3D Organoids for Regenerative Endodontics.

Apical periodontitis is the inflammation and destruction of periradicular tissues, mediated by microbial factors originating from the infected pulp space. This bacteria-mediated inflammatory disease is known to interfere with root development in immature permanent teeth. Current research on interventions in immature teeth has been dedicated to facilitating the continuation of root development as well as regenerating the dentin-pulp complex, but the fundamental knowledge on the cellular interactions and the role of periapical mediators in apical periodontitis in immature roots that govern the disease process and post-treatment healing is limited. The limitations in 2D monolayer cell culture have a substantial role in the existing limitations of understanding cell-to-cell interactions in the pulpal and periapical tissues. Three-dimensional (3D) tissue constructs with two or more different cell populations are a better physiological representation of in vivo environment. These systems allow the high-throughput testing of multi-cell interactions and can be applied to study the interactions between stem cells and immune cells, including the role of mediators/cytokines in simulated environments. Well-designed 3D models are critical for understanding cellular functions and interactions in disease and healing processes for future therapeutic optimization in regenerative endodontics. This narrative review covers the fundamentals of (1) the disease process of apical periodontitis; (2) the influence and challenges of regeneration in immature roots; (3) the introduction of and crosstalk between mesenchymal stem cells and macrophages; (4) 3D cell culture techniques and their applications for studying cellular interactions in the pulpal and periapical tissues; (5) current investigations on cellular interactions in regenerative endodontics; and, lastly, (6) the dental-pulp organoid developed for regenerative endodontics.

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.

Engineering a Novel Stem Cells from Apical Papilla-Macrophages Organoid for Regenerative Endodontics.

INTRODUCTION: A 3-dimensional (3D) tissue construct with a heterogeneous cell population is critical to understand the interactions between immune cells and stem cells from the apical papilla (SCAPs) in the periapical region for developing treatment strategies in regenerative endodontics. This study aimed to develop and characterize a 3D tissue construct with a binary cell system for studying the interactions between SCAPs and macrophages in the presence of lipopolysaccharide (proinflammatory) and interleukin 4 (anti-inflammatory) environments. METHODS: SCAPs and macrophages were seeded in the 3D-printed dumbbell-shaped molds to generate tissue constructs with a binary cell population. Two experimental (lipopolysaccharide and interleukin 4) and control (non-stimulation) conditions were applied to the tissue constructs to determine the characteristics of the tissue construct, the volume of viable cells, and their morphology using confocal laser scanning microscopy from a 0- to 7-day period. Experiments were conducted in triplicate, and data were analyzed with trend analysis and 2-way analysis of variance at a significance of P < .05. RESULTS: The tissue constructs revealed distinct SCAP-macrophage interaction in pro/anti-inflammatory environments. SCAPs displayed characteristic self-organization as a cap-shaped structure in the tissue construct. The growth of cells and cell-to-cell and cell-to-matrix interactions resulted in 70% and 30% decreased dimension of the tissue graft on the SCAP side and macrophage side, respectively, at day 7 (P < .0001). The tissue environments influenced SCAP-macrophage interactions, resulting in an altered viable cell volume (P < .05), morphology, and structural organization. CONCLUSIONS: This study developed and characterized an apical papilla organoid in a 3D collagen-based tissue construct for studying SCAP-macrophage crosstalk in tissue regeneration as well as repair.

Microtissue Engineering Root Dentin with Photodynamically Cross-linked Nanoparticles Improves Fatigue Resistance of Endodontically Treated Teeth.

