In vitro biocompatibility testing of 3D printing and conventional resins for occlusal devices.

OBJECTIVE: To assess and compare the in vitro biocompatibility of new resins (Keysplint Soft (Keystone Industries), NextDent Ortho Rigid (3D System), and Freeprint Splint (Detax)) and traditional resins (Orthocryl (Dentaurum)) used for dental splints. METHODS: Standardized discs (n = 40) and 1:1, 1:2, and 1:4 extracts of the tested materials were prepared. Human gingival fibroblasts (hGFs) were isolated from gingival tissues. Different biological tests were carried out, including MTT assays to assess cell metabolic activity, cell migration assays, cell cytoskeleton staining, cell apoptosis, generation of intracellular reactive oxygen species (ROS), and scanning electron microscopy (SEM). Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test (p<0.05). RESULTS: MTT experiments showed that Freeprint Splint significantly reduces the hGF metabolic activity (***p<0.001), whereas SEM analysis showed almost no cells adhered on its surface. Cell migration was significantly lower after exposure to undiluted extracts of Freeprint Splint at 48 and 72 h (***p<0.001). Cell cytoskeleton staining assays showed fewer attached cells in 1:1 and 1:2 dilutions of Freeprint Splint. Annexin-V and 7-AAD staining assays showed that only cells exposed to Keysplint Soft extracts displayed similar cell viability to the control group. Finally, ROS levels detected in undiluted extracts of all resins were significantly enhanced compared to the control group (***p<0.001). CONCLUSIONS: The 3D-printed resins and the conventional dental resin showed a similar biocompatibility, except for Freeprint Splint, which was the most cytotoxic on hGFs. CLINICAL SIGNIFICANCE: 3D printing has been on the rise in recent years and its use in daily clinical practice is expanding over time. Two of the three 3D-printed resins tested in this study performed as well in the cytotoxicity tests as the conventional one, supporting their use, but caution and further testing are required.

Influence of dual-cure and self-cure abutment cements for crown implants on human gingival fibroblasts biological properties.

AIMS: To analyze the biological effects of the cements Relyx Unicem 2, Panavia V5, Multilink Hybrid Abutment and SoloCem on human gingival fibroblast cells (HGFs). MATERIALS AND METHODS: HGFs were exposed to different eluates (n = 40) of the studied resin-based cements. Their cytotoxic effects and influence on cell migration were assessed using MTT and wound-healing assays, respectively. Level of HGF attachment, cell morphology and F-actin cytoskeleton content after exposition to the different eluates were analyzed by scanning electron microscopy (SEM) and confocal microscopy analysis, respectively. The levels of intracellular reactive oxygen species (ROS) produced by the eluates of the different cements were also determined by flow cytometry. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey´s test. RESULTS: Eluates of SoloCem significantly reduces the viability of HGFs (69% reduction compared to control at 48 h). Cell migration of HGFs in presence of undiluted SoloCem eluates was significantly lower than in the control (88% open wound area at 24 h). Contrarily, migration speed with Multilynk eluates was similar to that of the control group at all periods of time and all dilutions studied. SEM analysis showed very few cells in SoloCem group, and a moderate cell growth in Multilink, Panavia and Relyx groups were detected. Finally, ROS levels detected in HGFs treated with the more concentrated SoloCem and Relyx dilutions were significantly enhanced compared with that in the control cells or the other groups (44% and 11% ROS positive cells, respectively). CONCLUSIONS: The results obtained in the present work suggest that Multilink hybrid abutment has better biological properties and lower cytotoxicity for cementing implant crowns on abutments.

Biological interactions between calcium silicate-based endodontic biomaterials and periodontal ligament stem cells: A systematic review of in vitro studies.

