Cytotoxicity of dental cement on soft tissue associated with dental implants at different time intervals

Background To investigate and compare the effect of four commercially used dental cement at 24 hours, 48 hours,72 hours (h) and 6 days on the cellular response of human gingival fibroblast (HGF). Methods 3 cement pellet samples were made for each 4-test cement (n=12). The cement used for this study were zinc phosphate (ZP), zinc oxide non-eugenol (ZOE), RelyX U200 (RU200), and glass ionomer cement (GIC). The cytotoxicity of peri-implant tissues was investigated using one commercial cell line. All processing was done following International Organization for Standardization (ISO) methods 10993-5 and 10993-12 (MTT assay Test). Cell cultures without dental cement were considered as control. Standard laboratory procedures were followed to permit cell growth and confluence over 48 hrs after sub-cultivation. Before being subjected to analysis, the cells were kept in direct contact with the cement samples for the suggested time period. To validate the results the specimens were tested three times each. Cell death and inhibition of cell growth were measured quantitatively. Results were analyzed using 1-way ANOVA (a=0.05) followed by Tukey B post hoc test. Results The study showed the dental cement test material was cytotoxic. ZOE, ZP, GIC, and RU200 were cytotoxic in decreasing order, respectively, significantly reducing cell viability after exposure to HGF (p <0.001). Conclusions Within the limitations of this in-vitro cellular study, results indicated that HGF were vulnerable to the test the dental cement. The highest cytotoxicity was observed in ZOE, followed by ZP, GIC, and RU200.


Introduction
Endosseous implants to replace lost teeth have become the gold standard in dentistry.The implant-supported prosthetic reconstruction uses both cement-and screw-retained restorations.4][5] There are many kinds of luting dental cement that can be usedto lute bridges, crowns, veneers, and implant crowns, and dental materials that are biocompatible are gaining more attention from dentists and patients. 4,5This is on account of the contact of these cement with gingival tissues for extended periods of time eliciting a soft tissue response.This response is mainly on account of the cytotoxicity its components.The greater the content of the unreacted substances more is the cytotoxicity.The anatomic differences between natural teeth and implant supported prosthesis warrants greater attention to response of soft tissue to the unreacted components of the dental cement.
CRISP have both advantages and disadvantages.7][8] In these instances, it is very hard to remove all the dental cement. 91][12][13][14][15][16][17][18] One of the most important aspects of implant therapy success is the type of luting cement used to bond prostheses. 196][27][28] However, it has some disadvantages, such as being soluble, low pH, and inability to bond with the tooth chemically.Zinc oxide non eugenol (ZOE) is a temporary cement that uses different substitutes instead of eugenol, because eugenol can interfere with resin bonding.Traditional GICs have some benefits, such as being biocompatible, releasing fluoride, having a thermal expansion and an elasticity similar to dentin. 29aditional GICs have drawbacks such as dehydration, susceptibility, high solubility, and slow setting rate despite their benefits.Due to further developments in GICs, resin-modified GICs-which have greater physical and mechanical properties than regular GICs-have been introduced. 30Resin cement includes a significant amount of composite resin, which chemically attaches to the tooth. 31Despite their biocompatibility issues, dentists have been increasingly using this cement since they are said to improve restorative retention.3][34] Furthermore, despite GICs' increased mechanical qualities, only a few studies have shown their biocompatibility and harmful consequences. 30,31ntal cement can significantly influence the growth or suppression of various bacterial strains associated with periimplant disease.Furthermore, the effect of dental cements on the proliferation of host cells can provide additional insights for choosing the appropriate cement material.
In clinical scenarios where cement might be applied during the early healing phase, such as temporary restoration or abutment placement during surgery, or at a later stage in implant restoration where harmful bone alterations might occur if cement comes into contact with host tissues, it's particularly crucial to assess the effects of bacteria and host cells on dental component surfaces. 35ne loss around dental implants typically follows a specific pattern, moving from the crestal bone level towards the apex of the implant.This resorption profile may be primarily due to the interaction between soft tissue and remnants of cement from cement-retained prosthesis. 36val et al. conducted a study on the bacterial response of many late-stage Gram-negative colonisers, which are implicated in peri-implant disease. 37

