Deciphering 3D periodontal fibroblast-macrophage crosstalk in bioactive nanoparticle-guided immunomodulation for treating traumatic dental avulsion.

Prolonged extra-oral period in dental avulsion is often associated with loss of viability of Periodontal fibroblasts (PDLF) and increased risk of ankylosis. Root surface treatment with bioactive agents to reduce the risk of ankylosis can be a potential strategy. The objective of the study was to investigate the impact of an engineered chitosan nanoparticles (CSNP), photosensitizer Rose Bengal (RB) functionalized CSNP (CSRB) and sustained dexamethasone (CSDEX) releasing CSNP for application in management of delayed replantation of avulsed teeth. The 3D PDLF-macrophage (Mϕ) collagen model was developed and exposed to LPS, MCSF, RANKL with and without CSDEX/CSNP. Immunofluorescence and cytokine analysis was done at 2 and 7 days to assess cellular interactions. Maxillary right incisors in male Wistar rats were extracted, exposed to extraoral dry or LPS for 1 h and treated with or without CSDEX/CSRB for 1 min before replantation. Rats were euthanized after 21 days for micro-CT, TRAP, and immunofluorescence analysis. CSDEX/CSNP treatment in 3D model significantly reduced CD80, NFATc1, STAT6 and increased CD206 and periostin expression (p < 0.05). TNFα, MMP9 was downregulated and IL10, TGFß1, MMP2 upregulated with CSDEX/CSNP (p < 0.05). CSDEX/CSRB in animal study significantly reduced resorption, ankylosis, TRAP activity and osteocalcin expression and increased periostin (p<0.05). CSDEX demonstrated higher anti-inflammatory activity by downregulating TNFα, while CSNP upregulated TGFß1, periostin, and downregulated MMP9. The combination of matrix stabilization with CSRB with periostin upregulation and sustained releasing CSDEX showed potential for hampering root resorption and ankylosis in dental avulsion.

Engineered Immunomodulatory Nanoparticles Inhibit Root Resorption and Ankylosis.

INTRODUCTION: External root resorption following avulsion injury is a complex process wherein differentiation of macrophages (Mϕ) to multinucleated osteoclasts is temporally regulated by resident periodontal fibroblasts (PDLF). The current study aims to assess the effect of engineered bioactive chitosan nanoparticles (CSNP), sustained released dexamethasone conjugated CSNP (CS-DEX) and CSNP functionalized with photosensitizer Rose Bengal (CSRB) for application in root resorption using an in-vitro PDLF-Mϕ direct coculture model and in-vivo delayed reimplantation model. METHODS: PDLF-Mϕ direct coculture system was exposed to lipopolysaccharide (LPS), macrophage colony stimulating factor, receptor activator of nuclear factor kappa ß ligand with or without CSNP/CS-DEX for 7 days. Clastic differentiation was assessed by tartrate resistant acid phosphatase (TRAP) staining on day 7. On day 2 and 7, immunofluorescence analysis was conducted to assess the expression of Mϕ polarization markers (CD80, CD206), multinucleation markers (NFATc1, STAT6) in Mϕ and matricellular protein periostin in PDLF and cytokine profiling in cell culture supernatants. Delayed replantation model with extraoral air dry/LPS exposure for 1h followed by root surface treatment with CS-DEX/CSRB was used in Wistar rats. After 21 days, rats were euthanized for histologic and immunofluorescence analysis. Statistical analysis one-way ANOVA with Tukey's multiple comparisons was used to analyze the data (P < .05). RESULTS: CS-DEX significantly reduced TRAP+ multinucleated cells and CSNP treatment showed no TRAP+ cells. Immunofluorescence analysis showed that CSNP/CS-DEX reduced CD80, NFATc1 and STAT6 expression and increased periostin as expressed by fluorescence intensity. CSNP/CS-DEX significantly reduced TNFα, MMP9 and increased IL10, TGFß1. Osteoprotegerin was upregulated only by CSNP. Root surface treatment in delayed replantation model showed that CS-DEX and CSRB substantially reduced the degree of resorption and ankylosis. Further, CD80, CD206, and MMP2 expression in groups with root surface treatment with CS-DEX and CSRB was lower than airdry/LPS group and similar to healthy control and NFATc1, STAT6, and MMP9 expressions were lower than healthy control. CONCLUSION: The engineered nanosized immunomodulatory bioactive materials chitosan nanoparticles functionalized with photosensitizer and dexamethasone effectively reduced the clastic differentiation of Mϕ in in-vitro coculture and minimized the resorption and ankylosis in a delayed reimplantation model. These biomaterials have the potential to serve as root modification agents, promoting favorable healing outcomes in cases of dental avulsion.

