Synchronized Microbubble Photodynamic Activation to Disinfect Minimally Prepared Root Canals.

INTRODUCTION: The purpose of this study was to evaluate the antimicrobial efficacy of a novel irrigation strategy using synchronized microbubble photodynamic activation (SYMPA) in a minimally prepared single canal. METHODS: Single-canal mandibular incisors were inoculated with Enterococcus faecalis for 3 weeks and randomly allocated to 4 groups based on the irrigation protocols: (1) control (saline), (2) conventional needle irrigation (CI), (3) ultrasonic-assisted irrigation (UI), and (4) irrigation with SYMPA. The first 3 groups were instrumented to size 25.07v (WaveOne Gold Primary; Dentsply Sirona, Johnson City, TN), and the SYMPA group was minimally prepared to size 20.07v (WaveOne Gold Small, Dentsply Sirona). The apical 5 mm was resected for microbiological assessment using the culture technique (colony-forming unit), adenosine-5'-triphosphate-based viability assay (relative luminescence units), and the percentage of live bacteria using confocal laser scanning microscopy. RESULTS: Log colony-forming units from the UI (2.37 ± 0.66) and SYMPA (2.21 ± 0.86) groups showed a reduction compared with the control (5.16 ± 0.75) and CI (4.08 ± 1.19) groups. Relative luminescence unit reduction was significant for UI (619.08 ± 352.78) and SYMPA (415.25 ± 329.51) compared with the control (1213.2 ± 880.03) (P < .05). The percentage of live bacteria was significantly lower in the UI and SYMPA groups compared with the control and CI groups. Although higher microbial reduction was observed in SYMPA compared with UI, there was no statistical significance (P > .05). CONCLUSION: SYMPA in minimally prepared canals showed significant antimicrobial efficacy. The novel irrigation strategy using SYMPA could be an effective disinfection strategy for minimally prepared root canals.

Deciphering Stem Cell From Apical Papilla-Macrophage Choreography Using a Novel 3-dimensional Organoid System.

INTRODUCTION: Immune cell-mesenchymal stem cell crosstalk modulates the process of repair and regeneration. In this study, a novel heterogeneous cell containing a matrix-based 3-dimensional (3D) tissue construct was used to study the interactions between stem cells from apical papilla (SCAPs) and macrophage for a comprehensive understanding on the cellular signaling mechanisms guiding inflammation and repair. METHODS: SCAPs and macrophages were seeded with collagen in 3D-printed molds to generate self-assembled tissue constructs, which were exposed to 3 conditions: no stimulation, lipopolysaccharide (LPS), and interleukin (IL)-4 from 0 to 14 days. Specimens from each group were evaluated for cellular interactions, inflammatory mediators (IL-1ß, tumor necrosis factor [TNF]-α, macrophage-derived chemokine [MDC], macrophage inflammatory protein [MIP]-1ß, monocyte chemoattractant protein [MCP]-1, IL-6, IL-8, transforming growth factor [TGF]-ß1, IL-1RA, IL-10), expression of surface markers (CD80, 206), transcription factors (pSTAT1, pSTAT6), and SCAP differentiation markers (dentin sialophosphoprotein [DSPP], dentin matrix acidic phosphoprotein 1 [DMP-1], and alizarin red) using confocal laser scanning microscopy and multiplex cytokine profiling from 2 to 14 days. RESULTS: SCAP and macrophages displayed a cytokine-mediated interaction and differentiation characteristics. The increased pro-inflammatory cytokines/chemokines, IL-1ß, TNF-α, MDC, and MIP-1ß, in the earlier phase followed by the higher ratio of pSTAT6/pSTAT1 and decreased CD206 (P < .05), indicated a distinct polarization behavior in macrophages during repair in the LPS group. Conversely, the equal ratio of pSTAT6/pSTAT1, late increase in CD206, and amplified secretion of IL-1RA, IL-10, and TGF-ß1 (P < .05) in the anti-inflammatory environment, directed alternative macrophage polarization, promoting SCAP differentiation and tissue modeling in IL-4 group. CONCLUSIONS: The novel 3D organoid system developed in this study allowed a comprehensive analysis of the SCAP-macrophage interactions during inflammation and healing, providing a deeper insight on the periapical dynamics of the immature tooth.