REVISED Amendments from Version 1
The new version is the revised manuscript based on the suggestions made by the reviewer.The introduction and the references have been augmented based on her recommendations.
Any further responses from the reviewers can be found at the end of the article Different cement formulations (zinc oxide with eugenol, zinc oxide with noneugenol, zinc phosphate, and resin components), according to the authors' hypotheses, would produce various bacterial reactions.Several commercially available dental cements were exposed to Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis, and the bacterial viability was monitored to ascertain how the bacteria responded to the dental cement.The composition of dental cement has a substantial influence on whether certain bacterial strains linked to periimplant illness proliferate or are inhibited.A previous study by the authors studied the impact of dental cement on cell proliferation at 24 hrs. 38However, the impact of dental cement on host cellular proliferation in different time intervals may throw some light in choosing the right cement material as these cement may remain in contact with host tissues for varied time periods depending on the purpose and duration of the restoration.
Several factors may restrict the application of in vitro cell-based experiments to clinical scenarios.Primary cells, which maintain the same natural ploidy, gene expression regulation, stress response, and other biological parameters seen in humans, may provide more relevant data for clinicians compared to immortalised cell lines. 39man gingival fibroblasts are commensals of the oral environment and make interesting models for invitro evaluation of dental materials on account of the release of growth factors and cytokines suggesting immunological response.The current study therefore employed human gingival fibroblasts.
This study investigated and compared the effect of four commercially used dental cement at 24h, 48h, 72h, and six days on human gingival fibroblast (HGF) cellular response.The Null Hypothesis was that commercially used dental cement would have had no effect on the cellular response of HGF.

Methods
The test cement used in the study is presented in Table 1.

Preparation of the test cement
The preparation of the cement samples was done as follows: The powder was stirred into the liquid in modest increments as per the manufacturer's directions.Once the cement acquired the right consistency, it was transferred to three polytetrafluoroethylenes (PTFE) polymer moulds.The cement takes roughly 5-9 minutes to set.

Glass Ionomer Cement (GIC)
The GIC powder and liquid were combined as per the manufacturer's instructions.The mixture was immediately transferred to three PTFE moulds to preserve the gel structure.The GIC cement was left to cure for 24 hours.

Zinc oxide non-eugenol cement
The base and catalyst were mixed according to the manufacturer's guidelines on a mixing pad, then transferred to three PTFE moulds.The setting time for this cement was approximately 3 minutes and 30 seconds.

Resin cement
The required amount of resin was dispensed from the automix syringe's clicker dispenser, mixed, and poured into three PTFE moulds.The RU200 cement set in about 30 seconds.
All tests used a mould of size 7x3x3mm.The cement was kept at room temperature for two days, then washed with phosphate buffer saline pH7.4,and air-dried in a NUAIR biosafety cabinet hood to clean the surface of the solidified cement and remove any unsolidified items.
In brief, 1X104 HGF cells were grown in a 96-well plate and immediately exposed to the four test cements for a day at 37°C with 5% CO 2 .Direct contact testing was used to closely simulate an in vivo setting and assess the cytotoxicity of the dental cements under investigation.HGF cells unexposed to cement served as a control group.After the incubation period (24h, 48h, 72h, and six days), the cement was removed, and the cells were treated with MTT (5 mg/ml, Sigma, USA) for 4 hours at 37°C with 5% CO 2 .The formazan crystals formed were dissolved in 150 μL of DMSO (Sigma, USA), and the absorbance was measured using a microplate reader (Varioskan, ThermoFisher Scientific, USA) at 570 and 630 nm.All tests were performed in duplicate and repeated three times for validation.

Statistical analysis
The experiments were conducted with appropriate replicates and repeated three times.Intergroup comparison was done using the post-hoc Tukey Test.Statistical significance was defined as P > 0.05 using the Tukey post-hoc test.

Results
The present study was undertaken to determine the cytotoxic effect of four different dental cement on HGF.Cell viability was measured 24hrs, 48hrs, 72hrs, and 6 days post-exposure with four dental cements.The intergroup comparison was carried out using post-hoc Tukey Test.P > 0.05 by Tukey post-hoc test was considered statistically significant (Figure 1).