BACKGROUND: Most recently, the biological interactions, that is cytocompatibility, cell differentiation and mineralization potential, between calcium silicate-based biomaterials and periodontal ligament stem cells (PDLSCs) have been studied at an in vitro level, in order to predict their clinical behaviour during endodontic procedures involving direct contact with periodontal tissues, namely root canal treatment, endodontic surgery and regenerative endodontic treatment. OBJECTIVE: The aim of the present systematic review was to present a qualitative synthesis of available in vitro studies assessing the biological interaction of PDLSCs and calcium silicate-based biomaterials. METHODOLOGY: The present review followed PRISMA 2020 guidelines. An advanced database search was performed in Medline, Scopus, Embase, Web of Science and SciELO on 1 July 2020 and last updated on 22 April 2021. Studies assessing the biological interactions of PDLSCs with calcium silicate-based sealers (CSSs) and/or cements (CSCs) at an in vitro level were considered for inclusion. The evaluation of the 'biological interaction' was defined as any assay or test on the cytotoxicity, cytocompatibility, cell plasticity or differentiation potential, and bioactive properties of PDLSCs cultured in CSC or CSS-conditioned media. Quality (risk of bias) was assessed using a modified CONSORT checklist for in vitro studies of dental materials. RESULTS: A total of 20 studies were included for the qualitative synthesis. CSCs and CSSs, as a group of endodontic materials, exhibit adequate cytocompatibility and favour the osteo/cementogenic differentiation and mineralization potential of PDLSCs, as evidenced from the in vitro studies included in the present systematic review. DISCUSSION: The influence of the compositional differences, inclusion of additives, sample preparation, and varying conditions and manipulations on the biological properties of calcium silicate-based materials remain a subject for future research. CONCLUSIONS: Within the limitations of the in vitro nature of the included studies, this work supports the potential use of calcium silicate-based endodontic materials in stem cell therapy and biologically based regenerative endodontic procedures. REGISTRATION: OSF Registries; https://doi.org/10.17605/OSF.IO/SQ9UY.

Biocompatibility of a HA/ß-TCP/C Scaffold as a Pulp-Capping Agent for Vital Pulp Treatment: An In Vivo Study in Rat Molars.

Bioceramic materials possess desirable biological properties, highlighting their non-reactivity and osteoconductivity. Their use has been extended in vital pulp treatment. The purpose of this study was to evaluate and compare the effects of beta-tricalcium phosphate (ß-TCP), hydroxyapatite (HA), and collagen (C) scaffold with mineral trioxide aggregate (MTA) on the vital pulp of rat molars. Thirty-two molars of Sprague-Dawley rats underwent direct pulp capping with ß-TCP/HA/C (n = 16) and MTA (n = 16). After 30 days, the following parameters were evaluated in the tested samples: the degree of pulp inflammation and pulp vitality, the presence of reparative dentin, the homogeneity of the odontoblastic layer, and the presence of pulp fibrosis. No statistically significant differences were observed between HA/ß-TCP/C and MTA in terms of the degree of inflammation (p = 0.124). Significant differences were found in reparative dentin formation between the treatment groups (p = 0.0005). Dentin bridge formation was observed in the MTA-treated group. The local action of HA/ß-TCP/C is similar to that of MTA when used as an agent for pulp vital treatment in terms of absence of inflammation and maintenance of pulp vitality, although there are significant differences between both materials regarding the formation of dentin bridges.

Topical fluoride varnishes promote several biological responses on human gingival cells.

OBJECTIVE: To compare the in vitro cytotoxicity of four commercial topical fluoride varnishes widely used in daily dental practice for the prevention of caries on human fibroblasts: Cervitec F, Fixofluor, Fluor Protector S and Duraphat. MATERIAL AND METHODS: Human gingival fibroblasts (hGF) were exposed to different concentrations of fluoride varnishes extracts. Biological assays, including MTT and IC50 value determination, annexin-V/7-AAD staining, cell migration and F-actin staining with phalloidin were carried out. Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test. RESULTS: At 4% concentration, all of the fluoride varnishes extracts affected fibroblasts metabolic activity, exhibiting a high degree of cytotoxicity at all measured time points. At 0.1% and 1%, Duraphat and Fixofluor or Fluor Protector S and Cervitec F exerted the lowest or highest cytotoxic effects, respectively. Similar effects were evidenced when induction of apoptosis/necrosis and cell migration assays were analyzed. Immunocytochemical assays revealed a similar number of fibroblasts, without changes in the morphology and F-actin content at 0.1% concentration of all tested materials, while at 1% concentration, Fluor Protector S and Cervitec F showed few cells with aberrant morphology or non-adhered cells, respectively. CONCLUSIONS: Different commercial topical fluoride varnishes with the same therapeutic indication may exhibit different biological effects and cytotoxicity on fibroblasts.

Are Denture Adhesives Safe for Oral Cells?

