Sex-specific nociceptor modulation of the apical periodontitis transcriptome.

Introduction: Apical periodontitis (AP) is a painful disease that develops quickly following dental infections and is primarily characterized by robust inflammation surrounding the tissues of the affected tooth, resulting in disruption of bone homeostasis and periradicular bone loss. Moreover, there are distinct clinical presentations, symptoms, and responses to AP treatment between male and female subjects, creating a desperate need to further understand the sex-specific mechanisms of AP. Methods: With the growing evidence that nociceptors modulate AP development, we utilized RNA sequencing in nociceptor-ablated (Nav1.8 cre+/-, diphtheria toxin Alox+/-) transgenic mice to study the nociceptor regulation of the periapical lesion transcriptome using a rodent model of AP in female mice over 14 days. Results: Overall, we found that female mice exhibit unique patterns of differentially expressed genes throughout AP infection compared to male mice and that the expression of these genes is regulated by nociceptors. Additionally, nociceptor ablation results in a more significant enrichment of biological processes related to immune responses earlier compared to cre-control (Nav1.8 cre+/-) females and greater expression of genes involved in inflammatory processes and osteolytic activity. Discussion: Therefore, while nociceptor ablation augments inflammatory and bone resorption responses in both males and females in a mouse model of AP, transcriptomic analyses demonstrate that the mechanisms through which nociceptors modulate AP are distinct between sexes. These studies will provide the foundation needed to study further mechanisms of sex differences in AP, an area with a desperate need for investigation to treat current AP patients. Understanding these mechanisms can ultimately inform treatment options to alleviate suffering for millions of patients suffering from AP.

A Case of Respiratory Epithelium-Lined Cyst with Enriched Nociceptor Innervation.

Apical lesions of endodontic origin can be classified as either granulomas or cysts. In rare cases, respiratory epithelium can proliferate and encapsulate a lesion, forming a cyst. Moreover, the innervation of apical lesions has only been previously reported in animal models of apical periodontitis. This report demonstrates an unusual case in which tooth #15 was initially treated with nonsurgical root canal therapy. Still, the patient remained in moderate to severe pain for several days following the procedure. Next, an intentional replantation was performed in which a periapical cyst was curetted from the alveolus. The patient experienced immediate pain relief following the procedure. Histological analysis revealed that the periapical cyst was lined entirely with respiratory epithelium, and immunohistochemical analysis showed it to be densely innervated. In addition, these nerve fibers expressed the LPS receptor, TLR4. This is the first demonstration of the innervation pattern of a periapical cyst. Further studies are warranted to evaluate innervation in apical lesions and its correlation with pre- and intra-operative symptoms and their participation in the pathogenesis of apical periodontitis.

Expression of Toll-like Receptors in Stem Cells of the Apical Papilla and Its Implication for Regenerative Endodontics.

Regenerative therapies to replace cells and tissues damaged due to trauma and dental infections require temporal and spatial controlled recruitment and the differentiation of progenitor/stem cells. However, increasing evidence shows microbial antigens can interfere with this process. Toll-like receptors (TLRs) are crucial in recognizing pathogen-associated molecular patterns. Stem cells of the apical papilla (SCAP) are required for normal dental development and are intimately involved in the reparative and regenerative capacity of developing teeth. We hypothesized that TLRs are expressed in SCAP and that the activation of TLR2/TLR4 or TLR3 by different ligands results in differential cellular fate, impacting their differentiation into a mineralizing phenotype. We found that most TLRs are expressed as detected by PCR except TLR7 and TLR8; exposure to heat-killed E. coli results in upregulating TLR2 and TLR4 and reducing mineralization capacity. In addition, bacterial exposure resulted in the upregulation of 11 genes, of which 9 were chemokines whose proteins were also upregulated and released, promoting in vitro macrophage migration. On the other hand, TLR3 activation resulted in increased proliferation and a dramatic inhibition of osteogenic and odontoblastic differentiation, which was reversed by inhibition or the knockdown of TLR3 expression. The profound effects of TLR activation resulting in different cell fates that are ligand and receptor-specific warrants further evaluation and represents an important therapeutic target to make regenerative approaches more predictable following dental infections.

