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.

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.

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.

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.

Dental Treatments under General Anesthesia on Children with Special Health Care Needs Enrolled in the Spanish Dental Care Program.

The purpose is to analyze the medical characteristics of children with special health care needs (CSHCN) recommended for dental treatment under general anesthesia (GA), postoperative complications, and dental treatment outcomes under the regulation of the Spanish Dental Care Program (PADI). 111 clinical records were selected. The study population was divided into three age groups. The quantitative data was specified as the mean ± SD. For the qualitative variables, the Chi-Square test was used. One-way ANOVA and Bonferroni tests were used to examine the effect of the "age group" and the number of treatment procedures. A total of 1473 treatment procedures were performed, of which 110 (7.5%) were cleanings, 898 (61%) were restorative procedures, 332 (21.7%) were extractions, 22 (1.6%) were endodontic treatments, 62 (4.2%) were pulpotomies, and 59 (4%) were stainless steel crowns. Regarding the mean number of incisor root canal treatments (RCT), age group 3 received a significantly higher mean number of incisor RCTs than age group 1 (p = 0.02). Age group 1 received a higher average of pulpotomies and stainless-steel crowns (p = 0.00) compared to groups 2 and 3. GA is a safe procedure for the dental treatment of CSHCN, with minimal postoperative complications, which should be included among dental public programs.

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.

Laterally rotated flap for soft tissue augmentation around maxillary loaded osseointegrated dental implants: preliminary results of a pilot study.

A minimal width and thickness of keratinized and attached soft tissue is desirable to prevent peri-implant diseases. This report describes the preliminary results of a pilot study of a surgical approach for soft tissue augmentation around loaded dental implants in the partially or totally edentulous maxilla. Four patients presenting eight maxillary implants with a buccal peri-implant soft tissue deficiency received a laterally rotated flap. A buccal mesial and apical recipient area was created around each implant, and a pediculated keratinized graft was rotated 90° from the distopalatal and positioned and sutured on the peri-implant buccal aspect. All implants treated showed a gain in buccal clinical peri-implant attachment (1.37 ± 0.44 mm) and buccal soft tissue levels (2.06 ± 1.40 mm) and interproximal soft tissue levels (1 ± 0.75 mm). The technique provided quality soft tissue with a gain in soft tissue thickness (3.06 ± 0.68 mm) and keratinized wide tissue (4.69 ± 0.80 mm) with minimal morbidity (1575 ± 549.67 mg of ibuprofen) and maintenance of prosthetic loading. Peri-implant soft tissue stability was maintained for 13.5 ± 1.87 months. Laterally rotated flap can be applied and provide clinical benefits to compromised implants due to the presence of buccal peri-implant soft tissue deficiency. Further studies are required to confirm these preliminary results.

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.

Thermo-setting glass ionomer cements promote variable biological responses of human dental pulp stem cells.

OBJECTIVE: To evaluate the in vitro cytotoxicity of Equia Forte (GC, Tokyo, Japan) and Ionostar Molar (Voco, Cuxhaven, Germany) on human dental pulp stem cells (hDPSCs). METHODS: hDPSCs isolated from third molars were exposed to several dilutions of Equia Forte and Ionostar Molar eluates (1/1, 1/2 and 1/4). These eluates were obtained by storing material samples in respective cell culture medium for 24h (n=40). hDPSCs in basal growth culture medium were the control. Cell viability and cell migration assays were performed using the MTT and wound-healing assays, respectively. Also, induction of apoptosis and changes in cell phenotype were evaluated by flow cytometry. Changes in cell morphology were analysed by immunocytofluorescence staining. To evaluate cell attachment to the different materials, hDPSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy (SEM). The chemical composition of the materials was determined by energy dispersive X-ray (EDX) and eluates were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). Statistical analysis was performed with analysis of variance (ANOVA) and Student's t-test (α<0.05). RESULTS: Undiluted Equia Forte extracts led to a similar cell proliferation rates than the control group from 72h onwards. There were no significance differences between Equia Forte and Ionostar Molar in terms of cell apoptosis and phenotype. However, in presence of Equia extracts the migration capacity of hDPSCs was higher than in presence of Ionostar Molar (p<0.05). Also, SEM studies showed a higher degree of cell attachment when Equia Forte extracts were used. Finally, EDX analysis pointed to different weight percentages of C, O and Ca ions in glass ionomer cements, while other elements such as La, Al, Si, W, Mo and F were also detected. SIGNIFICANCE: In summary, Equia Forte promoted better biological responses in hDPSCs than Ionostar Molar.