A Novel Genetic Variant in MBD5 Associated with Severe Epilepsy and Intellectual Disability: Potential Implications on Neural Primary Cilia.

Disruptions in the MBD5 gene have been linked with an array of clinical features such as global developmental delay, intellectual disability, autistic-like symptoms, and seizures, through unclear mechanisms. MBD5 haploinsufficiency has been associated with the disruption of primary cilium-related processes during early cortical development, and this has been reported in many neurodevelopmental disorders. In this study, we describe the clinical history of a 12-year-old child harboring a novel MBD5 rare variant and presenting psychomotor delay and seizures. To investigate the impact of MBD5 haploinsufficiency on neural primary cilia, we established a novel patient-derived cell line and used CRISPR-Cas9 technology to create an isogenic control. The patient-derived neural progenitor cells revealed a decrease in the length of primary cilia and in the total number of ciliated cells. This study paves the way to understanding the impact of MBD5 haploinsufficiency in brain development through its potential impact on neural primary cilia.

Scaffolds for Dentin-Pulp Complex Regeneration.

Background and Objectives: Regenerative dentistry aims to regenerate the pulp-dentin complex and restore those of its functions that have become compromised by pulp injury and/or inflammation. Scaffold-based techniques are a regeneration strategy that replicate a biological environment by utilizing a suitable scaffold, which is considered crucial for the successful regeneration of dental pulp. The aim of the present review is to address the main characteristics of the different scaffolds, as well as their application in dentin-pulp complex regeneration. Materials and Methods: A narrative review was conducted by two independent reviewers to answer the research question: What type of scaffolds can be used in dentin-pulp complex regeneration? An electronic search of PubMed, EMBASE and Cochrane library databases was undertaken. Keywords including "pulp-dentin regeneration scaffold" and "pulp-dentin complex regeneration" were used. To locate additional reports, reference mining of the identified papers was undertaken. Results: A wide variety of biomaterials is already available for tissue engineering and can be broadly categorized into two groups: (i) natural, and (ii) synthetic, scaffolds. Natural scaffolds often contain bioactive molecules, growth factors, and signaling cues that can positively influence cell behavior. These signaling molecules can promote specific cellular responses, such as cell proliferation and differentiation, crucial for effective tissue regeneration. Synthetic scaffolds offer flexibility in design and can be tailored to meet specific requirements, such as size, shape, and mechanical properties. Moreover, they can be functionalized with bioactive molecules, growth factors, or signaling cues to enhance their biological properties and the manufacturing process can be standardized, ensuring consistent quality for widespread clinical use. Conclusions: There is still a lack of evidence to determine the optimal scaffold composition that meets the specific requirements and complexities needed for effectively promoting dental pulp tissue engineering and achieving successful clinical outcomes.

Effect of restorative timing on shear bond strength of composite resin/calcium silicate-based cements adhesive interfaces.

OBJECTIVES: The aim of this study was to evaluate the shear bond strength inherent to immediate and delayed definitive composite resin restorations, over three different biomaterials. METHODS: Fifty-four aluminum blocks were randomly assigned to six experimental groups (n = 9) according to the biomaterial (Biodentine, TotalFill BC Putty, or PCM) and restoration timing (immediate or 7 days delayed). Regarding the restorative procedures, a two-step self-etch bonding system (Clearfil SE Bond) was applied and subsequently restored using a flowable composite resin (SDR). After shear bond strength testing, fracture pattern analysis was performed. The significance level was set at α = 0.05. RESULTS: Within the immediate groups, Biodentine showed the highest mean shear bond strength value, with no statistically significant differences (P > 0.05) being detected when compared with both TotalFill (P = 0.072) and PCM (P = 0.232) groups. Regarding the delayed restoration groups, Biodentine and TotalFill presented the highest mean shear bond strength value, with statistical differences when compared with PCM (P = 0.002 and P = 0.043, respectively). No statistically significant differences (P > 0.05) were verified between both immediate and delayed groups for each tested biomaterial. Also, a statistically significant association was found between the fracture type and the biomaterial (P < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that all tested biomaterials present suitable alternatives which allow to perform restorative procedures immediately after pulp capping biomaterial placement (3 or 12 min, depending on the bioactive cement), therefore requiring one single appointment to complete the procedure. Biodentine and TotalFill BC Putty have shown superior shear bond strength results when compared with PCM, within a delayed timeframe (7 days).