PURPOSE: To compare the cytotoxicity of six commercially available denture adhesives on human gingival cells: Poligrip Flavour Free Fixative Cream, Fixodent Pro Duo Protection, Novafix cream, FittyDent, Polident Total Action, and Fixodent Pro Plus Duo Protection. MATERIAL AND METHODS: Eluates of denture adhesives were brought into contact with human gingival cells and compared to untreated cells (w/o any dental adhesive elute). Cell toxicity was assessed by measuring cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assays), cell morphology (immunofluorescence assays), induction of apoptosis/necrosis and production of reactive oxygen species (ROS) (flow cytometry assays). In addition, the pH of each sample was determined. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. RESULTS: All denture adhesives tested led to a reduction in pH, especially Fixodent Pro Duo Protection and Fixodent Pro Plus Duo Protection. The cell viability assays showed that Fixodent Pro Duo Protection (1:1 72 hours, p = 3.04 × 10-6 ; 1:2 72 hours, p = 2.07 × 10-6 ; 1:4 72 hours, p = 2.04 × 10-6 ) and Fixodent Pro Plus Duo Protection (1:1 72 hours, p = 2.01 × 10-6 ; 1:2 72 hours, p = 3.03 × 10-6 ; 1:4 72 hours, p = 2.02 × 10-6 ) significantly decreased cell viability at all dilutions. Compared to the control group and the rest of the adhesives, Poligrip Flavour Free Fixative Cream (PFF 1:1 72 hours, p = 2.24 × 10-6 ; 1:2 72 hours, p = 2.44 × 10-6 ; 1:4 72 hours, p = 2.04 × 10-6 ) showed a significantly higher cell viability score at all dilutions. Fixodent Pro Duo Protection and Fixodent Pro Plus Duo Protection, both adhesives containing zinc salts in their composition, were responsible for necrosis, and the number of cells was much reduced, with aberrant morphology and pyknotic nucleus. Finally, Fixodent (1:2, p = 2.04 × 10-6 , 1:4, p = 0.00036; 1:2, p = 8.82 × 10-6 , 1:4, p = 2.30 × 10-6 ) products significantly promoted ROS production in gingival cells. CONCLUSIONS: The results suggest that denture adhesives containing zinc in their composition could be responsible of the decrease of cell viability, ROS production, aberrant cell morphology, and induction of apoptosis and cell death. However, other possible additional cytotoxic factors must be considered. Thus, more studies are necessary to confirm this hypothesis.

Comparative Surface Morphology, Chemical Composition, and Cytocompatibility of Bio-C Repair, Biodentine, and ProRoot MTA on hDPCs.

Biocompatibility is an essential property for any vital pulp material that may interact with the dental pulp tissues. Accordingly, this study aimed to compare the chemical composition and ultrastructural morphology of Biodentine (Septodont, Saint Maur-des-Fosses, France), ProRoot MTA (Dentsply Tulsa Dental Specialties, Johnson City, TN, USA), and Bio-C Repair (Angelus, Londrina, PR, Brazil), as well as their biological effects on human dental pulp cells. Chemical element characterization of the materials was undertaken using scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDX). The cytotoxicity was assessed by analyzing the cell viability (MTT assay), cell morphology (immunofluorescence assay), and cell attachment (flow cytometry assay). The results were statistically analyzed using ANOVA and Tukey's test (p < 0.05). EDX revealed that ProRoot MTA and Biodentine were mostly composed of calcium, carbon, and oxygen (among others), whereas Bio-C Repair evidenced a low concentration of calcium and the highest concentration of zirconium. SEM showed adequate attachment of human dental pulp cells (hDPCS) to vital pulp materials and cytoskeletal alterations were not observed in the presence of material eluates. Remarkably, the undiluted Biodentine group showed higher viability than the control group cells (without eluates) at 24 h, 48 h, and 72 h (p < 0.001). Based on the evidence derived from an in vitro cellular study, it was concluded that Bio-C Repair showed excellent cytocompatibility that was similar to Biodentine and ProRoot MTA.

Melatonin as an Agent for Direct Pulp-Capping Treatment.