Cell viability at 24h time interval
HGF viability was 99.99% in the control cement at 24 hours, 10.84% in the ZOE cement, 20.82% in the ZP cement, 81.73% in the GIC, and 107.78% in the RU200 cement, respectively (Figure 1).Compared to the other three cement it was tested, RU200 had the highest cell viability, and ZOE cement had the lowest cell viability.There is a statistically significant difference between all of the groups (P <0.0001) except for the control and RU200 cement group (P=0.5635).

Cell viability at 48-hour time interval
HGF viability in control, ZOE, ZP, -GIC and RU200 was 99.99 percent, 11.03 percent, 26.76 percent, 30.79 percent, and 48.29 percent, respectively, after 48 hours.Compared to the other three cements, RU200 has the highest cell viability.
Cell viability is lowest in ZOE and ZP cement.There is a statistically significant difference between all groups, P< 0.0012, except for the zinc phosphate and GIC group (P=0.7575).

Cell viability at 72 hours time interval
HGF viability was 99.99 percent, 35.11 percent, 11.86 percent, 56.34 percent, and 51.71 percent at 72 hours in control, ZOE, ZP, GIC, and RU200, respectively.GIC has the most excellent cell viability compared to the other three cement groups, while ZP cement has the lowest.Of the four cement groups, ZOE and ZP cement had the lowest cell vitality.The results were statistically significant between all of the groups.(P< 0.0001) except ZOE and ZP (p=0.7954) and ZOE and GIC(P=0.0669).

Cell viability at sixth day time interval
On 6 th day the HGF viability in control, ZOE, ZP, GIC cement and RU200 cement was 99.99%,7.14%,4.53%,23.51%,and 45.85%, respectively.RU200 cement shows the highest cell viability, while zinc oxide non-eugenol cement shows the least cell viability among all 4 cement.The results were statistically significant between all of the groups.(P<0.0001)except for ZOE and ZP (P =0.7811).This study evaluated the cytotoxic impact of four distinct dental cement on Human Gingival Fibroblasts (HGF).The viability of the cells was assessed at 24 hours, 48 hours, 72 hours, and 6 days after exposure to the cements.The Tukey posthoc test was used for intergroup comparisons.A p-value greater than 0.05 was deemed statistically significant (Figure 1).
At the 24-hour mark, the viability of HGF was highest in the RU200 cement and lowest in the ZOE cement, compared to the other three cements tested.The difference between all groups was statistically significant (P <0.0001), except for the control and RU200 cement group (P=0.5635).
After 48 hours, RU200 cement continued to show the highest cell viability among the four cements.The lowest cell viability was observed in ZOE and ZP cement.All groups showed a statistically significant difference (P< 0.0012), except for the zinc phosphate and GIC group (P=0.7575).
At the 72-hour interval, GIC showed the highest cell viability among the four cements, while ZP cement showed the lowest.The results were statistically significant between all groups (P< 0.0001), except between ZOE and ZP (p=0.7954) and ZOE and GIC (P=0.0669).
On the sixth day, RU200 cement demonstrated the highest cell viability, while zinc oxide non-eugenol cement showed the least among all four cements.The results were statistically significant between all groups (P<0.0001),except for ZOE and ZP (P =0.7811).

