Abstract
Shanthini Kalimuthu, Prasanna Neelakantan
Intracellular Bacteria in Stem Cells of the Apical Papilla: A “Trojan Horse” for Regenerative Endodontic Failure
Regenerative endodontic procedures show unpredictable outcomes in infected teeth due to persistent infection. While current disinfection protocols reduce but do not eliminate root canal bacteria, the fate of residual microorganisms during stem cell recruitment remains unclear. This study tested the hypothesis that endodontic pathogens invade and survive within stem cells of the apical papilla (SCAP), diminishing its biomineralization potential. Internalization of SCAP by Gram-positive (Enterococcus faecalis, Actinomyces naeslundii) and Gram-negative (Porphyromonas gingivalis, Fusobacterium nucleatum) bacteria was characterized using colony-forming unit assays, confocal microscopy and flow cytometry. Intracellular survival of E. faecalis was monitored for 14 days in both planktonic and biofilm phases. The impact of intracellular E. faecalis on SCAP biomineralization and odontogenic differentiation was assessed using alkaline phosphatase activity, alizarin red staining, and quantitative real-time PCR. Statistical analyses were performed using one-way ANOVA with Dunnett's multiple comparison test or two-way ANOVA with Tukey's multiple comparison test. P < .05 was considered statistically significant. All tested bacteria invaded SCAP with differential efficiencies. E. faecalis and F. nucleatum demonstrated robust internalization (∼5-log colony forming unit), while P. gingivalis and A. naeslundii showed reduced invasion (∼2.5 log colony forming unit). E. faecalis persisted intracellularly for up to 14 days, with biofilm-phase bacteria showing enhanced survival in monolayer SCAP cultures. Intracellular E. faecalis abrogated SCAP mineralization while maintaining cell viability, accompanied by significant downregulation of all tested odontogenic genes (P < .05). Bacteria invade and establish persistent intracellular infections within SCAP, creating protected reservoirs. These intracellular bacteria severely compromise odontogenic differentiation without killing the host cells, representing a potentially unrecognized mechanism of regenerative endodontic procedure failure. This "Trojan horse" strategy may explain the unpredictable outcomes of regenerative endodontics in infected teeth.
Phachara Promchouy, Kanet Chotvorrarak, Sittichoke Osiri
Prevalence of Vertical Root Fractures in Root Filled Teeth and Their Clinical and Radiographic Risk Factors: A Modern Case-Control Analysis Based on Surgical Diagnosis
Vertical root fractures (VRFs) are challenging in endodontic diagnosis. This study determined VRF prevalence and risk factors in root-filled teeth (RFT) requiring endodontic surgery. From 411 RFT that underwent endodontic surgery (2005–2022), 130 VRF cases and 260 randomly selected controls were analyzed. Demographic, clinical, and radiographic data were collected from dental records and images. Multiple imputations addressed missing data. Univariable and multivariable logistic regression analyses with robust standard errors to account for the clustering of multiple teeth within the same patient were performed, with subanalyses for teeth with intraradicular posts and molars. VRF characteristics were analyzed in individuals with available cone beam computed tomography images. VRF prevalence was 31.63% among RFT undergoing endodontic surgery. Significant risk factors included age (adjusted odds ratio [aOR] = 1.02), probing depth ≥5 mm (aOR = 2.37), isolated perilateral radiolucency (aOR = 4.05), halo radiolucency (aOR = 2.70), overfilled root canal (aOR = 2.11), and root canal space-to-root-width ratio >1/3 (aOR = 1.52). For teeth with posts, a post space-to-root-width ratio >1/3 increased VRF risk (unadjusted OR [uOR] = 2.46). Cone beam computed tomography findings revealed the absence of the buccal plate (uOR = 1.74) and space between the cortical plate and root surface (uOR = 2.73) as significant factors. VRFs affected nearly one-third of RFT requiring endodontic surgery; major risk factors included older age, overfilled root canals, and excessive root canal space-to-root-width ratio. VRF-associated clinical and radiographic signs included probing depth ≥5 mm, isolated perilateral radiolucency, and halo radiolucency.