Dynamic Photoelastic Analysis of Stress Distribution in Simulated Canals Using Rotary Instruments with Varied Tip and Taper Sizes: A Quasi-3D Approach.

INTRODUCTION: To compare the stress produced on the walls of simulated canals by rotary instruments with varied tip and taper sizes. METHODS: Ninety isotropic transparent blocks, each containing a 60-degree curved canal, were distributed into 18 groups (n = 5) based on the instrument tip (sizes 10, 15, 20, 25, 30, and 35) and taper (sizes 0.02, 0.04, and 0.06). The blocks were fixed in a circular polariscope setup for dark field analysis. A digital camera was employed to capture the real-time birefringence patterns generated by each instrument. Digital image frames, corresponding to the instrument reaching the end of each canal third, were extracted and evaluated by 2 independent observers for the stress generation on canal walls. The data analysis employed a semi-quantitative scale ranging from 0 to 5. Cohen's Kappa coefficient test was used to determine the inter-observer agreement while the results were compared using Kruskal-Wallis test followed by an all-pairwise posthoc procedure (α = 5%). RESULTS: Inter-observer agreement was 0.95. A significant influence of the tip size on stress was observed across the coronal (P = .011), middle (P = .006), and apical (P = .026) thirds. In contrast, taper size did not affect the stress induced at the coronal (P = .509), middle (P = .958), or apical (P = .493) thirds. The variations in tip and taper sizes did not result in a significant stress differences among the thirds (P = .181). CONCLUSIONS: The stress significantly increased across all canal thirds with larger tip sizes of rotary instruments, whereas the taper sizes did not influence the stress when compared to the canal thirds.

Bioarchitectural Design of Bioactive Biopolymers: Structure-Function Paradigm for Diabetic Wound Healing.

Chronic wounds such as diabetic ulcers are a major complication in diabetes caused by hyperglycemia, prolonged inflammation, high oxidative stress, and bacterial bioburden. Bioactive biopolymers have been found to have a biological response in wound tissue microenvironments and are used for developing advanced tissue engineering strategies to enhance wound healing. These biopolymers possess innate bioactivity and are biodegradable, with favourable mechanical properties. However, their bioactivity is highly dependent on their structural properties, which need to be carefully considered while developing wound healing strategies. Biopolymers such as alginate, chitosan, hyaluronic acid, and collagen have previously been used in wound healing solutions but the modulation of structural/physico-chemical properties for differential bioactivity have not been the prime focus. Factors such as molecular weight, degree of polymerization, amino acid sequences, and hierarchical structures can have a spectrum of immunomodulatory, anti-bacterial, and anti-oxidant properties that could determine the fate of the wound. The current narrative review addresses the structure-function relationship in bioactive biopolymers for promoting healing in chronic wounds with emphasis on diabetic ulcers. This review highlights the need for characterization of the biopolymers under research while designing biomaterials to maximize the inherent bioactive potency for better tissue regeneration outcomes, especially in the context of diabetic ulcers.

Physico-chemical and biological characterization of synthetic and eggshell derived nanohydroxyapatite/carboxymethyl chitosan composites for pulp-dentin tissue engineering.

Hybrid nanohydroxyapatite/carboxymethyl chitosan (nHAp-CMC) scaffolds have garnered significant attention in the field of regenerative engineering. The current study comparatively analyzed the physicochemical and biological properties of synthetic nanohydroxyapatite (SnHA)- and eggshell-sourced nanohydroxyapatite (EnHA)- based CMC biocomposites for pulp-dentin regeneration. EnHA and CMC were synthesized through a chemical process, whereas SnHA was commercially obtained. Composite scaffolds of SnHA-CMC and EnHA-CMC (1:5 w/w) were prepared using freeze-drying method. All biomaterials were characterized by FTIR, micro-Raman, XRD, HRSEM-EDX, and TEM analyses, and their in vitro bioactivity was assessed by immersing them in simulated body fluid for 21 days. The biological properties of the composite scaffolds were evaluated by assessing cytocompatibility using MTT assay and biomineralization potential by analyzing the odontogenic gene expressions (ALP, DSPP, DMP-1 and VEGF) in human dental pulp stem cells (DPSCs) using RT-qPCR method. Characterization studies revealed that EnHA displayed higher crystallinity and superior surface morphology compared to SnHA. The composite scaffolds showed a highly interconnected porous microstructure with pore sizes ranging between 60 and 220 µm, ideal for cell seeding. All tested materials, SnHA, EnHA, and their respective composites, displayed high cytocompatibility, increased ALP activity and degree of mineralization with significant upregulation of odontogenic-related genes on DPSCs (p < 0.05). Nevertheless, the odontogenic differentiation potential of EnHA-CMC on DPSCs was significantly higher when compared to SnHA-CMC. The findings from this study highlight the potential of EnHA-CMC as a promising candidate for pulp-dentin engineering.

