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

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. Regarding cost, aesthetics, ease of fabrication and passivity, cement-retained implant supported prosthesis (CRISP) restorations function better than screw-retained ones.¹ In addition they provide better occlusion, loading characteristics streamlined clinical and treatment procedures.²

Dental professionals often choose dental cement for CRISP based on preferences of the product’s characteristics, such as material properties, mixing method, delivery system, or consistency.^3–5 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.^4,5 This 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. One of the demerits is related to the leftover cement in subgingival areas.^6–8 In these instances, it is very hard to remove all the dental cement.⁹ Peri-implant diseases are complex, inflammatory conditions caused by a group of bacteria that are usually anaerobic and Gram-negative and proliferate subgingivally.^10–18 One of the most important aspects of implant therapy success is the type of luting cement used to bond prostheses.¹⁹ It has been proved that residual cement left in CRISP can be an etiological factor for peri-implantitis in 8.6-14.4% of cases.^20–24

ZP cement is a dental cement that has been shown to be very effective in dental practice.^25–28 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.²⁹

Traditional 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.³⁰ Resin cement includes a significant amount of composite resin, which chemically attaches to the tooth.³¹ Despite their biocompatibility issues, dentists have been increasingly using this cement since they are said to improve restorative retention. However, certain material components can triggers a pulpal or gingival reaction, raising concerns about cytotoxicity.^30,32–34 Furthermore, despite GICs' increased mechanical qualities, only a few studies have shown their biocompatibility and harmful consequences.^30,31

Dental cement can significantly influence the growth or suppression of various bacterial strains associated with peri-implant 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.³⁵

Bone 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.³⁶

Raval et al. conducted a study on the bacterial response of many late-stage Gram-negative colonisers, which are implicated in peri-implant disease.³⁷

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 peri-implant illness proliferate or are inhibited. A previous study by the authors studied the impact of dental cement on cell proliferation at 24 hrs.³⁸ However, 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.³⁹

Human 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.

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).

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

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.⁴⁰

The evaluation of the cytotoxic potential of dental cement was carried out using a standardized technique in this experimental investigation. Utilizing in-vitro cytotoxic assays offers straightforward control over experimental parameters that are complex to manage in in-vivo studies.^41–43

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. They yield findings comparable to primary human gingival fibroblasts, potentially serving as a model for in-vitro studies on gingival toxicity.^44–46

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.⁴⁷ A 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.³⁸ Discrepancies 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.²⁸

Leirskar 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.^23–25

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).^34,49,50

The 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.^22,23 Kanjevac et al. conducted a study linking cytotoxicity to fluoride leakage in current GIC cement.⁴⁵ They 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).^51,52

Previous 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.⁵³ Dentin 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.⁵⁶ This aligns with Schmid-Schwap et al., who found that self-adhesive cement (Rely-XTM Plus) was more cytotoxic than adhesive resin cement.⁴⁷

However, certain monomers in resin composite cement can enter dentin tubules, potentially causing pulpal damage and hindering pulp tissue healing.^57–59 These monomers exhibit cytotoxicity in vitro for pulp and gingival cells, and ions could trigger cell changes.³⁶ Bakopoulou et al. demonstrated that the cytogenetic effects were caused by released chemicals like TEGDMA found in the composition of resin cement. The cytotoxicity ranking of commonly used monomers was identified as Bis-GMA > UDMA > TEGDMA > HEMA > MMA.^58,60–62 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.^63,64 Additionally, 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.⁶⁵

The 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: