was in accordance with the current study where cytotoxicity of cement was noted even at 48 h.[22] Becher et al. concluded triethylene glycol dimethacrylate, HEMA, and glycerol dimethacrylate monomer, and extracts from various compomers had the ability to cause cell death in macrophages in vitro.[19] The results of this work are consistent with research by Schwap et al.[23] and Trumpate et al.,[11] which found that resin based products were hazardous when tested in cell culture. The results of this study proved that cementation of fixed dental prosthesis needs proper cement selection in order to reduce the effect of leached out/cytotoxic components from luting cement on the gingiva which is in accordance with other in vitro studies.

According to the results of the current investigation, when the least cytotoxic RC was evaluated for the release of growth factor and cytokine after 48-h time interval which has an impact on periodontium, no significant release of PDGF and TGF growth factor was observed when compared to the control group. In contrast to the control group, cytokines IL-1α and IL-8 were significantly released. Oral mucosa cells have been demonstrated to produce both IL-1α and IL-8 in other ex vivo and in vivo animal experiments.[34] The study’s findings could potentially be attributed to the continuous release of IL-1α, the initial regulator, in the presence of harmful substances.

Limitation of the study

In the current study, cytotoxicity of luting agents, release of growth factor, and cytokines from the HGMSCs was evaluated for a limited period of 48 h which marks the limitation of the study. Second, dental luting cement exposure would result in different responses in vivo due to the presence of oral environment as compared to this study which did not simulate any oral environment.

Future scope of the study

Oral environment should be simulated while testing the cytotoxicity of dental luting cement in vitro. Furthermore, cytotoxicity of luting agents, release of growth factor, and cytokines from the HGMSCs could be evaluated for a long period of time interval.

CONCLUSION

Conclusions drawn from the present study are:

1. This study concluded that the viability of GMSCs shows a decline in cell number with the increase in the time interval from 1 h, 24 h, and 48 h for all the test cement

2. Amongst the selected cements, the Resin Cement was found to be the least cytotoxic to GMSCs, a moderate cytotoxic effect of RMGIC was noted while GIC had the maximum depleting effect on GMSCs due to the release of fluoride ions initially and leached out polyacrylic liquid which is of acidic nature

3. The least cytotoxic RC had a positive impact on the cytokine release while no significant release of growth factors was observed from the HGMSC after 48 h.

Several protective mechanisms present in the oral environment may be able to offset the cytotoxic effects of luting substances in contrast to their stated cytotoxicity. Concomitantly, the present study also showed the significant positive biological response of GMSCs with the release of ILs which helps in wound healing when exposed to the RC. Dental professionals should therefore handle luting cement with extreme caution and attention, keeping in mind the possibility of cytotoxicity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.