Knowledge of undergraduate and postgraduate dental students about managing traumatic dental injuries based on the 2020 International Association of Dental Traumatology guidelines: A online survey of 10 dental schools from 10 countries.

BACKGROUND/AIM: The knowledge of dental students about managing traumatic dental injuries (TDIs) may not be uniform, depending on global location and dental education. The aim of this study was to evaluate the level of knowledge of undergraduate and postgraduate students specializing in endodontics and pediatric dentistry at 10 dental schools in 10 countries about the 2020 International Association of Dental Traumatology (IADT) guidelines regarding the management of TDIs. MATERIALS & METHODS: A previously published questionnaire was used in the current survey. It was an online survey with 12 questions regarding the management of TDIs and some additional questions regarding sociodemographic and professional profiles of the participants were added. The survey was distributed to final-year undergraduate students and postgraduate students in pediatric dentistry and endodontics from 10 dental schools. Simple frequency distributions and descriptive statistics were predominantly used to describe the data. Differences in the median percentage scores among the student categories were assessed using the Kruskal-Wallis test followed by Dwass-Steel-Critchlow-Fligner pairwise comparisons. RESULTS: A total of 347 undergraduates, 126 postgraduates in endodontics, and 72 postgraduates in pediatric dentistry from 10 dental schools participated in this survey. The postgraduates had a significantly higher percentage score for correct responses compared with the undergraduates. No significant difference was observed between the endodontic and pediatric dentistry postgraduates. CONCLUSION: The knowledge possessed by undergraduate and postgraduate students concerning the IADT-recommended management of TDIs varied across the globe and some aspects were found to be deficient. This study emphasizes the critical importance of reassessing the teaching and learning activities pertaining to the management of TDIs.

Pericervical Dentin Metrics in Mandibular First Molars Determined with Digital Periapical Radiography and Cone-beam Computed Tomography.

INTRODUCTION: Pericervical root dentin is decisive for the long-term mechanical integrity of root-filled teeth. Current treatment protocol does not include a customized step to determine the pretreatment residual pericervical root dentin. OBJECTIVE: To determine and compare the residual root dentin and canal width using digital periapical radiography (DPR) and cone-beam computed tomography (CBCT) at the apical limit of the pericervical area (PCA) in mandibular first molars. METHODS: DPR and CBCT images of 60 patients with age between 22 and 76 years were used to determine (a) the mesiodistal widths of the root canal (pericervical dimensions [PCL]-C) and the root (PCL-R) of mandibular first molars at the apical limit of the PCA and (b) the intracanal distance from the apical limit of the PCA to the radiographic apex (intracanal distance [ICD]). The correlation between the PCL and ICD measurements obtained from DPR and CBCT were evaluated. RESULTS: Values between 0.10-0.80 mm and 0.00-1.10 mm were obtained for PCL-C using DPR and CBCT respectively (95% CI). The PCL values between 0.90-2.30 mm and 0.00-2.30 mm were obtained from DPR and CBCT respectively (95% CI). The ICD ranged between 4.6-12.3 mm in DPR and 4.40-12.0 mm in CBCT (95% CI). The comparative analysis showed differences from -0.9 to 0.5 mms for PCL and -2.00 to 1.5 mms for ICD between DPR and CBCT techniques respectively. CONCLUSION: The PCL and ICD determined from DPR and CBCT provided the pericervical dentin metrics that could be utilized clinically as a guideline for decision-making in endodontic treatment.

Impact of Type 2 Diabetes Mellitus on the Occurrence of Vertical Root Fracture: A Case Control Study.