Regeneration of pulp-dentin complex using human stem cells of the apical papilla: in vivo interaction with two bioactive materials.

OBJECTIVES: To compare the regenerative properties of human stem cells of the apical papilla (SCAPs) embedded in a platelet-rich plasma (PRP) scaffold, when implanted in vivo using an organotypic model composed of human root segments, with or without the presence of the bioactive cements - ProRoot MTA or Biodentine. MATERIAL AND METHODS: SCAPs were isolated from third molars with incomplete rhizogenesis and expanded and characterized in vitro using stem cell and surface markers. The pluripotency of these cells was also assessed using adipogenic, chondrogenic, and osteogenic differentiation protocols. SCAPs together with a scaffold of PRP were added to the root segment lumen and the organotypic model implanted on the dorsal region of immunodeficient rats for a period of 4 months. RESULTS: Presence of SCAPs induced de novo formation of dentin-like and pulp-like tissue. A barrier of either ProRoot MTA or Biodentine did not significantly affect the fraction of sections from roots segments observed to contain deposition of hard material (P > 0.05). However, the area of newly deposited dentin was significantly greater in segments containing a barrier of Biodentine compared with ProRoot MTA (P < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: SCAPs offer a viable alternative to other dental stem cells (DSCs) in their regenerative properties when enclosed in the microenvironment of human tooth roots. The present study also suggests that the presence of bioactive materials does not hinder or impede the formation of new hard tissues, but the presence of Biodentine may promote greater mineralized tissue deposition.

Vital Pulp Therapy in Permanent Mature Posterior Teeth with Symptomatic Irreversible Pulpitis: A Systematic Review of Treatment Outcomes.

Background and Objectives: Symptomatic irreversible pulpitis in permanent mature teeth is a common indication for nonsurgical root canal treatment (NSRCT), but contemporary studies have reported on vital pulp therapy (VPT) applied in such teeth as a less invasive treatment. This systematic review assessed the outcomes of VPT, including partial and full pulpotomy performed with hydraulic calcium silicate cements (HCSCs) in permanent mature posterior teeth diagnosed with symptomatic irreversible pulpitis. Materials and Methods: The PRISMA guidelines were followed. The search strategy included PubMed®, EMBASE, Cochrane library and grey literature electronic databases. The quality assessment of the identified studies followed the Cochrane Collaboration Risk of Bias, ROBINS-I and Newcastle-Ottawa Scale tools. Results: The search of primary databases identified 142 articles, of which 9 randomized controlled trials and 3 prospective cohort studies were selected for review. The risk-of-bias was assessed as 'high' or 'serious', 'fair', and 'low' for three, seven and two articles, respectively. One to five years after VPT using HCSCs, the success rates mostly ranged from 78 to 90%. Based on two articles, the outcomes of the VPT and NSRCT were comparable at one and five years. Despite the necessity for the intra-operative pulp assessment in VPT procedures, the majority of the studies did not fully report on this step or on the time needed to achieve hemostasis. Small sample sizes, of under 23 teeth, were reported in three studies. Conclusions: The reviewed 12 articles reported favorable outcomes of the VPT performed with HCSCs in permanent mature posterior teeth with symptomatic irreversible pulpitis, with radiographic success in the range of 81 to 90%. Two articles suggested comparable outcomes of the VPT and root canal treatment. Universal case selection and outcome criteria needs to be established for VPT when considered as an alternative to NSRCT. This evidence supports the need for further research comparing longer-term outcomes of both of the treatment modalities.

Analytical protocols for separation and electron microscopy of nanoparticles interacting with bacterial cells.