Nociceptors regulate osteoimmune transcriptomic response to infection.

Osteoimmune diseases, such as apical periodontitis, are prevalent, often painful, inflammatory conditions resulting in bone loss and reduced quality of life. There is growing evidence that the nociceptive fibers densely innervating affected tissues regulate disease progression; therefore, we hypothesized that nociceptors regulate the transcriptomic profile of the periapical osteolytic lesion in a mouse model of apical periodontitis. Male control and nociceptor-ablated mice underwent pulp exposures, and after 0, 7, or 14 days, total RNA from periapical tissues was submitted for sequencing and bioinformatic analysis. Pulp exposure triggers the differential expression of hundreds of genes over the course of infection. At 14 days post pulp exposure, 422 genes, including Tnf, Il1a, and Il1b, were differentially expressed between nociceptor-ablated and control mice with greater enrichment of biological processes related to inflammation in nociceptor-ablated mice. Nociceptor ablation regulates the transcriptomic profile of periapical lesions in a mouse model of apical periodontitis, shifting the gene expression profile to a greater enrichment of inflammatory genes, suggesting nociceptors play a role in the kinetics of the immune response. This newly uncovered neuro-immune axis and its mechanisms in apical periodontitis can be an important therapeutic target for the treatment of this prevalent disease.

Effect of Dentin Conditioning with EDTA and Diode Lasers on Expression of Odontoblast-like Cell Markers of Dental Pulp Stem Cells.

Regenerative endodontic procedures rely on the delivery of mesenchymal stem cells into the root canal and on the effect of local growth factors from the dentin and blood clot. The aim of this study was to assess the effect of dentin conditioning with ethylenediamine tetraacetic acid (EDTA) and diode lasers with different wavelengths (808 nm and 980 nm) on the expression of odontoblast-like cell markers. Forty dentin cylinders were divided into four groups according to the irrigation protocol: EDTA, EDTA + 808 nm diode laser, EDTA + 980 nm diode laser, and phosphate-buffered saline as the control group. Dental pulp stem cells were seeded into the previously conditioned cylinders and incubated for 14 days. The quantitative real-time polymerase chain reaction was used to evaluate the expression of dentin sialophosphoprotein (DSPP), dentin morphoprotein-1 (DMP-1), and transforming growth factor-beta 1 (TGF-ß1). Data analysis was performed using the Kruskal-Wallis test. The activation of EDTA with 980 nm and 808 nm diode lasers resulted in lower DSPP and DMP-1 expression than that for EDTA alone (p < 0.05 and p < 0.01, respectively). The expression of TGF was similar among all groups. The highest level of expression of odontoblast-like differentiation markers was observed with EDTA alone. However, the use of an 808 nm diode laser during EDTA irrigation reduced the expression of odontoblastic differentiation markers.

A randomized controlled trial evaluating the effect of epithelial removal on free soft tissue autograft healing.

BACKGROUND: The purpose of this study is to determine if there is a difference in dimensional change of a free soft tissue autograft (FSTA) with epithelium compared to without epithelium. The secondary aim is to determine the patient and professional evaluation of color match and graft texture between the two groups. METHODS: Patients with ≤2 mm keratinized tissue indicated for a FSTA were randomly assigned to control group (FSTA with epithelium) or test group (de-epithelialized FSTA). The vertical and horizontal measurements of the grafts were taken at surgery, and 1, 3, and 6 months postoperatively. Patients were asked to evaluate the color match at each postoperative time point on a 21-step Numeric Rating Scale (NRS-21). Professional assessment of color match and graft texture were evaluated on images at the same time points. RESULTS: Forty-six patients and 55 grafts were included in the study. For change in graft height, width, and area, there were no significant differences between the treatment groups at any time point. Graft height and area in both groups decreased significantly from baseline to month 1 (p < .001), but no other difference was significant over time. When patients and professionals used the NRS-21 for evaluation of color match between the graft site and the surrounding soft tissue, there was no significant difference between the treatment groups. Similarly, evaluation of texture match on color images and black-and-white images revealed no significant differences between or within groups. CONCLUSION: De-epithelialized FSTA showed no difference in dimensional change or color and texture match compared to FSTA with epithelium.