Effect of taxifolin and epigallocatechin-3-gallate on biomineralization potential of stem cells from dental apical papilla.

OBJECTIVE: Considering the variety of pharmacological activities and the potential to mediate biomineralization, the flavonoids taxifolin and epigallocatechin-3-gallate (EGCG) could be explored as biomolecules in scaffolds for regenerative endodontic procedures. The aim of this study was to evaluate the effect of taxifolin and EGCG on the cell viability, differentiation, and expression of biomineralization markers in stem cells from the apical papilla. DESIGN: Stem cells from the apical papilla were exposed to single or continuous treatments with taxifolin at 200, 100 and 50 µM and EGCG at 50, 25 and 12.5 µM for 48 h, 8 and 14 days, in regular or mineralizing media. Cell viability, alkaline phosphatase activity and calcium deposition were analyzed using resazurin, p-nitrophenylphosphate and alizarin-based assays. RESULTS: None of the flavonoid groups affected cell viability at 48 h, however at 8 and 14 days, Taxifolin 200 µM and EGCG 50 µM were cytotoxic. Cells did not express alkaline phosphatase activity when grown in regular medium, even in the presence of flavonoids. Alkaline phosphatase activity and biomineralization potential were higher in cells treated with Taxifolin 50 µM and EGCG 12.5 µM. CONCLUSION: Taxifolin and EGCG exhibited a concentration, time, and therapeutic mode dependent bioactivity on stem cells from the apical papilla. Both flavonoids at the lower concentrations tested exhibited cytocompatibility and increased expression of mineralization markers in the presence of mineralizing agents.

Local Immunomodulatory Effects of Intracanal Medications in Apical Periodontitis.

The immune system is an extremely complex biological network that plays a crucial role in the hemostasis of periapical tissue, pathogenesis of apical periodontitis (AP), and periapical tissue healing. The successful elimination of microbial infections remains a significant challenge, mostly because of the ever-growing development of antimicrobial-resistant pathogens. The bacterial endurance in the root canal system contributes to features ranging from altered posttreatment healing to exacerbation of chronic periradicular immune response, which compromise the outcome of endodontic treatment. A highly effective strategy for combating infectious diseases and the associated inflammation-mediated tissue damage is to modulate the host immune response in conjunction with antimicrobial therapy. There are several medications currently used in endodontic treatment; however, they suffer various levels of microbial resistance and do not deliver all the required characteristics to simultaneously address both intracanal bacteria and periapical inflammation. The interaction of antimicrobial agents with the immune system can impact its function, leading to immune-suppressive or immune-stimulatory effects. The group of nonconventional antimicrobial medications, such as antimicrobial peptides, propolis, and nanomaterials, are agents that provide strong antimicrobial effectiveness and concomitant immunomodulatory and/or reparative effect without any host tissue damages. In this review, we provide an overview of local immune modulation in AP and a comprehensive review of the immunomodulatory effect of antimicrobial intracanal medications applied in endodontics with specific emphasis on the antimicrobial nanomaterial-based approaches that provide immunomodulatory potential for successful clinical deployment in endodontics.

Proteomic profiling reveals engineered chitosan nanoparticles mediated cellular crosstalk and immunomodulation for therapeutic application in apical periodontitis.