An important step toward understanding interactions between nanoparticles (NPs) and bacteria is the ability to directly observe NPs interacting with bacterial cells. NP-bacteria mixtures typical in nanomedicine, however, are not yet amendable for direct imaging in solution. Instead, evidence of NP-cell interactions must be preserved in derivative (usually dried) samples to be subsequently revealed in high-resolution images, for example, via scanning electron microscopy (SEM). Here, this concept is realized for a mixed suspension of model NPs and Staphylococcus aureus bacteria. First, protocols for analyzing the relative colloidal stabilities of NPs and bacteria are developed and validated based on systematic centrifugation and comparison of colony forming unit (CFU) counting and optical density (OD) measurements. Rate-dependence of centrifugation efficiency for each component suggests differential sedimentation at a specific predicted rate as an effective method for removing free NPs after co-incubation; the remaining fraction comprises bacteria with any associated NPs and can be examined, for example, by SEM, for evidence of NP-bacteria interactions. These analytical protocols, validated by systematic control experiments and high-resolution SEM imaging, should be generally applicable for investigating NP-bacteria interactions.

From trihalomethanes chronic daily intake through multiple exposure routes to cancer and non-cancer health risk assessment: Evidence from public Portuguese indoor swimming pools facilities using a probabilistic approach.

This study aimed to estimate chronic daily intake (CDI) and to predict the attributable lifetime cancer risk (LCR) and hazard index (HI) from concurrent exposure to four trihalomethanes (THMs; chloroform, bromodichloromethane, dibromochloromethane and bromoform), via multiple exposure routes (oral ingestion, dermal contact and inhalation), among 238 non-competitive attendees of 10 Portuguese public indoor swimming pools (SPs), using a probabilistic approach based on Monte Carlo simulations. Exposure parameters of study participants were collected via questionnaires and THMs levels in SPs water were determined according the respective normative standards. The CDI for total THMs calculated for male and female participants considering all routes was 7.52 and 8.97 mg/kg/day, respectively. SP attendees presented higher CDI through inhalation than via the other two exposure routes, and chloroform was the compound contributing the most to total THMs CDI. The risk analysis indicated that the total LCR and HI from the targeted THMs were higher than the negligible risk levels (1 × 10-6 and 1, respectively) in the scenarios examined (central tendency exposure and reasonable maximum exposure), and the health risk for females was slightly higher than for males. This study suggests that there are possible adverse health risks, thus, to protect pool attendees, adequate mitigation measures, and comprehensive regulatory guidelines on individual THMs concentrations are needed.

Influence of Preoperative Pulp Inflammation in the Outcome of Full Pulpotomy Using a Dog Model.

INTRODUCTION: This study aimed to evaluate the impact of preoperative pulp inflammation on the histologic outcome of full pulpotomy performed in mature permanent posterior teeth using 4 different biomaterials. METHODS: Five beagle dogs (providing a total of 120 roots) were selected. Dentin exposure was performed in teeth from the second and third quadrants. One week later, full pulpotomy procedures were performed using 4 different bioactive materials (ProRoot MTA [MTA], TotalFill BC Putty [BC], Biodentine [BIO], and an experimental cement [ie, pulp capping material]). The hemostasis time was registered. After 14 weeks, the animals were killed. Pulp-dentin tissues were histologically and radiographically assessed. The significance level was set at .05. RESULTS: Teeth with previously exposed dentin revealed a statistically significant increase in the time required to achieve hemostasis (P < .001), therefore confirming the pulp inflammation status induced by 1-week exposure of occlusal dentin before performing full pulpotomy. There was no radiographic evidence of root resorption, periapical radiolucency, or lamina dura alterations. No statistically significant differences were observed between normal and inflamed pulp regardless of the evaluated histologic parameters. Moreover, histologic data concerning calcified barrier formation and the pulp tissue response show better results for BIO without statistical differences compared with MTA or BC (P > .05). The pulp capping material presented a lower performance, with statistically significant differences being detected in regard to the remaining 3 tested materials (P < .001). CONCLUSIONS: Radiographic and histologic outcomes of full pulpotomy are not jeopardized by short-term preoperative pulp inflammation. Moreover, BIO, MTA, and BC cements present suitable alternatives to be used as pulp capping agents.

Subcutaneous Implantation Assessment of New Calcium-Silicate Based Sealer for Warm Obturation.