Golriz Rostami, Rajeshwari Hadagalau Revana Siddappa, Anil Kishen
Signaling Pathways in Root Resorption: Linking Inflammation, Odontoclastogenesis, and Tissue Remodeling
Root resorption is a pathologic process characterized by the breakdown of dentin and cementum by odontoclasts, mirroring the mechanisms of osteoclast-driven bone resorption. Osteoclastogenesis is tightly regulated by the RANK/RANKL/OPG axis and other signaling pathways, including Wnt, ATP-P2RX7-IL-1, programmed cell death, and inflammasome activation. This review provides a comprehensive analysis of the key signaling pathways and molecular mediators orchestrating root resorption in orthodontic, traumatic, and inflammatory conditions. A literature-based analysis was conducted, focusing on molecular and cellular mechanisms involved in root resorption. Key pathways such as RANK/RANKL/OPG, Wnt signaling, ATP-P2RX7-IL-1, and inflammasome activation were examined. The role of proinflammatory and anti-inflammatory cytokines, matrix metalloproteinases, and periostin were also analyzed. Proinflammatory mediators such as IL-1, IL-6, IL-8, tumor necrosis factor-alpha, IL-17, IL-22, and IL-23 drive odontoclastic differentiation, whereas anti-inflammatory cytokines, including IL-4, IL-10, and transforming growth factor-beta, counteract resorptive activity. Additionally, matrix metalloproteinases and periostin modulate extracellular matrix remodeling, impacting resorption progression. The balance between resorptive and reparative processes is influenced by the inflammatory microenvironment, fibroblast-macrophage interactions, and mechanotransduction. While the molecular mechanisms underlying odontoclastogenesis parallel bone resorption, unique features of root structures, such as the cementoid layer, contribute to resistance against resorption. The intricate cross talk between pro-resorptive and antiresorptive factors, emphasizing their roles in odontoclast activation and extracellular matrix remodeling, dictate the extent of root degradation.
Malika Malik, Elizabeth Perkins, Alyssa Nedell, Hitesh Chopra, James Sugai, Darnell Kaigler
Viability of Dental Pulp Derived Stem Cells After Long-Term Cryopreservation
Dental pulp-derived stem cells (DPSCs) have promise for use in regenerative therapies due to their regenerative and immunomodulatory potential. Following their isolation from dental pulp tissue, DPSCs can be cryopreserved and banked for future use, however, it remains unclear to what extent long-term storage affects their properties. This study evaluated the properties of DPSCs cryopreserved for up to 13 years. DPSCs from 12 patients cryopreserved for 5 (DPSC-5 YR), 10 (DPSC-10 YR), and 13 (DPSC-13 YR) years were analyzed for viability, immunophenotype (CD34, CD45, CD73, CD90, CD105), proliferation, and stemness. Additionally, senescence was evaluated through gene expression and senescence-associated β-galactosidase activity. All cryopreserved DPSCs showed high expression of stem cell markers CD73, CD90, and CD105 (>90%) with low expression of hematopoietic markers CD34 and CD45 (<4%). Proliferative capacity and proliferation rate demonstrated no significant differences in population doubling time among groups, with values of 1.32 ± 0.41 for DPSC-5 YR, 1.36 ± 0.44 for DPSC-10 YR, and 1.38 ± 0.53 for DPSC-13 YR, all comparable to DPSCs cryopreserved for less than 1 year (1.37 ± 0.57). Osteogenic and adipogenic differentiation of DPSCs confirmed their ability to retain multipotency. Finally, senescence-associated β-galactosidase staining revealed an absence of senescent cells up to passage 6 and while stemness and senescence gene expression profiles varied, no significant differences were found in expression of these genes between DPSCs, regardless of numbers of years of cryopreservation. DPSCs can maintain viability, proliferative capacity, and stemness following long-term (up to 13 years) cryopreservation. These data support their long-term banking for clinical therapies aimed at tissue regeneration and immunomodulation.