Macrophages (MQ) are major constituents of chronically inflamed periapical tissues in apical periodontitis. This study aimed to investigate the immunomodulatory effect of engineered bioactive chitosan-based nanoparticles (CSnp) antibiofilm medication on MQ cocultured with periodontal ligament fibroblasts (PdLF). Cells viability, spreading, PdLF migration, and intracellular CSnp uptake were characterized. Tandem Mass Tag-based proteomics was applied to analyze MQ global protein expression profiles after interaction with Enterococcus faecalis biofilm, CSnp-treated biofilm, and CSnp. Secreted inflammatory mediators were analyzed. Following bioinformatics analyses, candidate proteins were validated via targeted proteomics. CSnp maintained cells viability, increased MQ spreading, and PdLF migration (p < 0.05). Transmission electron micrographs demonstrated CSnp internalization via macropinocytosis, clathrin-mediated endocytosis, and phagocytosis. Proteomic analysis revealed that CSnp-treated biofilm upregulated proteins (>1.5-folds, p < 0.05) showed functional enrichment in the pathway of metal sequestration by antimicrobial proteins, while downregulated proteins showed enrichment in ferroptosis. CSnp upregulated proteins exhibiting antioxidant and immunoregulatory properties. Upregulation of SERPINB1 by CSnp (>1.5-folds, p < 0.05) was validated. CSnp-treated biofilm reduced pro-inflammatory IL-1ß and nitric oxide but enhanced anti-inflammatory IL-10 and TGF-ß1 (p < 0.05). Internalized engineered bioactive CSnp reprogrammed MQ proteomic and cytokine profiles to modulate biofilm-mediated inflammation, and prompted PdLF migration, emphasizing its potential to regulate healing process in the treatment of apical periodontitis.

Engineered Chitosan-based Nanoparticles Modulate Macrophage-Periodontal Ligament Fibroblast Interactions in Biofilm-mediated Inflammation.

INTRODUCTION: Crosstalk between immune cells and tissue-resident cells regulates the pathophysiology and posttreatment healing of apical periodontitis. This investigation aimed to understand the influence of residual root canal biofilm on macrophage (MQ)-periodontal ligament fibroblast (PdLF) interaction and evaluate the effect of engineered chitosan-based nanoparticles (CSnp) on MQ-PdLF interactions in residual biofilm-mediated inflammation. METHODS: Six-week-old Enterococcus faecalis biofilms in root canal models were disinfected conventionally using sodium hypochlorite alone or followed by calcium hydroxide medication or CSnp dispersed in carboxymethylated chitosan (CMCS). The effect of the treated biofilms (n = 25/group) on the inflammatory response of THP-1-differentiated MQ monoculture versus coculture with PdLF was evaluated for cell viability, MQ morphometric characterization, inflammatory mediators (nitric oxide, tumor necrosis factor alpha, interleukin [IL]-1 beta, IL-1RA, IL-6, transforming growth factor beta 1 [TGF-ß1], and IL-10), and the expression of transcription factors (pSTAT1/pSTAT6)/cluster of differentiation markers (CD80/206) after 24, 48, and 72 hours of interaction. PdLF transwell migration was evaluated after 8 and 24 hours. Unstimulated cells served as the negative control, whereas untreated biofilm was the positive control. RESULTS: Biofilm increased nitric oxide and IL-1ß but suppressed IL-10, IL-1RA, and PdLF migration with significant cytotoxic effects. CSnp/CMCS reduced nitric oxide and IL-1ß (P < .01) while maintaining ≥90% cell survival up to 72 hours with evident M2-like MQ phenotypic changes in coculture. CSnp/CMCS also increased the IL-1RA/IL-1ß ratio and enhanced TGF-ß1 production over time (P < .05, 72 hours). In coculture, CSnp/CMCS showed the highest IL-10 level at 72 hours (P < .01), reduced the pSTAT1/pSTAT6 ratio, and enhanced PdLF migration (P < .01, 24 hours). CONCLUSIONS: CSnp/CMCS medication facilitated MQ switch toward M2 (regulatory/anti-inflammatory) phenotype and PdLF migration via paracrine signaling.

A Novel Self-Mineralizing Antibacterial Tissue Repair Varnish to Condition Root-end Dentin in Endodontic Microsurgery.