Endocannabinoids Modulate Production of Osteoclastogenic Factors by Stem Cells of the Apical Papilla In Vitro.

INTRODUCTION: Many mediators are produced during pulp inflammation and necrosis, including endocannabinoids (ECbs), which might affect the function of stem cells of the apical papilla (SCAP), cells of paramount importance for root formation, and regenerative endodontic treatment. The aim of this study was to evaluate the production of osteoclastogenesis-related mediators by SCAP modulated by ECbs and lipopolysaccharide (LPS) in vitro. METHODS: SCAP were cultured and treated with ECb anandamide (AEA), 2-arachidonoylglycerol, or N-arachidonoylaminophenol. All groups were incubated in the presence of a vehicle or LPS and the antagonist of transient receptor potential cation channel subfamily V member 1, capsazepine. After 24 hours, the culture medium supernatants were collected for further quantification of tumor necrosis factor alpha, CCL2, macrophage colony-stimulating factor, osteoprotegerin, and receptor activator of nuclear factor kappa B ligand. RESULTS: Small amounts of tumor necrosis factor alpha and receptor activator of nuclear factor kappa B ligand were detected in SCAP supernatants, and none of the experimental conditions altered their production. A down-regulation in constitutive CCL2 production was observed in the AEA group compared with that in the LPS group. The production of macrophage colony-stimulating factor was significantly increased in all groups treated with AEA compared with the control and LPS-treated groups. Osteoprotegerin was significantly increased by AEA alone and by 2-arachidonoylglycerol and N-arachidonoylaminophenol in the presence of LPS and capsazepine. CONCLUSIONS: AEA modulates some of the osteoclastogenic factors produced by SCAP in a bone resorption protective fashion.

Microbiome Changes during Regenerative Endodontic Treatment Using Different Methods of Disinfection.

INTRODUCTION: The purpose of this study was to characterize qualitatively and quantitatively the changes in the endodontic microbiome, in teeth with necrotic pulp, open apexes, and apical periodontitis, with 3 antimicrobial protocols, undertaken in a multicenter clinical trial. METHODS: Microbiological samples were collected from 116 regenerative endodontic teeth, and 97 qualified for inclusion. The teeth were randomly divided into 3 treatment groups: apexification (APEX), regeneration (REGEN), and revascularization (REVASC), all in 2 appointments. The group variables in the first appointment irrigants, and second appointment irrigants and medicaments were as follows: APEX: 5.25%-6% NaOCl, 5.25%-6% NaOCl + 17% EDTA and calcium hydroxide; REGEN: 1.25% NaOCl, 17% EDTA, and 0.1 mg/mL triple antibiotic paste (TAP); and REVASC 5.25% NaOCl, saline, and 1 g/mL TAP, respectively. Sampling was done upon access (S0), after irrigation in the first appointment (S1), and after using medication and irrigation in the second appointment (S2). RESULTS: Quantitative polymerase chain reaction analysis of the 16S ribosomal RNA gene showed significant reduction in bacterial load from S0 to S2 in all groups; however, the APEX and REVASC groups had significantly less residual DNA than the REGEN group (P = .0045). The relative abundance of Bacteroidetes, Fusobacteria, Spirochaetes, and Synergistetes were reduced with the treatment rendered. However, relative abundance of Firmicutes and Actinobacteria was not changed, and that of Proteobacteria increased. LEfSe analysis showed that reduction in bacterial taxa was more in REVASC than APEX, which in turn was more than in REGEN. CONCLUSION: Enhanced antimicrobial protocols lead to better reduction in quantitative and qualitative parameters of the endodontic microflora.