Discussion
The experimental arrangement of this research displayed that human gingival fibroblast displayed susceptibility to alterations in viability when subjected to commercial dental cement.Furthermore, these fibroblasts can trigger biological responses.The Null Hypothesis was invalidated due to significant fluctuations in fibroblast viability, which suggests that the proximity of cement to periimplant tissue during cementation plays a role in causing potential toxic tissue damage.The extent of this damage is proportional to the quantity of cement in proximity with oral tissues and the leaching of components.Variations in individual sensitivity could exist, emphasizing the crucial necessity of removal of excess cement. 40e evaluation of the cytotoxic potential of dental cement was carried out using a standardized technique in this experimental investigation.[43] The impact of ZP, ZOE, GIC, and RU200 cement on HGF cells was investigated using the MTT test.Chemical constituents released from tooth restorative materials target fibroblasts.HGF cells were chosen for this study due to their ease of production and cultivation.These cells offer benefits in terms of in-vitro growth efficiency and result reproducibility.[46] The MTT test, a widely recognized method for assessing cell vitality, was employed.In the current investigation, when the MTT assay was conducted on cell cultures exposed to the test cement for 24 hours, the highest cell viability, at 107.78%, was observed with resin cement at 24 hours 48 hours, and 66 days.Conversely, the lowest viability was found with ZOE and ZP cement, respectively.Significant differences were found between the control, ZOE, and ZP groups.
At all the various time intervals, ZOE and ZP cement exhibited the most pronounced cytotoxicity.ZOE cement samples displayed severe cell cytotoxicity, with cell viability measuring below 30%.In the case of ZP cement, cell viability stood at 10.84%, 11.03%, 11.86%, and 4.53% at 24, 48, 72 hours, and the sixth day, respectively.This occurrence can be attributed to the release of leachable substances from the materials, an impact that progressively diminishes and eventually falls below detectable levels after a span of six weeks. 47A preceding investigation conducted by the researchers revealed GIC to exhibit the highest cytotoxicity. 38However, it's noteworthy that the time frame analyzed in that study was limited to 24 hours. 38Discrepancies in methodology could play a role in yielding differing outcomes.Potential explanations for the cytotoxic impacts of these cements include the liberation of zinc and fluoride ions, as well as acidity and the discharge of other substances. 28irskar and Helgeland conducted an analysis of the culture conditions involving ZP cement and observed the released zinc had detrimental effects on the studied cell line.In addition since cell death was noted from the first to the third day, it indicated that factors apart from acidification were incriminated.These findings aligned with research by Welker and Neupert, as well as Leirskar et al. 41,42 During cell death, proinflammatory cytokines generally increase, making the suppression of inflammatory responses a key aspect of effective anti-inflammatory ingredients.The extent of cytotoxic effects was linked to the quantity of zinc released from ZP cement, suggesting that other elements, like the acid produced by the cement, might amplify the zinc's impact.Earlier studies revealed that zinc absorption in certain cell types reduced as pH dropped. 48 Zn2+ acts as an anti-inflammatory ingredient by regulating proinflammatory cytokines, which is why a higher zinc dose is recommended to lower these cytokine levels in the blood plasma of inflammation patients.Another study found that Harvard ZP cement exhibited greater cytotoxicity compared to Fuji PLUS cement (RM-GIC).Fuji PLUS had varying levels of cytotoxic effects, ranging from mild to severe, while Fuji I GIC showed the least cytotoxicity.
Lewis et al. suggested that the leachable components of GICs might affect the rate of cell cycle progression rather than causing immediate cell death. 48According to Oliva et al., RM-GIC has notable cytotoxic effects attributed to its leaching poly-acidic phase.The higher HEMA content in Fuji PLUS cement which quickly diffuses into dentine might contribute to its greater cytotoxicity compared to Fuji I. [23][24][25] The levels of HEMA permeating into pulpal tissue are considerably lower than those causing acute toxicity.Thus, RM-GIC's cytotoxicity could be linked to the leaching of a hazardous mix of components, including resin monomers and fluoride ions.Kanjevac et al. established a connection between cytotoxicity and fluoride leakage in current GIC cement, also noting that other components like strontium and aluminum ions had more harmful effects on cell cultures when leached.Due to higher fluoride release, Fuji PLUS (RMGIC) exhibited more cytotoxicity than Fuji I (GIC). 34,49,50e quantities of HEMA capable of permeating into pulpal tissue are notably lower than those causing acute toxicity.Consequently, the leaching of a hazardous mixture of components, including resin monomers and fluoride ions, could be suspected in the case of RM-GIC. 22,23Kanjevac et al. conducted a study linking cytotoxicity to fluoride leakage in current GIC cement. 45They also highlighted that other elements like strontium (Sr2+) and aluminum ions (Al3+) had more pronounced harmful effects on cell cultures when leached.Due to increased fluoride release, Fuji PLUS (RMGIC) exhibited greater cytotoxicity than Fuji I (GIC). 51,52evious in-vitro investigations have shown that when diffusates are extracted from cells using a dentin barrier test device, the apparent cytotoxicity of materials is reduced. 53Dentin can absorb chemicals in the tubules and hinder the diffusion of harmful compounds into the pulp, as explained by Hanks et al. 54 Self-adhesive resin cements, composed of filled polymers, are designed to bond to tooth structure without requiring additional adhesives or etchants.These cements might undergo a slower rate of polymerization and attain a lower final polymerization degree compared to traditional resin cements, whether in dual-or self-cured modes, as noted by Moraes et al. 55 Their final polymerization degree often proved higher in the dual-cure mode.Dual-cured specimens of resin-based cement (Duo-Link and BisCem) were demonstrated to be more harmful than chemically set cement.BisCem exhibited more cytotoxicity than Rely-XTM Unicem in the study by Ulker and Sengun. 56This aligns with Schmid-Schwap et al., who found that self-adhesive cement (Rely-XTM Plus) was more cytotoxic than adhesive resin cement. 478][59] These monomers exhibit cytotoxicity in vitro for pulp and gingival cells, and ions could trigger cell changes. 36Bakopoulou et al. demonstrated that the cytogenetic effects were caused by released chemicals like TEGDMA found in the composition of resin cement.1][62] The cytotoxic effects of Duo-Link cement could be attributed to the presence of UDMA and inorganic fluoride.The most severe cytotoxic effects in this study were associated with BisCem composite resin cement, which comprises TEGDMA and HEMA. 63,64dditionally, BisCem lowered the pH to 3-4, potentially explaining the decreased cell viability compared to Duo-Link cement's impact.Reduced drying time increases the cytotoxicity of resin cement, emphasizing the need for adequate curing time. 65e findings also suggest that the choice of cell system may impact results in advanced biocompatibility assays, such as protein expression or -omics studies, or assessments of cell-cell interactions.This should be explored in future research with larger sample sizes for each experimental group.This is particularly relevant considering the need to prioritize more relevant and mechanistic insights into hazardous pathways over isolated cytotoxicity endpoints.Therefore, while the clinical relevance of in vitro assays needs careful consideration, the results of this study must be approached cautiously.Despite resin cement's strong retention and preferred status as a luting agent in CRISP, clinical reports indicate a high incidence of peri-implantitis and difficulties in prosthesis retrieval in complication cases.Thorough clinical practices are necessary to reduce soft tissue exposure to these cements during attempts to remove excess cement.

