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.

Production of TNF-𝛼 by macrophages stimulated with endodontic pathogens and its effect on the biological properties of stem cells of the apical papilla.

OBJECTIVES: The first objective of the present study was to investigate TNF-𝛼 secretion by macrophages stimulated with endodontic pathogens and bacterial cell surface components. The second objective was to assess the in vitro effects of TNF-𝛼 on periostin, cytokine, and matrix metalloproteinase (MMP) secretion by and the viability, proliferation rate, and mineralization potential of stem cells of the apical papilla (SCAP). METHODS: TNF-𝛼 secretion by macrophages stimulated with either endodontic pathogens or bacterial surface components was assessed using an enzyme-linked immunosorbent assay (ELISA). The viability and proliferation rate of SCAP treated with TNF-𝛼 were assessed using a colorimetric MTT assay. The mineralization potential of TNF-𝛼-treated SCAP was determined by Alizarin Red staining. Periostin secretion by SCAP was determined by ELISA while cytokine and MMP secretion were assessed using a multiplexing laser bead assay. RESULTS: TNF-𝛼 secretion by macrophages increased following a stimulation with Gram-negative and Gram-positive endodontic pathogens. Lipopolysaccharide and lipoteichoic acid also dose-dependently increased the secretion of TNF-𝛼 by macrophages. The viability, proliferation rate, and mineralization activity of SCAP were negatively affected by a TNF-𝛼 treatment. Treating SCAP with TNF-𝛼 attenuated the secretion of periostin and upregulated the secretion of several cytokines and MMPs. CONCLUSIONS: TNF-𝛼 exerts deleterious effects on SCAP by affecting their viability, proliferation rate, and mineralization potential. By its ability to induce the secretion of pro-inflammatory cytokines and MMPs by SCAP, TNF-𝛼 can contribute to creating an inflammatory environment, promoting tissue destruction, and consequently interfering with the success of regenerative endodontic therapy. CLINICAL RELEVANCE: TNF-𝛼 has deleterious impacts on stem cells of the apical papilla and may compromise the outcome of regenerative endodontic therapy.

Author Correction: Stem cells from human apical papilla decrease neuro­inflammation and stimulate oligodendrocyte progenitor differentiation via activin­A secretion.

In the original publication, sixth author's surname was incorrectly published as "Llyod" instead of "Lloyd". The correct name should read as "Amy Lloyd".

Stem cells from human apical papilla decrease neuro-inflammation and stimulate oligodendrocyte progenitor differentiation via activin-A secretion.

Secondary damage following spinal cord injury leads to non-reversible lesions and hampering of the reparative process. The local production of pro-inflammatory cytokines such as TNF-α can exacerbate these events. Oligodendrocyte death also occurs, followed by progressive demyelination leading to significant tissue degeneration. Dental stem cells from human apical papilla (SCAP) can be easily obtained at the removal of an adult immature tooth. This offers a minimally invasive approach to re-use this tissue as a source of stem cells, as compared to biopsying neural tissue from a patient with a spinal cord injury. We assessed the potential of SCAP to exert neuroprotective effects by investigating two possible modes of action: modulation of neuro-inflammation and oligodendrocyte progenitor cell (OPC) differentiation. SCAP were co-cultured with LPS-activated microglia, LPS-activated rat spinal cord organotypic sections (SCOS), and LPS-activated co-cultures of SCOS and spinal cord adult OPC. We showed for the first time that SCAP can induce a reduction of TNF-α expression and secretion in inflamed spinal cord tissues and can stimulate OPC differentiation via activin-A secretion. This work underlines the potential therapeutic benefits of SCAP for spinal cord injury repair.

Mechanisms of transient signaling via short and long prolactin receptor isoforms in female and male sensory neurons.

Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ε (PKCε) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3-5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCε, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.

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.

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.

Peri-implantitis induction and resolution around zirconia versus titanium implants.

BACKGROUND: This study compared titanium and zirconia implant ligature-induced peri-implant defect progression and response to regenerative surgical intervention. METHODS: Eight tissue-level endosseous implants were placed in 6 mixed-breed foxhounds, with 2 zirconia and 2 titanium alternating in each hemimandible. Cotton ligatures were placed subgingivally for 16 weeks followed by 8 weeks of spontaneous progression. Standardized radiographs were captured every 2 weeks to evaluate the rate of bone loss. Regenerative surgery was performed utilizing water-jet decontamination, enamel matrix derivative, and locally harvested autogenous bone. After 16 weeks of healing, final radiographic bone levels as well as probing depths, recession, and clinical attachment levels were assessed. RESULTS: All 48 implants integrated successfully. The final average post-ligature radiographic defects were 2.88 and 3.05 mm for titanium and zirconia implants, respectively. There was no significant difference between materials in the rate of radiographic bone loss (p = 0.09). Following regenerative surgery, the total average amount of radiographic bone gain was 1.41 and 1.20 mm for titanium and zirconia, respectively. The percentage of defect fill was 51.56% and 37.98% (p = 0.03) for titanium and zirconia, respectively. Inter-group differences were minimal for clinical parameters at the time of sacrifice including periodontal pocket depths (p = 0.81), recession (p = 0.98), or clinical attachment levels (p = 0.51). CONCLUSIONS: No significant difference was found in the rate of peri-implant defect development between titanium and zirconia implants. Both materials gained significant radiographic bone following regenerative surgery with significantly greater defect percentage fill in titanium implants. The final clinical parameters were similar in both groups.

Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration.

Traditional bone regeneration strategies relied on supplementation of biomaterials constructs with stem or progenitor cells or growth factors. By contrast, cell homing strategies employ chemokines to mobilize stem or progenitor cells from host bone marrow and tissue niches to injured sites. Although silica-based biomaterials exhibit osteogenic and angiogenic potentials, they lack cell homing capability. Stromal cell-derived factor-1 (SDF-1) plays a pivotal role in mobilization and homing of stem cells to injured tissues. In this work, we demonstrated that 3-dimensional collagen scaffolds infiltrated with intrafibrillar silica are biodegradable and highly biocompatible. They exhibit improved compressive stress-strain responses and toughness over nonsilicified collagen scaffolds. They are osteoconductive and up-regulate expressions of osteogenesis- and angiogenesis-related genes more significantly than nonsilicified collagen scaffolds. In addition, these scaffolds reversibly bind SDF-1α for sustained release of this chemokine, which exhibits in vitro cell homing characteristics. When implanted subcutaneously in an in vivo mouse model, SDF-1α-loaded silicified collagen scaffolds stimulate the formation of ectopic bone and blood capillaries within the scaffold and abrogate the need for cell seeding or supplementation of osteogenic and angiogenic growth factors. Intrafibrillar-silicified collagen scaffolds with sustained SDF-1α release represent a less costly and complex alternative to contemporary cell seeding approaches and provide new therapeutic options for in situ hard tissue regeneration.

Biomimetic silicification of demineralized hierarchical collagenous tissues.

Unlike man-made composite materials, natural biominerals containing composites usually demonstrate different levels of sophisticated hierarchical structures which are responsible for their mechanical properties and other metabolic functions. However, the complex spatial organizations of the organic-inorganic phases are far beyond what they achieved by contemporary engineering techniques. Here, we demonstrate that carbonated apatite present in collagen matrices derived from fish scale and bovine bone may be replaced by amorphous silica, using an approach that simulates what is utilized by phylogenetically ancient glass sponges. The structural hierarchy of these collagen-based biomaterials is replicated by the infiltration and condensation of fluidic polymer-stabilized silicic acid precursors within the intrafibrillar milieu of type I collagen fibrils. This facile biomimetic silicification strategy may be used for fabricating silica-based, three-dimensional functional materials with specific morphological and hierarchical requirements.

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.

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.

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.

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.

Preserving the Neurovascular Bundle in Targeted Endodontic Microsurgery: A Case Series.

Endodontic microsurgery encompasses the use of microscopy, specialized instruments, and advanced imaging with cone-beam computed tomographic (CBCT) imaging. This treatment modality results in high clinical success rates and facilitates the enucleation of osteolytic lesions, the resection of apical root canal complexities harboring persistent bacterial biofilms, and the evaluation of possible root defects and fractures. However, there is the risk of injury to important anatomic structures, particularly when treating posterior teeth. Neurovascular bundles are among these structures at risk for injury. Fortunately, high-resolution CBCT scans can be used to detect these structures that are known to have a high anatomic variability. In addition, CBCT information can be combined with high-resolution intraoral scans to plan, design, and fabricate surgical guides to be used in a targeted endodontic microsurgery (TEMS) approach. We report 3 cases with previous endodontic treatment having persistent apical periodontitis that were treated with TEMS to avoid damage to the neurovascular bundles at risk of injury. In the first case, the palatal root of tooth #14 was adjacent to the greater palatine artery. In the second case, the mental nerve exited through 2 separate foramina close to the predictive osteotomy site for the mesial root of tooth #19. In the third case, the posterior superior alveolar artery was in close proximity to the mesiobuccal root of tooth #14. Collectively, these cases illustrate the diagnostic value of CBCT imaging for detecting neurovascular bundles and the use of TEMS to mitigate the risk of injury to these important structures. Thus, the combination of CBCT imaging and TEMS can potentially minimize the risk of intraoperative complications and postoperative sequelae while increasing the predictability of endodontic microsurgeries in complex cases.

Endocannabinoids Regulate Stem Cells of the Apical Papilla via a Cannabinoid Receptor and TRPV1-Independent Mechanism.

INTRODUCTION: Endogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential. METHODS: Gene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days. RESULTS: The enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group. CONCLUSIONS: SCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.