INTRODUCTION: Microtissue engineering root canal dentin with biopolymeric nanoparticles has the potential to improve mechanical properties of iatrogenically compromised root dentin. This study aims to characterize the surface mechanical property, bulk biomechanical response, and fatigue resistance of microtissue-engineered root dentin using photodynamically (photodynamic-activated [PDA]) cross-linked chitosan nanoparticles (CSnps). METHODS: Experiments were conducted in 3 parts: part 1, root canal dentin sections were subjected to nanoindentations before/after treatment with CSnps and chemically (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide [EDC] cross-linked CSnps) and photodynamically cross-linked CSnps to determine the properties of treated surfaces (n = 84 points/group); part 2, root canal dentin specimens treated with PDA cross-linked CSnps were subjected to strain analysis using customized moiré interferometry (n = 5/group); and part 3, root canal dentin specimens treated with EDC cross-linked CSnps, PDA cross-linked CSnps, and instrumented controls were tested using an accelerated fatigue loading protocol to evaluate the sustained loads and cycles at failure (n = 15/group). Data were analyzed using the paired sample t test, trend analysis, and Kaplan-Meier with log-rank tests at a significance of .05 in each experiment. RESULTS: Root dentin microtissue engineered with PDA cross-linked CSnps showed a 16.8% increase in elastic modulus and a conspicuous decrease in strain distribution in cervical root dentin (P < .01). There was a significant reduction in the tensile strain formed at the apical region of the instrumented root dentin after treatment (P < .05). Survival analysis showed a statistically significant difference (P < .05) among evaluated conditions in fatigue resistance (ie, PDA cross-linked CSnps > EDC cross-linked CSnps > control). CONCLUSIONS: This study highlighted the potential of root canal dentin microtissue engineering with PDA cross-linked CSnps to diminish radicular strain distribution and improve resistance to fatigue loads in endodontically treated teeth.

Novel Activated Microbubbles-based Strategy to Coat Nanoparticles on Root Canal Dentin: Fluid Dynamical Characterization.

INTRODUCTION: Activated microbubbles (MBs) have the potential to deliver nanoparticles in complex microspaces such as root canals. The objective of the study is to determine the fluid dynamical parameters associated with ultrasonic, sonic, and manual activation of MBs in simulated root canals and to assess the effectiveness of surface coating formed by delivering chitosan nanoparticles using activated MBs within root canals in extracted teeth. METHODS: In stage 1, polydimethylsiloxane models were fabricated to determine the physical effects of MBs agitated manually (MM), sonically (MS), and ultrasonically (MU). Spherical tracer particles were used to visualize and record the fluid motion using an inverted microscope linked to a high-speed camera. The velocity, wall stress, and penetration depth were analyzed at regions of interest. In stage 2, 35 extracted human incisors were divided into 7 groups to evaluate the effectiveness of chitosan nanoparticle delivery using activated MBs (MM, MS, and MU groups). Field emission scanning electron microscopy and energy-dispersive X-rays were used to characterize the nanoparticle coating on root canal dentin and the degree of dentinal tubule occlusion. RESULTS: In stage 1, velocity, wall stress, and penetration depth increased significantly in the MB groups compared with the control (P < .01). In stage 2, 70% of the dentin surface was coated, and 65% of the dentinal tubule was occluded with nanoparticle-based coating in the MM, MU, and water ultrasonic groups. Element analysis displayed the presence of dentin smear on the root canal surface for the MU and water ultrasonic groups. CONCLUSIONS: Activated MBs enhanced fluid dynamical parameters when compared with water in simulated root canal models. Manual activation of MBs resulted in uniform and significant nanoparticle-based surface coating and tubule blockage in root canal dentin without dentin smear formation.

Biomechanical Effects of Bonding Pericervical Dentin in Maxillary Premolars.

INTRODUCTION: Pericervical dentin (PCD) loss may increase root fracture propensity in root-filled teeth. This study evaluated the impacts of bonding PCD with composite resin (CR) on radicular microstrain distribution and load at failure of root-filled maxillary premolars. METHODS: Ten single-canal maxillary premolars decoronated 2 mm coronal to the cementoenamel junction (CEJ) had canals enlarged with ProTaper Universal instruments (Dentsply Tulsa Dental Specialties, Tulsa, OK) to F3. They were root filled with gutta-percha (GP) to the CEJ and restored with Cavit (3M Deutschland GmbH, Neuss, Germany) (GP group, n = 5) or 6 mm apical to the CEJ and restored with bonded CR to simulate bonding of PCD (bonded PCD group, n = 5). Digital moiré interferometry was used to evaluate pre- and postoperative whole-field microstrain distribution in the root dentin under physiologically relevant loads (10-50 N). Another 30 premolars, similarly treated as groups 1 and 2 or left untreated as controls (n = 10/group), were subjected to cyclic loads (1.2 million cycles, 45 N, 4 Hz) followed by uniaxial compressive load to failure. Mechanical data were analyzed with 1-way analysis of variance and the post hoc Tukey test at a 5% level of significance. RESULTS: Microstrain distribution showed bending and compressive patterns at the coronal and apical root dentin, respectively. In the GP group, microstrain distribution was unaltered. In the bonded-PCD group, different microstrain distribution suggested stiffening at the PCD. The load at failure did not differ significantly for the GP, bonded PCD, and control groups (P > .05). CONCLUSIONS: CR bonding of PCD might impact the biomechanical responses in maxillary premolar roots at low-level continuous loads. The effect of this impact on root fracture loads when subjected to cyclic load warrants further investigation.