Conclusion
Within the limitations of this in-vitro study, the following conclusions were drawn: • All the luting cement is cytotoxic to the gingival fibroblast cells.
• Maximum cytotoxicity was demonstrated in ZOE, followed by ZP, GIC and RU200 cement.
The article has scientific merit.However, the corrections as indicated and questions posed in the manuscript using annotations need to be addressed.The repetition of information in the results section should be deleted.The source of HGF should be mentioned.The results section should be written in past tense.Note other minor corrections in grammar.
Please view the additional review comments in the file found here.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genotoxicity, Cytotoxicity, Mutagenicity I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Bheema setty Manasali
Taif University, Taif, Saudi Arabia "Cytotoxicity of dental cement on soft tissue associated with dental implants at different time intervals" In my opinion, there isn't much literature on the topic and the article should be well-received.Moreover, it studies the impact of dental cement on soft tissue in association with implants which is most relevant clinically.I also notice vast improvements in the writing style, recent references, and discussion.I notice that the discussion has been augmented with additional references, particularly contradictory findings.I do not have any major corrections to be suggested.However, as minor recommendations, I would be happy to see the authors suggesting clinical tips when dealing with resin cement considering the incidence of peri-implantitis.I recommend this article for approval.Thanking You

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?

Figure 1 .
Figure 1.The effect of direct cement exposure on the viability of HGF cells at different time intervals.

Reviewer Report 30
May 2024 https://doi.org/10.5256/f1000research.159945.r234384© 2024 Manasali B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 1 .
Test cement used in the study.