INTRODUCTION: The aim of this case-control study was to examine the relationship between type 2 diabetes mellitus (DM) and the occurrence of VRFs. The crack extension, dentin sclerosis, and chemical characteristics of root dentin in teeth with VRF from patients with/without DM were also compared. METHODS: One hundred and thirty-two patients diagnosed with VRF in crowned root filled posterior teeth were selected. The study was conducted in 2 parts. In Part-1: The cases were matched with control teeth (1:1) for age (±5 years), sex, tooth type, apical extent of root filling, time period after root filling to a diagnosis of VRF, presence or absence of intracanal post and abutment status. The presence or absence of type 2 DM (HbA1c > 6.5) was recorded. In Part-2: The extracted teeth with VRF from the case control study were used to evaluate the extension of VRF, presence of sclerotic dentin and isthmus using a microscopic analysis; while the levels of pentosidine, collagen cross-linking ratio and mineral-collagen ratio were determined by Fourier-transform infrared spectroscopy. The distribution of DM between cases and controls was analyzed using Pearson Chi-Square test and Odds Ratio estimated. Chemical composition data was analyzed using Mann-Whitney test. The extent of sclerotic dentin was analyzed using Pearson Chi-Square test. RESULTS: When compared to patients without DM, patients with DM had 2.67 (95% CI: 1.6-4.45) folds higher odds for occurrence of VRF. Pentosidine (P = .014), collagen cross-linking ratio(P = .047), mineral-collagen ratio (P = .009) and sclerotic dentin extent (P = .0009) were significantly higher in patients with DM and VRF. CONCLUSIONS: Type 2 DM was more often associated with VRFs in root canal treated teeth with crowns. Root dentin from patients with type 2 DM and VRF had higher levels of pentosidine, collagen cross-linking ratio, mineral to collagen ratio and sclerotic dentin.

Irrigants and irrigation activation systems in Endodontics.

Root canal infections are typically polymicrobial and involve strong bacterial interactions. The goal of endodontic treatment is to remove infected content from the root canal system to allow the healing of a pre-existing periapical lesion or to prevent infection of the periradicular tissues. Instrumentation alone is not capable of touching all of the root canal walls. Therefore, the irrigation process is an essential step in the endodontic treatment. However, due to the complex anatomy of the root canal system, this cleaning is very challenging. Although syringe and needle irrigation associated with the use of chemical substances is still the most used method, it does not guarantee optimal cleaning of the root canals. As a result, not only alternative irrigating substances but also numerous activation systems - which are technologies that aim to optimize the action of irrigating substances, both chemically and physically - have been developed. This work aimed to review the characteristics of both classic and current alternatives of irrigating substances and irrigation activation systems.

Impact of 3D collagen-based model and hydrostatic pressure on periodontal ligament fibroblast: A morpho-biochemical analysis.

This study developed a customized hydrostatic pressure-based loading environment to investigate the effect of static hydrostatic pressure on the periodontal ligament fibroblasts (PDLf) in a three-dimensional (3D) collagen-based model. The cylindrical tissue constructs were comprised of PDL fibroblast cells seeded in type I collagen matrices and divided into three experimental groups: Control (no load), low-load (∼0.07 kPa), and high-load (∼60 kPa), all subjected to 24 h of experimental duration. Cells in the 3D construct were stained with fluorophore-conjugated antibodies for cytoskeletal protein F-actin and matricellular protein periostin. Cell culture supernatant was assessed for receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) expression. Transmission electron microscopy examined the contact between the cells and the collagen matrix. Ultrastructural changes in the 3D collagen matrix were also analyzed using scanning electron microscopy. Experiments were performed in triplicates, and data was analyzed using one-way ANOVA (p < 0.05). The 3D PDLf constructs from the low-load group demonstrated the highest levels of homogeneous cell distribution and higher expression of F-actin and periostin with enhanced interaction with the matrix. The collagen matrix in this group showed more closely packed fibers forming thicker bundles when compared to the control and the high-load 3D PDLf constructs. Nonuniform cell distribution with decreased expression of F-actin and periostin was observed in the control and high-load PDLf constructs. The high-load group showed the highest RANKL/OPG expression. This study demonstrated low-level hydrostatic pressure's role in regulating PDLf functions and extracellular matrix response, while excessive hydrostatic pressure may be detrimental to PDL fibroblast cell function.

Nociceptor-Macrophage Interactions in Apical Periodontitis: How Biomolecules Link Inflammation with Pain.

Periradicular tissues have a rich supply of peripheral afferent neurons, also known as nociceptive neurons, originating from the trigeminal nerve. While their primary function is to relay pain signals to the brain, these are known to be involved in modulating innate and adaptive immunity by initiating neurogenic inflammation (NI). Studies have investigated neuroanatomy and measured the levels of biomolecules such as cytokines and neuropeptides in human saliva, gingival crevicular fluid, or blood/serum samples in apical periodontitis (AP) to validate the possible role of trigeminal nociceptors in inflammation and tissue regeneration. However, the contributions of nociceptors and the mechanisms involved in the neuro-immune interactions in AP are not fully understood. This narrative review addresses the complex biomolecular interactions of trigeminal nociceptors with macrophages, the effector cells of the innate immune system, in the clinical manifestations of AP.