Microbial biomarkers as a predictor of periodontal treatment response: A systematic review.

To evaluate the prognostic accuracy of microbial biomarkers and their associations with the response to active periodontal treatment (APT) and supportive periodontal therapy (SPT). Microbial dysbiosis plays a crucial role in the disease processes of periodontitis. Biomarkers based on microbial composition may offer additional prognostic value, supplementing the limitations of current clinical parameters. While these microbial biomarkers have been clinically evaluated, there is a lack of consensus regarding their prognostic accuracy. A structured search strategy was applied to MEDLINE (PubMed), Cochrane Library, and Embase on 1/11/2022 to identify relevant publications. Prospective clinical studies involving either APT or SPT, with at least 3-month follow-up were included. There were no restrictions on the type of microbial compositional analysis. 1918 unique records were retrieved, and 13 studies (comprising 943 adult patients) were included. Heterogeneity of the studies precluded a meta-analysis, and none of the included studies had performed the sequence analysis of the periodontal microbiome. Seven and six studies reported on response to APT and SPT, respectively. The prognostic accuracy of the microbial biomarkers for APT and SPT was examined in only two and four studies, respectively. Microbial biomarkers had limited predictive accuracy for APT and inconsistent associations for different species across studies. For SPT, elevated abundance of periodontal pathogens at the start of SPT was predictive of subsequent periodontal progression. Similarly, persistent high pathogen loads were consistently associated with progressive periodontitis, defined as an increased pocket probing depth or clinical attachment loss. While there was insufficient evidence to support the clinical use of microbial biomarkers as prognostic tools for active periodontal treatment outcomes, biomarkers that quantify periodontal pathogen loads may offer prognostic value for predicting progressive periodontitis in the subsequent supportive periodontal therapy phase. Additional research will be required to translate information regarding subgingival biofilm composition and phenotype into clinically relevant prognostic tools.

Accuracy of the American Association of Endodontists diagnostic criteria for assessing pulp health in primary teeth.

OBJECTIVES: There is a lack of studies evaluating the accuracy of the 2009 American Association of Endodontists (AAE) diagnostic criteria for diagnosing pulpal health in primary teeth. This study aimed to estimate and correlate the diagnostic accuracy of clinical diagnosis of reversible and irreversible pulpitis using the 2009 AAE criteria with histological findings in primary teeth. METHODS: Eighty primary teeth that were clinically diagnosed with normal pulp (n = 10), reversible pulpitis (n = 30), irreversible pulpitis (n = 30) and pulp necrosis (n = 10) were collected. The teeth were histo-processed, and pulp tissues were diagnosed histologically as uninflamed pulp, reversible or irreversibly inflamed and necrosis based on previously proposed criteria. RESULTS: The clinical diagnosis of pulp necrosis (sensitivity 70%, specificity 96%) and normal pulp (sensitivity 91%, specificity 100%) matched the histological diagnosis of necrosis and uninflamed pulp in 70% and 100%, respectively. The clinical diagnosis of irreversible pulpitis (sensitivity 64%, specificity 72%) matched the histological diagnosis of irreversible pulp inflammation for 47% of teeth evaluated. For the clinical diagnosis of reversible pulpitis (sensitivity: 65%, specificity: 86%), 80% matched the histological diagnosis of reversible pulp inflammation. Teeth with histologically diagnosed irreversible pulp inflammation were more likely to have lingering (OR 5.08; 95% CI 1.48-17.46, P = 0.010) and nocturnal tooth pain (OR 15.86; 95% CI 1.57-160.47, P = 0.019) when compared to teeth with reversible pulp inflammation. Using the classification and regression tree model, the presence of widened periodontal ligament space and nocturnal tooth pain were useful predictors of irreversible pulp inflammation with an accuracy of 78%. CONCLUSION: The 2009 AAE criteria was acceptable for primary teeth with pulp necrosis and normal pulp but poor for reversible pulpitis and irreversible pulpitis.

A critical analysis of research methods and biological experimental models to study pulp regeneration.

With advances in knowledge and treatment options, pulp regeneration is now a clear objective in clinical dental practice. For this purpose, many methodologies have been developed in attempts to address the putative questions raised both in research and in clinical practice. In the first part of this review, laboratory-based methods will be presented, analysing the advantages, disadvantages, and benefits of cell culture methodologies and ectopic/semiorthotopic animal studies. This will also demonstrate the need for alignment between two-dimensional and three-dimensional laboratory techniques to accomplish the range of objectives in terms of cell responses and tissue differentiation. The second part will cover observations relating to orthotopic animal studies, describing the current models used for this purpose and how they contribute to the translation of regenerative techniques to the clinic.