Kristen Hasegawa, Anna Vincek, Janny V Evenhuis, Stephanie Goldschmidt, Maria Soltero-Rivera, Natalia Vapniarsky, Kathryn Good, Claudio J Gutierrez, Boaz Arzi
Clinical features and computed tomography findings of retrobulbar disease in cats
The objective of this study was to describe the clinical features and computed tomography (CT) findings of cats affected with retrobulbar disease. The medical records of 37 client-owned cats diagnosed with retrobulbar disease between the years 2009–2024 were reviewed. Clinical information, signalment, the presenting specialty service, clinical signs, diagnostic results, treatment, and outcomes were documented. CT features of retrobulbar disease as well as cytology and histopathology were reviewed. Fifteen cats (40.5%) were diagnosed with primary disease in the retrobulbar space and 22 cats (59.5%) were diagnosed with secondary retrobulbar disease. Out of the 15 cats with primary retrobulbar disease, 9 were diagnosed with neoplasia, 3 were diagnosed with an infectious/inflammatory process, 2 were traumatic in origin, and 1 cat had a cyst. Of the 22 cats with secondary retrobulbar disease, 21 cats were diagnosed with neoplasia and 1 cat was diagnosed with an infectious disease. CT findings of orbital osteolysis and reduction of retrobulbar fat were significantly associated with neoplasia. Survival outcomes for cats diagnosed with a primary retrobulbar infectious/inflammatory disease were significantly better than for those diagnosed with primary or secondary retrobulbar neoplasia. This study found that neoplasia is the most common primary and secondary retrobulbar disease in cats. Due to the significantly different prognostic implications between cats with primary infectious/inflammatory retrobulbar disease, primary neoplasia and secondary neoplasia, we also confirmed that CT is an essential part of diagnosis and characterization of the extent of the disease, and that additional diagnostics such as histopathology, cytology, culture and susceptibility, or fungal cultures are needed to further support and guide treatment options. Finally, cats that present with either primary infectious/inflammatory or traumatic retrobulbar disease carry favorable prognosis with either medical or surgical intervention.
Seok Bin Yang, Ji Hoi Moon
Microbial therapeutics for canine periodontal disease: current status and future perspectives
Periodontal disease in dogs arises from ecological disruption of the oral microbiome. Sequencing-based studies and quantitative molecular analyses reveal characteristic dysbiotic transitions in affected dogs, with coordinated increases in Porphyromonas gulae, Prevotella, and Treponema, along with Treponema denticola and Tannerella forsythia, together with a loss of commensal-dominated genera such as Moraxella, Capnocytophaga, and members of the Neisseriaceae family. Rather than being driven by a single dominant pathogen, accumulating evidence indicates that canine periodontitis is driven by polymicrobial synergy within dysbiotic biofilms. This ecological perspective has stimulated growing interest in microbial therapeutics aimed at modulating community structure and function instead of relying solely on broad-spectrum antibiotics. Probiotics and postbiotics show potential in reducing halitosis and modulating epithelial innate immune responses. Bacteriophage-based approaches and predatory bacteria exhibit strain-specific antimicrobial activity in preclinical human or in vitro models, although their relevance to canine oral disease remains unvalidated. Synthetic biology and CRISPR-based antimicrobial systems provide conceptual frameworks for genotype-targeted modulation of virulence. Remaining challenges include transient microbial persistence, limited veterinary clinical evidence, biosafety concerns, and the absence of standardized regulatory pathways. Collectively, emerging microbial therapeutics highlight the potential but also the current limitations of ecology-guided, non-antibiotic strategies for canine periodontal therapy.
Marie Pia Paoloa Häußling, Tim Steinberg, Kathrin Büttner, Hannig Christian, Hannig Matthias, Lilia Lemke, Oliver Zierau, Carsten Staszyk
Equine dental destructive disorders: An epidemiological survey in northern Germany
Destructive lesions of the peripheral and infundibular cementum are increasingly recognized in equine dentistry. While similarities to human caries have been noted, current evidence does not conclusively support this classification. This study aims to assess the prevalence, distribution, and potential risk factors associated with these lesions in a defined horse population. Material and Methods A total of 114 horses from northern Germany underwent standardized oral examination between 2021 and 2023. Clinical data on peripheral and infundibular lesions, diastemata, and influencing factors such as age and sex were collected and statistically analyzed using adapted grading systems and multiple comparative tests. Results Peripheral cemental lesions were present in 77.2% and infundibular changes in 61.4% of the horses. While molars were more frequently affected than premolars, no significant correlation was found between the two lesion types. Infundibular disease increased significantly with age, whereas peripheral lesions showed a non-significant age-related trend. Female horses exhibited a higher prevalence of peripheral cemental lesions. Discussion / Conclusion Despite similar cemental involvement, peripheral and infundibular lesions differ markedly in clinical relevance and progression, necessitating distinct diagnostic approaches. Peripheral changes are often benign, whereas infundibular lesions may lead to pulpitis or fractures. These findings underline the need for species-specific diagnostic criteria and further research into modifiable risk factors.