INTRODUCTION: A novel 2-part varnish system containing chitosan nanoparticles (part 1) and chitosan-grafted hydroxyapatite precursor nanocomplex (part 2) was developed to condition the root-end dentin using the principle of biomineralization while rendering both antimicrobial efficacy and bioactivity. This in vitro study aimed to characterize and assess the effectiveness of the chitosan nanoparticles and chitosan-grafted hydroxyapatite precursor nanocomplex containing self-mineralizing antibacterial tissue repair varnish to condition as well as seal root-end dentin during endodontic microsurgery. METHODS: In phase 1, the antibacterial properties and cellular response of the varnish were characterized. The antibacterial activity and cellular responses were evaluated using Enterococcus faecalis and periodontal ligament (PDL) fibroblasts, respectively. In phase 2, a resected root-end model was used to apply the 2-part varnish and examine the dentin-varnish interface using transmission electron microscopy. The percentage of root end sealed with time was determined using scanning electron microscopy (n = 6/time point). Statistical analysis was performed using 1-way analysis of variance where applicable. RESULTS: The part 1 and 2 of the varnish displayed significant antibacterial activity and reduced bacterial adherence/survival (P < .01). The attachment and spreading of PDL fibroblasts on the varnish-conditioned dentin was enhanced compared with unconditioned dentin (P < .01). In the resected root-end model, the 2-part varnish displayed a biomineralized varnish layer with close interaction with the subsurface dentin. Root-end coverage with the biomineralized layer increased with incubation time (P < .01), reaching approximately 95% coverage after 24 hours. CONCLUSIONS: The 2-part varnish system effectively conditioned/sealed the root end with a biomineralized layer while reducing bacterial load and promoting PDL fibroblast attachment. This therapeutic modification of root-end dentin could provide optimal conditions to enhance healing and improve prognosis in teeth with root-end cracks after endodontic microsurgery.

Bioactive molecule carrier systems in endodontics.

INTRODUCTION: Bioactive molecule carrier systems (BACS) are biomaterial-based substrates that facilitate the delivery of active signaling molecules for different biologically based therapeutic applications, which include regenerative endodontic procedures. Tissue regeneration or organized repair in regenerative endodontic procedures is governed by the dynamic orchestration of interactions between stem/progenitor cells, bioactive molecules, and extracellular matrix. BACS aid in mimicking some of the complex physiological processes, overcoming some of the challenges faced in the clinical translation of regenerative endodontic procedures. AREAS COVERED: This narrative review addresses the role of BACS in stem/progenitor cell proliferation, migration, and differentiation with the application for dentin-pulp tissue engineering both in vitro and in vivo. BACS shield the bioactivity of the immobilized molecules against environmental factors, while its design allows the pre-programmed release of bioactive molecules in a spatial and temporal-controlled manner. The polymeric and non-polymeric materials used to synthesize micro and nanoscale-based BACS are reviewed. EXPERT OPINION: Comprehensive characterization of well-designed and customized BACS is necessary to be able to deliver multiple bioactive molecules in spatiotemporally controlled manner and to address the release kinetics required for potential in vivo application. This warrants further laboratory-based experiments and rigorous clinical investigations to enable their clinical translation for regenerative endodontic procedures.

Antibiofilm and Immune Response of Engineered Bioactive Nanoparticles for Endodontic Disinfection.

The biological aim of root canal treatment is to facilitate periapical tissue healing following endodontic therapy. This study aimed to develop an organotypic infected root canal model to understand the interaction of bacterial biofilm with macrophages and study the therapeutic effect of engineered bioactive chitosan nanoparticles (CSnp) on macrophages. Ex-vivo experiments were conducted in two phases; Phase-1: Enterococcus faecalis biofilms (two and six weeks old) developed in organotypic root canal model were used to characterize residual biofilm after conventional chemical treatment alone and combined with CSnp utilizing Confocal Laser Scanning Microscopy, Scanning Electron Microscopy and colony-forming units from pulverized dentin. Phase-2: The interaction of post-treatment biofilm and RAW macrophages was evaluated regarding pro/anti-inflammatory markers, cell viability and spreading at 24, 48 and 72 h. Compared to conventionally disinfected six-week-old biofilm, CSnp resulted in less viable bacteria (p < 0.01). Scanning electron micrographs demonstrated disruption of the biofilm. CSnp exhibited less residual bacterial load in pulverized dentin (p < 0.001). Macrophage interaction with CSnp-treated biofilm reduced proinflammatory markers (nitric oxide, TNF-α, IL-1ß, and IL-6), increased anti-inflammatory marker (TGF-ß1) and enhanced cell survival and spreading over time (p < 0.01 at 72 h). Engineered chitosan nanoparticles concurrently inactivated biofilm and altered the inflammatory response of macrophages that would promote healing.