3D bioprinting and microscale organization of vascularized tissue constructs using collagen-based bioink.

Bioprinting three-dimensional (3D) tissue equivalents have progressed tremendously over the last decade. 3D bioprinting is currently being employed to develop larger and more physiologic tissues, and it is of particular interest to generate vasculature in biofabricated tissues to aid better perfusion and transport of nutrition. Having an advantage over manual culture systems by bringing together biological scaffold materials and cells in precise 3D spatial orientation, bioprinting could assist in placing endothelial cells in specific spatial locations within a 3D matrix to promote vessel formation at these predefined areas. Hence, in the present study, we investigated the use of bioprinting to generate tissue-level capillary-like networks in biofabricated tissue constructs. First, we developed a bioink using collagen type-1 supplemented with xanthan gum (XG) as a thickening agent. Using a commercial extrusion-based multi-head bioprinter and collagen-XG bioink, the component cells were spatially assembled, wherein the endothelial cells were bioprinted in a lattice pattern and sandwiched between bioprinted fibroblasts layers. 3D bioprinted constructs thus generated were stable, and maintained structural shape and form. Post-print culture of the bioprinted tissues resulted in endothelial sprouting and formation of interconnected capillary-like networks within the lattice pattern and between the fibroblast layers. Bioprinter-assisted spatial placement of endothelial cells resulted in fabrication of patterned prevascularized constructs that enable potential regenerative applications in the future.

Cellular ageing of oral fibroblasts differentially modulates extracellular matrix organization.

BACKGROUND AND OBJECTIVES: Ageing is associated with an impaired cellular function that can affect tissue architecture and wound healing in gingival and periodontal tissues. However, the impact of oral fibroblast ageing on the structural organization of the extracellular matrix (ECM) proteins is poorly understood. Hence, in this study, we investigated the impact of cellular ageing of oral fibroblasts on the production and structural organization of collagen and other ECM proteins. METHODS: Oral fibroblasts were serially subcultured, and replicative cellular senescence was assessed using population doubling time, Ki67 counts and expression of P21WAFI . The production and structural organization of ECM proteins were assessed at early (young-oFB) and late (aged-oFB) passages. The thickness and pattern of collagen produced by live cultures of young- and aged-oFB were assessed using a label-free and non-invasive second harmonic generation (SHG)-based multiphoton imaging. Expression of other ECM proteins (fibronectin, fibrillin, collagen-IV and laminins) was evaluated using immunocytochemistry and confocal microscopy-based depth profile analysis. RESULTS: Aged-oFB displayed a higher population doubling time, lower Ki67+ cells and higher expression of P21WAFI indicative of slower proliferation rate and senescence phenotype. SHG imaging demonstrated that young-oFB produced a thick, interwoven network of collagen fibres, while the aged-oFB produced thin and linearly organized collagen fibres. Similarly, analysis of immunostained cultures showed that young-oFB produced a rich, interwoven mesh of fibronectin, fibrillin and collagen-IV fibres. In contrast, the aged-oFB produced linearly organized fibronectin, fibrillin and collagen-IV fibres. Lastly, there was no observable difference in production and organization of laminins among the young- and aged-oFB. CONCLUSION: Our results suggest that oral fibroblast ageing impairs ECM production and more importantly the organization of ECM fibres, which could potentially impair wound healing in the elderly.

Pluripotent stem cells: An in vitro model for nanotoxicity assessments.

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd.

Stearic acid nanoparticles increase acyclovir absorption by oral epithelial cells.

OBJECTIVE: Acyclovir (ACY) is used to treat oral viral herpes but has low solubility and bioavailability. Stearic acid (SA) is lipophilic and can be combined with drugs. Therefore, this study aimed to characterize the properties of SA nanoparticles in increasing the cellular uptake of ACY by oral epithelial cells. The hypothesis was that SA nanoparticles increase sustained ACY release, are stable, and increase drug uptake. METHODS: The production parameters (duration and amplitude of sonication) were optimized to produce solid lipid nanoparticles (SLN) of SA-containing ACY. Particle stability was characterized under different storage conditions (4 °C and 37 °C for 1, 15, and 45 days). SLN were further characterized for their pharmacokinetic profile, cytotoxicity, in vitro permeability, and ability to modulate gene expression and promote ACY uptake by oral epithelial cells. RESULTS: Pharmacokinetic studies revealed sustained and diffusional release of ACY from the SLN, with an initial burst release of 15 min. After 45 d of storage, SLN kept at both 4 °C and 37 °C showed a maximum release of > 90 % of the drug at 120 min. Cells treated with SLN presented a significantly higher intracellular drug content than those treated with ACY and significantly increased the genetic expression of TJP-1, OCLN, and ECAD. SIGNIFICANCE: The hypothesis was accepted as SA nanoparticles containing ACY can sustain drug delivery and enhance its absorption into epithelial cells. Therefore, SA nanoparticles are promising for improving ACY uptake in treating oral herpes and other infections caused by HSV-1.