Residual Microstrain in Root Dentin after Canal Instrumentation Measured with Digital Moiré Interferometry.

INTRODUCTION: Residual microstrain influences the resistance to crack propagation in a biomaterial. This study evaluated the residual microstrain and microdefects formed in dentin after canal instrumentation in teeth maintained in hydrated and nonhydrated environments. METHODS: Canals of 18 extracted human premolars with single-root canals were instrumented in accordance with 3 groups: the ProTaper Universal (Dentsply Maillefer, Ballaigues, Switzerland) group: ProTaper Universal (S1, S2, F1, and F2) used in rotation, the WaveOne Primary (Dentsply Maillefer) group: the WaveOne (Primary) used in reciprocal motion, and the control group: hand files. Half the specimens (3/group) were maintained in deionized water (hydrated) and half in ambient relative humidity conditions (22°C, 55% RH) for 72 hours (nonhydrated). Customized high-sensitivity digital moiré interferometry was used to qualitatively evaluate pre- and postinstrumentation dentinal microstrain. Subsequently, specimens were examined for dentinal microdefects with micro-computed tomographic imaging and polarized light microscopy. RESULTS: Digital moiré interferometry showed only minor changes in postinstrumentation microstrain in hydrated dentin in all groups, suggestive of a stress relaxation behavior. Nonhydrated dentin in all groups showed localized concentration of postinstrumentation microstrain, which appeared higher in the WaveOne group than in the other groups. No dentinal microdefects were detected by micro-computed tomographic imaging and polarized light microscopy in hydrated and nonhydrated specimens in all groups. CONCLUSIONS: This study suggested that the biomechanical response of root dentin to instrumentation was influenced by hydration. Reciprocating, rotary, and hand instrumentation of well-hydrated roots did not cause an increase in residual microstrain or the formation of microdefects in root dentin.

Repair of a perforating internal resorption: two case reports.

Internal resorption is a rare condition in permanent teeth, and may result from trauma, caries, or restorative procedures. Internal resorption is usually asymptomatic and is first identified as a round-shaped enlargement of a root-canal space on routine radiographs. Large resorption defects may result in penetration of the tooth into the periodontium through the cementum. The gold-standard treatment consists of debridement and obturation of the pulp space, sealing of the external communication, and restoration of the normal function of the tooth through a nonsurgical or surgical method. In this case presentation, we report on two methods for repair of internal resorption with perforation. The first method consisted of treating the lower right second premolar by conventional endodontic therapy under a microscope, followed by repair with mineral trioxide aggregate. The second method consisted of surgical treatment of the upper right central incisor. The choice of treatment depends on the size of the perforation, its location, and the ability to approach it for repair.

Organic thin-film-transistor Au/poly(3-hexylthiophene)/ (bilayer dielectrics)/Si having carbon nanotubes chemically bonded to poly(3-hexylthiophene) in the active layer.

Using poly(3-hexylthiophene) (P3HT) covalently bonded with carbon nanotubes (CNT) as an active layer in a bottom-gate/top contact, Au/(P3HT)/(bilayer dielectric)/Si OTFT device has resulted in an enhanced charge transport. The CNTs were firstly functionalized via ligand exchange with ferrocene, next lithiated by sec-butyllithium (s-BuLi) and then linked anionically with P3HT which has been synthesized via the modified Grignard metathesis method. Compared to the pristine P3HT, the CNTs-containing P3HT composite material has a higher energy level of HOMO and a smaller electrochemical bandgap E(g)(chem). The smaller bandgap enhances the charge carrier transport and the higher HOMO energy level indicates a reduced barrier and an increased injection rate for charge carriers at the source contact. Furthermore, the threshold voltage V(T) of CNTs-containing P3HT samples is lower and its saturation current I(D) and the the field-effect mobility are higher. An OTFT device fabricated with such a composite sample containing 1.16% CNTs has a carrier mobility and saturation current three to five times higher than pristine P3HT.