Calcium silicate-based sealers were recently introduced as a new class of endodontic sealers, with potential further benefits due to their bioactivity. The aim of this study was to evaluate the biocompatibility of two new hydraulic calcium silicate-based sealers, TotalFill BC Sealer (FKG, La Chaux-des-Fonds, Switzerland) and TotalFill BC Sealer HiFlow (FKG, La Chaux-des-Fonds, Switzerland) through subcutaneous implantation in connective tissue of rats. Subcutaneous implantation was performed in 16 young Wistar rats. Four polyethylene tubes were implanted in each animal, one empty to serve as a control, and three filled with tested sealers: AH Plus as reference (Dentsply DeTrey, Konstanz, Germany), TotalFill BC Sealer (BC) and TotalFill BC Sealer HiFlow (HiFlow). Eight rats were euthanized at 8 days and the remaining eight at 30 days. Hematoxylin-eosin staining was used to score the inflammatory reaction, macrophage infiltrate and to measure the thickness of the fibrous capsule. von Kossa staining was performed to evaluate the mineralization level. Kruskal-Wallis test followed by Dunn's post hoc test was used to analyze non-parametric data. To analyze the influence of the implantation time within each material, a Mann-Whitney U test was performed. At eight days post-implantation, AH Plus induced a more intense inflammatory reaction when compared both with the control (p ≤ 0.001) and BC (p ≤ 0.01). HiFlow presented a higher score of macrophage infiltrate than control (p ≤ 0.01) and BC (p ≤ 0.05). The fibrous capsule thickness in this period was significantly higher for the BC group when compared to control (p ≤ 0.01) and AH Plus (p ≤ 0.05). The mineralization potential was higher for the HiFlow group when compared with the control (p ≤ 0.001) and AH Plus (p ≤ 0.001). At 30 days post-implantation, the score for the inflammatory reaction remained higher for the AH Plus group when compared both to control (p ≤ 0.01) and BC (p ≤ 0.001). The macrophage infiltrate of the HiFlow was significantly higher than control (p ≤ 0.001) and AH Plus groups (p ≤ 0.01), additionally, the fibrous capsule of the BC (p ≤ 0.001) and HiFlow (p ≤ 0.01) groups were both thicker than control. Mineralization potential was observed only on BC (p ≤ 0.05) and HiFlow groups (p ≤ 0.001), when compared to control). BC exhibited the best biocompatibility performance of all tested sealers and HiFlow provided the greatest induction of mineralized tissues. Both TotalFill BC Sealer and TotalFill BC Sealer HiFlow are biocompatible and show potential bioactivity when implanted in the subcutaneous tissue. Bioactivity was not found in AH Plus.

Biocompatibility of a bioceramic silicone-based sealer in subcutaneous tissue.

This study evaluated the biocompatibility of a new silicone-based sealer (GuttaFlow Bioseal) in rat subcutaneous tissue and compared the results with those for GuttaFlow2 and AH Plus. Each of 16 Wistar rats received four subcutaneous tissue implants, namely, GuttaFlow Bioseal, GuttaFlow2, AH Plus, and one empty polyethylene tube. Eight rats were euthanized at day 8 and the remaining eight at day 30. Histological sections were stained with haematoxylin and eosin and analysed with a light microscope. Scores were established for inflammatory reaction, macrophage infiltrate, thickness of the fibrous capsule, and vascular changes. Differences between groups were assessed by using the Friedman test with Bonferroni correction. Histological analysis showed that GuttaFlow Bioseal had the lowest inflammatory reaction of all tested sealers at day 8. At day 30, the silicone-based sealers had similar inflammation profiles, but inflammation scores were nonsignificantly higher for AH Plus than for the negative control. The inflammatory reaction decreased from day 8 to day 30 in all sealers. GuttaFlow Bioseal had the most macrophage infiltrate. Under the present experimental conditions, GuttaFlow Bioseal induced limited inflammatory reactions at days 8 and 30, and initial inflammatory reactions to GuttaFlow2 and AH Plus subsided within 30 days. All tested sealers exhibited satisfactory biocompatibility at day 30 after subcutaneous implantation.

Effects of a New Bioceramic Material on Human Apical Papilla Cells.

