Influence of surface treatment and curing mode of resin composite cements on fibroblast behavior

The soft tissue compartments around teeth and dental implants comprise an epithelial and a connective tissue attachment. In healthy periodontal and peri-implant conditions these attachments provide an effective barrier against penetrating bacteria or bacterial toxins from the oral environment, and protect the underlying alveolar bone [1‐3]. Fixed dental prostheses retained on natural abutment teeth, dental implants, or implant abutments, are in contact with the soft tissue compartments. Smooth restoration surfaces are intended to minimize biofilm adherence, ease mechanical biofilm removal, and to facilitate soft tissue attachment. Removal of excess cement is challenging with intrasulcular preparation margins along tooth abutments and with intraoral cementation of implant restorations. A high surface roughness of residual cement promotes biofilm adhesion and subsequently the development of peri-implant diseases [4‐8].

Several factors influence the clinicians’ choice of the cement material, such as the restoration material, the abutment preparation design, the retention on the abutment, the position of the restoration margin, and drying possibilities. When using conventional cements such as zinc phosphate or glass ionomer cements, retentive preparations and thin cement margins are required to avoid hydrolysis of exposed cement material. Resin composite cements facilitate adhesion to tooth or implant abutments so that retention is not required and noninvasive preparation designs are feasible [6, 9, 10]. Resin composite cements are characterized by higher strength, lower cement wear, and improved esthetics compared to conventional cements [11‐14]. These cements are composed of an organic resin matrix, inorganic and organic filler particles, and silanes [15‐17]. The application of an acidic agent and priming system for bonding to tooth structure is required for most adhesive cement systems. Curing of resin composite cements can be catalyzed by either a chemical (autopolymerization), a photo (light-curing) activated initiator, or both (dual-curing), depending on the product. A higher degree of conversion of dual-curing cement is to be expected when light-curing is performed [16, 18, 19]. During radical chain polymerization, the reaction is severely retarded or even stopped by the oxygen in the air causing the formation of a superficial sticky layer on the surface [20, 21]. This so-called oxygen inhibition layer is primarily composed of unreacted monomers, which have been associated with potential cytotoxic effects [22, 23]. Elimination of the oxygen inhibition layer is particularly difficult in the intrasulcular and interdental area where access with hand instruments or rotating polishers is impeded [6], and possibly influences the surrounding soft tissue.

The purpose of this in-vitro study was to evaluate the influence of the curing mode and the removal technique of the oxygen inhibition layer using two different resin composite cements on the viability and morphology of HGF cells. The null-hypotheses were that viability of HGF cells is not affected (i) by light-cured compared to autopolymerized cement surfaces, and (ii) by polished or scaled surfaces compared to specimens with the oxygen inhibition layer left intact.

Viability and morphology of human gingival fibroblast (HGF) cells were evaluated after 24 h cultivation on discs of an adhesive (Multilink Automix, Ivoclar Vivadent, Ellwangen, Germany [MLA]) and a self-adhesive resin composite cement (RelyX Unicem 2 Automix, 3 M, Neuss, Germany [RUN]) (Table 1). Cement specimens were produced using either autopolymerization or light-curing and with three different surfaces treatments.

This in-vitro study investigated the impact of the curing mode and surface treatment on HGF cell viability and morphology for two resin composite cements. The results showed that the cell viability was mainly affected by the curing mode but also the surface treatment of the cement. Higher cell viability values were measured for light-cured specimens compared to autopolymerized specimens, and for polished or scaled surfaces compared to surfaces with the oxygen inhibition layer left intact. Both hypotheses were rejected and supported by matching SEM images which showed that the morphology of HGF cells was influenced by the curing mode and surface treatment.

The surface treatment protocols in this study were selected to reflect those encountered in clinical situations, comprising no treatment due to insufficient access to cement margins resulting in the oxygen inhibition layer remaining, polishing of excess cement using a rubber polisher [24], and cement removal with a scaler. The impact of the application of a glycerin paste to prevent oxygen inhibition layer formation was considered in a pre-test, but was not pursued due to highly uneven surfaces with great variability and an unpredictable outcome. As the specimens were rinsed with ethanol prior to cell experiments to limit the risk of contamination, cytotoxic components such as monomers may have been partially removed prior to the cell experiments. Therefore, the cytotoxic effect may be even higher in a clinical situation [22].

Further, cell activity and cell proliferation, which affect cell viability assays are very complex metabolic processes, and in-vitro data cannot be directly transferred to the clinical setting.