Trigeminal neurons control immune-bone cell interaction and metabolism in apical periodontitis.

Apical periodontitis (AP) is an inflammatory disease occurring following tooth infection with distinct osteolytic activity. Despite increasing evidence that sensory neurons participate in regulation of non-neuronal cells, their role in the development of AP is largely unknown. We hypothesized that trigeminal ganglia (TG) Nav1.8+ nociceptors regulate bone metabolism changes in response to AP. A selective ablation of nociceptive neurons in Nav1.8Cre/Diphtheria toxin A (DTA)Lox mouse line was used to evaluate the development and progression of AP using murine model of infection-induced AP. Ablation of Nav1.8+ nociceptors had earlier progression of AP with larger osteolytic lesions. Immunohistochemical and RNAscope analyses demonstrated greater number of macrophages, T-cells, osteoclast and osteoblast precursors and an increased RANKL:OPG ratio at earlier time points among Nav1.8Cre/ DTALox mice. There was an increased expression of IL-1α and IL-6 within lesions of nociceptor-ablated mice. Further, co-culture experiments demonstrated that TG neurons promoted osteoblast mineralization and inhibited osteoclastic function. The findings suggest that TG Nav1.8+ neurons contribute to modulation of the AP development by delaying the influx of immune cells, promoting osteoblastic differentiation, and decreasing osteoclastic activities. This newly uncovered mechanism could become a therapeutic strategy for the treatment of AP and minimize the persistence of osteolytic lesions in refractory cases.

Effect of intravenous dexamethasone on postoperative pain and swelling following periodontal flap surgery: A randomized controlled trial of patient-centered outcomes.

BACKGROUND: This randomized, crossover trial sought to determine if a preoperative intravenous (IV) dose of dexamethasone reduces pain, swelling, and analgesic usage following periodontal surgery. METHODS: Thirty-seven patients planned for two similar periodontal flap surgeries under IV sedation were enrolled. Patients were randomized to receive either 2 mL (8 mg) dexamethasone sodium phosphate or 2 mL of IV solution (placebo) before the first surgery, and 2 mL of the other solution before the second surgery. Postoperative discomfort was managed with a standardized regimen of 600 mg ibuprofen and 325 mg acetaminophen. A smartphone application was used to record self-assessed pain and swelling scores using 21-point numerical (NRS-21) and 4-point verbal (VRS-4) rating scales as well as the number of analgesic medications taken at 12-, 24-, 48-, 72-, 168-, and 336-hours following each surgery. RESULTS: IV dexamethasone was associated with a significant reduction in pain at 12, 24, 48, and 72 hours (P <0.05), and swelling at 12, 24, 48, and 168 hours (P < 0.05) postoperatively when compared with placebo based on NRS-21 responses. VRS-4 data showed significant reductions in pain at 12, 72, and 168 hours and swelling at 12, 24, and 168 hours postoperatively with dexamethasone. No significant differences were found in the number of tablets of ibuprofen or acetaminophen between dexamethasone and placebo surgeries. CONCLUSIONS: Preoperative, intravenously administered dexamethasone reduces pain and swelling within the first postoperative week following periodontal flap surgery and should be considered a useful adjunct for perioperative management.

Pharmacological Management of Acute Endodontic Pain.

Pain associated with infections of the tooth pulp and periapical tissues is intense and often the most common reason for patients seeking emergency dental care. Effective management of acute dental pain requires a deep understanding of pain mechanisms, which enables accurate diagnosis and definitive treatment. While drugs are only used as an adjunct to definitive dental treatment, a thorough understanding of their mechanism of action and effectiveness enables clinicians to effectively control intra-operative and post-operative pain and prevent persistent pain. This review describes how pain is detected, processed, and perceived. It also provides information on evidence-based strategies on the use of different classes of drugs to effectively manage endodontic pain.