BACKGROUND: The development of materials with bioregenerative properties is critically important for vital pulp therapies and regenerative endodontic procedures. The aim of this study was to evaluate the cytocompatibility and cytotoxicity of a new endodontic biomaterial, PulpGuard, in comparison with two other biomaterials widely used in endodontic procedures, ProRoot Mineral Trioxide Aggregate (MTA) and Biodentine. METHODS: Apical papilla cells (APCs) were isolated from third molars with incomplete rhizogenesis from patients with orthodontic indication for dental extraction. Cultured APCs were incubated for 24, 48, or 72 h with different dilutions of eluates prepared from the three materials. Cellular viability, mobility, and proliferation were assessed in vitro using the Alamar Blue assay and a wound-healing test. The cells were also cultured in direct contact with the surface of each material. These were then analyzed via Scanning Electron Microscopy (SEM), and the surface chemical composition was determined by Energy-Dispersive Spectroscopy (EDS). RESULTS: Cells incubated in the presence of eluates extracted from ProRoot MTA and PulpGuard presented rates of viability comparable to those of control cells; in contrast, undiluted Biodentine eluates induced a significant reduction of cellular viability. The wound-healing assay revealed that eluates from ProRoot MTA and PulpGuard allowed for unhindered cellular migration and proliferation. Cellular adhesion was observed on the surface of all materials tested. Consistent with their disclosed composition, EDS analysis found high relative abundance of calcium in Biodentine and ProRoot MTA and high abundance of silicon in PulpGuard. Significant amounts of zinc and calcium were also present in PulpGuard discs. Concerning solubility, Biodentine and ProRoot MTA presented mild weight loss after eluate extraction, while PulpGuard discs showed significant water uptake. CONCLUSIONS: PulpGuard displayed a good in vitro cytocompatibility profile and did not significantly affect the proliferation and migration rates of APCs. Cells cultured in the presence of PulpGuard eluates displayed a similar profile to those cultured with eluates from the widely used endodontic cement ProRoot MTA.

Is the chlorophyll derivative Zn(II)e6Me a good photosensitizer to be used in root canal disinfection?

BACKGROUND: The aim of this study was to assess antimicrobial efficacy and cytotoxic outcomes of a chlorophyll based photosensitizer (PS) Zn(II)chlorin e6 methyl ester (Zn(II)e6Me), when applied to human dentin discs and root blocks infected with 48 h biofilms. The results were compared with the ones obtained with FotoSan® (commercial Toluidine Blue O formulation) and 3% sodium hypochlorite (NaOCl). METHODOLOGY: Dentin and root blocks were infected with mixed biofilms of Enterococcus faecalis and Candida albicans; exposed for 15 min to 0.1 mg/mL of Zn(II)e6Me or Fotosan® and then irradiated with red light (627 nm, 75 mW, 3150 J/cm2) for 90 s or treated with NaOCl. Biofilm removal was calculated with safranin red assay and biofilm cells viability with XTT® assay. The PSs cytotoxicity was evaluated over human apical papilla primary cell line (hAPCs) with AlamarBlue® assay and cell morphology assessed with widefield fluorescence microscopy. RESULTS: At dentin discs, the chlorophyll derivative performed better in biofilm removal (59.1%) than FotoSan® agent (57.5%), however, with lower efficacy than NaOCl (68.1%) (P = 0.0185). Conversely, at the root block, the chlorophyll Zn(II)e6Me (79.7%) present better antimicrobial efficacy than NaOCl (75.5%) and the disinfection pattern was more consistent at inner and outer samples for the former. No dark or photoinduced cytotoxic outcomes were detected for Zn(II)e6Me over human cells at 24 and 48 h when compared with other PSs (FotoSan®, Rose Bengal and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin - TMPyP). CONCLUSIONS: The chlorophyll derivative Zn(II)e6Me showed adequate antimicrobial efficacy, performing better in mixed biofilm removal than FotoSan® in both experimental conditions. No cytotoxic effects over human apical papilla cells were identified for this chlorophyll derivative, subsequently it deserves further evaluation to assess its potential use in endodontic therapy.