Melatonin plays an essential role in the regulation of bone growth. The actions that melatonin exerts on odontoblasts may be similar to its action on osteoblasts. This research aimed to evaluate the pulp response to melatonin used for direct pulp capping to evaluate the antioxidant effect of melatonin administered orally and its influence on dental pulp. Direct pulp capping was performed on the upper molars of Sprague Dawley rats using melatonin or Mineral Trioxide Aggregate (MTA). The study groups were: MTA; Melatonin; MTA + Melatonin administered orally; and Melatonin + Melatonin administered orally. In the latter two groups, the animals drank water dosed with melatonin ad libitum (10 mg/100 mL). After 30 days, the animals were sacrificed, and 5 ml of blood, the kidneys, and the liver were extracted in order to evaluate oxidative stress using thiobarbituric acid reactive substances testing (TBARS). Fragments of the maxilla containing the study molars were prepared for histological evaluation. The degree of pulp inflammation and pulp necrosis, the presence of reparative dentin and dentin bridging the pulp chamber, the presence and regularity of the odontoblastic layer, and the presence of pulp fibrosis were evaluated. No significant differences were found between the four study groups for any of the studied histological variables. The oral administration of melatonin did not modify the local effects of MTA or melatonin on dental pulp, or reduce basal-level oxidative stress. The effect of melatonin on pulp is similar to that of MTA and may be used as an agent for direct pulp capping.

In Vitro Evaluation of the Biological Effects of ACTIVA Kids BioACTIVE Restorative, Ionolux, and Riva Light Cure on Human Dental Pulp Stem Cells.

This study aimed to analyze the biological effects of three new bioactive materials on cell survival, migration, morphology, and attachment in vitro. ACTIVA Kids BioACTIVE Restorative (Pulpdent, Watertown, MA, USA) (Activa), Ionolux (Voco, Cuxhaven, Germany), and Riva Light Cure UV (SDI, Bayswater, Australia) (Riva) were handled and conditioned with a serum-free culture medium. Stem cells from human dental pulp (hDPSCs) were exposed to material extracts, and metabolic activity, cell migration, and cell morphology were evaluated. Cell adhesion to the different materials was analyzed by scanning electron microscopy (SEM). The chemical composition of the materials was evaluated by energy-dispersive X-ray (EDX). One-way analysis of variance followed by a Tukey test was performed (p < 0.05). Ionolux promoted a drastic reduction in metabolic activity and wound closure compared to the control (p < 0.05), whereas Activa induced adequate metabolic activity and cell migration. Moreover, SEM and immunofluorescence analysis showed abundant cells exposed to Activa. The materials showed different surface morphologies, and EDX spectra exhibited different peaks of C, O, Si, S, Ca, and F ions in glass ionomer cements. The results showed that Activa induced cell migration, cell attachment, and cell viability to a greater extent than Riva and Ionolux.

Comparative Cytocompatibility and Mineralization Potential of Bio-C Sealer and TotalFill BC Sealer.

The aim of this study was to investigate the cytocompatibility and mineralization potential of two premixed hydraulic endodontic sealers compared with an epoxy resin-based root canal sealer. The cellular responses and mineralization capacity were studied in human periodontal ligament stem cells (hPDLSCs) that were exposed to premixed hydraulic sealers, Bio-C Sealer (Angelus, Londrína, PR, Brazil), TotalFill BC Sealer (FKG Dentaire SA, La-Chaux-de-fonds, Switzerland) and an epoxy resin-based material, AH Plus (Dentsply De Trey, Konstanz, Germany). Non-exposed cultures served as the control. The endodontic sealers were assessed using scanning electron microscopy (SEM) and energy dispersive X-ray microanalysis (EDX). Statistical analyses were done using Analisis of Variance (ANOVA), with Bonferroni adjusted pairwise comparison (p = 0.05). AH Plus reduced cell viability and cell migration, whereas increased cell viability and cell migration were observed in the Bio-C Sealer and the TotalFill BC Sealer (p < 0.05). The lowest cell attachment and spreading were observed for all concentrations of AH Plus, whereas the highest were observed for TotalFill BC Sealer. At the end of 21 days, only the Bio-C Sealer and the TotalFill BC Sealer supported matrix mineralization (p < 0.05). Additionally, SEM-EDX revealed high content of calcium, oxygen, and silicon in the Bio-C Sealer and the TotalFill BC Sealer. Based on the results from this study, Bio-C Sealer and TotalFill BC Sealer demonstrated better cytocompatibility in terms of cell viability, migration, cell morphology, cell attachment, and mineralization capacity than AH Plus.