Robert Marx, Ana Nemec, Andraž Kocjan, Metka Voga
In Vitro Effects of Different Biomaterials on Canine Dental Pulp Stem Cells
Regenerative endodontic treatments are being developed in veterinary dentistry. The aim of this study was to evaluate the biocompatibility and odontogenic potential of three biomaterials, ProRoot® MTA (MTA), RS+™ (RS+), and CellFoam™ (CF), on canine dental pulp stem cells (cDPSCs) under conditions simulating early and clinically relevant exposures. Methods: cDPSCs were isolated from three healthy dog teeth extracted for clinical reasons and characterized by flow cytometry (CD44⁺/CD90⁺/CD29⁺/CD34⁻) and multilineage differentiation. Cells were cultured with material suspensions (acute cytotoxic effect) or conditioned medium (physiologically relevant effect). Metabolic activity and cell viability were assessed by MTT and live/dead assays. Osteogenic/odontogenic differentiation was evaluated by Alizarin Red S staining and RT‒qPCR for RUNX2, ALPL, and MMP13 expression. Results: In suspension cultures, compared with MTA and RS+, CF maintained significantly higher metabolic activity and cell viability across several dilutions, indicating lower acute cytotoxicity. Under conditioned exposure, no significant differences among materials were observed, reflecting the dilution and buffering effects that mitigate early reactivity. All the materials supported Alizarin Red S-positive mineral deposition, with a significant difference at D3, when ARS staining of cDPSCs was greater in cells conditioned with MTA than in those conditioned with CF. Gene expression analysis revealed lower RUNX2 and ALPL expression in MTA-conditioned cells, suggesting, together with ARS staining, progression toward a more advanced osteogenic or odontogenic differentiation stage. MMP13 expression remained comparable across materials. Conclusions: MTA, RS+, and CF demonstrated overall biocompatibility with cDPSCs and supported odontogenic differentiation under clinically relevant conditions. CF exhibited the lowest acute cytotoxicity, indicating its potential as a carrier for DPSC-based regenerative endodontic applications. These findings support the translational importance of in vitro cDPSC models for evaluating biomaterial performance in veterinary regenerative endodontics.
Rachelle Fisher, Stephanie Lynne Goldschmidt, Marguerite F Knipe, Maria M Soltero-Rivera, Natalia Vapniarsky, Boaz Arzi
Differential Causes of Masticatory Muscle Disorders in Dogs: A Review of Diagnosis, Treatment and Long-Term Management
Masticatory muscle disorders in the dog are complex and challenging cases to manage. Patients may present with inability or difficulties in opening or closing the mouth, making dysphagia and respiratory compromise major concerns. Time is of the essence in these circumstances, and prompt and accurate diagnosis and treatment are required to prevent potentially life-threatening complications from occurring. Several local and systemic disease processes may cause masticatory muscle dysfunction further complicating the diagnostic process. Current literature on masticatory muscle disorders is widespread with many recommendations in veterinary medicine extrapolated from human medicine. The goal of Masticatory Muscle Disorders 2 this paper is to synthesize the current literature and illuminate areas that require additional research.
Boaz Arzi, Stephanie Goldschmidt, Emilia Chrostek, Minh Vo Duong, Barbro Filliquist, Po-Yen Chou
A needle arthroscopy approach to the temporomandibular joints in dogs
The temporomandibular joint (TMJ) has been identified as an important contributor to orofacial pain and dysfunction in dogs. Although TMJ disorders have been partially characterized, a more comprehensive understanding and development of diagnostic and therapeutic options are needed. Arthroscopy of the TMJ is a technique that allows for direct visualization and inspection of the internal joint structures, in addition to the collection of biopsies and the performance of select surgical procedures under the operator’s direct visual control. This Methods article, illustrated by two clinical cases, demonstrates the practical and efficient use of needle arthroscopy in dogs’ TMJs. Understanding the fundamental aspects of TMJ arthroscopy is important for diagnosing and treating TMJ conditions, especially given the TMJ’s unique anatomical features in dogs. A semi-open approach was found to be more practical and less likely to cause iatrogenic damage than a single stab incision. The ability to visualize joint structures in health and disease and to perform arthroscopy-guided biopsies is valuable and carries clinically relevant implications.