Previously, it has been shown that cell viability was significantly higher on smooth cement surfaces with Ra values between 0.2 and 0.8 μm [6]. These results are not confirmed by the findings of this study where polished and scaler-treated surfaces displayed strong variations in surface roughness values, but this variation in roughness parameters did not seem to affect the cell viability. Therefore, it can be assumed that the impact of the different surface treatments (or leaching components) on the cell viability was stronger than the impact of the surface roughness. Interestingly, the microscopic imaging revealed that light-curing can apparently generate smoother surfaces compared with autopolymerized surfaces. Autopolymerized surfaces of RUN displayed cracks while surfaces of MLA appeared smooth. Those cracks may have occurred during production when the samples were moved to 37 °C after being stored for 60 minutes at room temperature. Rising temperature during storage from 23 °C to 37 °C significantly increased the indirect tensile strength of RUN but not MLA, which indicates that the polymerization process of RUN is strongly affected by temperature [25].

Surfaces that were treated with the scaler appeared rougher when autopolymerized and higher Rz values were measured for both cement materials than for light-cured specimens, which may indicate that the material remains softer on the surface due to insufficient polymerization and the scaler treatment thus leads to deeper scratches on the surface. The thickness of the formed oxygen inhibition layer on the surface of light-cured compared to autopolymerized specimens should be further analyzed and hardness measurements may be performed.

Rohr et al. [6] found that RUN contains silica particles and aluminum fluoride fillers with a filler size of 12.5 μm. MLA showed a filler size of 0.25–3.0 μm and additionally, zirconia fillers, ytterbium trifluoride, and barium glass were added. With the energy dispersive X-ray analysis analyses of previous studies, only the inorganic components, such as the fillers, could be analyzed. In none of the six tested resin composite cements did the cement type or composition correlate with cell viability [6]. In addition, the biocompatibility did not seem to be affected by the fillers of resin composite material [26]. An in-vitro study compared the cytotoxic effects of different types of monomers and observed the following range of increased toxicity: hydroxyethylenethylmethacrylate (HEMA) < triethyleneglycoldimethacrylate (TEGDMA) < urethanedimethacrylate (UDMA) < bisglycidylmethacrylate (Bis-GMA). The increase in toxicity was proportional to the increase in molecular mass [23]. RUN is composed of TEGDMA and other methacrylates [27, 28], but the manufacturers did not specify the quantity of monomers the cement contains [27]. As described in MLA’s safety data sheets, the base paste contains 10–25% Bis-GMA and ≥ 2.5 to < 10% HEMA [29]. The catalyst paste contains 2.5 to < 10% UDMA and 2.5 to < 10% HEMA [30]. The cements analyzed in the present study contained different monomers, which could possibly explain the significant difference in cell viability between both cements when not sufficiently polymerized for the specimens left with the oxygen inhibition layer intact.

In the present study, higher viability was observed for cells on light-cured specimens compared to autopolymerized specimens. Hence, the potentially higher degree of polymerization with light-curing was found to have a more severe impact on cell viability than the surface roughness. Dual-cured cements generally reached a higher degree of conversion when light-curing was performed [16, 18, 19].

Previous studies have shown that monomer release had a toxic effect on L^− 929 mouse fibroblasts, especially if cements were not sufficiently polymerized [31, 32]. Additionally, a reduction of curing time significantly promoted the cytotoxicity of methacrylate-based materials [31]. Although neither the degree of conversion nor the amount of monomer leaking from the surface have been quantified, it can be assumed that light-cured specimens displayed a higher degree of conversion than autopolymerized specimens. Additionally, the presence of residual monomers on the untreated specimens with the oxygen inhibition layer was probably higher than that on the surfaces treated subtractively by polishing or with the scaler. The relative cell viability values for autopolymerized specimens left with the oxygen inhibition layer intact (RUN 46.6 ± 13.3%, MLA 25.7 ± 20.2%) are < 70% and have to be interpreted as cytotoxic effects according to ISO standard 10,993-5 [33]. However, when the surface of resin composite cements is light-cured and the oxygen inhibition layer is manually removed, a cell viability of fibroblasts even higher than on the control surface polystyrene can be expected.

In conclusion, the oxygen inhibition layer is an inhibitory factor for the viability of fibroblasts; hence if accessible, the surface of the cement margins should be finished with a rubber polisher or scaler to remove the superficial layer, which is probably not sufficiently polymerized. Clinicians should perform light-curing of the cement margin of a restoration to increase fibroblast viability.