Biofabrication Strategies for Oral Soft Tissue Regeneration.

Gingival recession, a prevalent condition affecting the gum tissues, is characterized by the exposure of tooth root surfaces due to the displacement of the gingival margin. This review explores conventional treatments, highlighting their limitations and the quest for innovative alternatives. Importantly, it emphasizes the critical considerations in gingival tissue engineering leveraging on cells, biomaterials, and signaling factors. Successful tissue-engineered gingival constructs hinge on strategic choices such as cell sources, scaffold design, mechanical properties, and growth factor delivery. Unveiling advancements in recent biofabrication technologies like 3D bioprinting, electrospinning, and microfluidic organ-on-chip systems, this review elucidates their precise control over cell arrangement, biomaterials, and signaling cues. These technologies empower the recapitulation of microphysiological features, enabling the development of gingival constructs that closely emulate the anatomical, physiological, and functional characteristics of native gingival tissues. The review explores diverse engineering strategies aiming at the biofabrication of realistic tissue-engineered gingival grafts. Further, the parallels between the skin and gingival tissues are highlighted, exploring the potential transfer of biofabrication approaches from skin tissue regeneration to gingival tissue engineering. To conclude, the exploration of innovative biofabrication technologies for gingival tissues and inspiration drawn from skin tissue engineering look forward to a transformative era in regenerative dentistry with improved clinical outcomes.

Development and characterization of nitazoxanide-loaded poly(ε-caprolactone) membrane for GTR/GBR applications.

OBJECTIVE: Guided tissue/guided bone regeneration (GTR/GBR) membranes are widely used for periodontal bone regeneration, but their success depends on a bacteria-free environment. Systemic antibiotic treatment often proves inadequate, moreover, the increasing prevalence of antibiotic resistance in oral infections exacerbates this challenge. This study aimed to fabricate antibacterial membranes using a new class of antibiotics for local drug delivery, to eradicate infections and promote tissue regeneration. METHODS: Membranes loaded with nitazoxanide (NTZ) were fabricated via electrospinning using poly(ε-caprolactone) (PCL) with varying concentrations of NTZ (0 %, 2.5 %, and 5 % w/w) relative to the polymer weight. Morphochemical of NTZ-loaded membranes were assessed using scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and Fourier Transform Infrared spectroscopy (FTIR). Mechanical properties were evaluated using universal testing machine and NTZ release profile from membranes was determined by spectrophotometer (λmax = 444) for 14 days. Antimicrobial efficacy against periodontal pathogens, cell compatibility and mineralization were evaluated using periodontal ligament stem cells (PDLSCs). RESULTS: Optimized spinning parameter maintained a uniform fiber diameter and successful loading of NTZ was confirmed by SEM-EDS and FTIR. NTZ incorporation did not significantly affect mechanical properties, whereas the drug release kinetics showed an initial burst, followed by sustained release over 14 days. NTZ-loaded membranes demonstrated antibacterial activity against Aggregatibacter actinomycetemcomitans (Aa) and Fusobacterium nucleatum (Fn). Importantly, the presence of NTZ showed minimal cell toxicity; however, it reduced the mineralization potential compared with that of the pure PCL membrane, which increased over time. SIGNIFICANCE: Taken together, these findings established that NTZ-loaded membranes could be promising barrier membrane to counteract microbial environment and aid periodontal bone regeneration.

Waterpipe smoke condensate induces epithelial-mesenchymal transformation and promotes metastasis of oral cancer by FOXD1 expression.

BACKGROUND/PURPOSE: Smoking is a major contributor to global oral cancer cases, necessitating urgent intervention. FOXD1, involved in developmental processes and various cancers, shows promise as a prognostic marker in oral squamous cell carcinoma (OSCC). This study investigates the impact of waterpipe smoke condensate (WPSC) on OSCC, focusing on FOXD1 role in inducing epithelial-mesenchymal transition (EMT) and metastasis. METHODS: The study involved using OSCC cells treated with WPSC to evaluate their proliferation, colony formation, gene expression, and protein levels. The researchers also explored the clinical relevance of their findings using online databases to analyze FOXD1 expression in cancer tissues and its correlation with clinicopathological features and patient survival. Additionally, in silico tools were employed for functional analysis, pathway enrichment, and network exploration. RESULTS: The study found that WPSC increased the expression of FOXD1 in OSCC cells, which led to increased cell growth. The study also showed that FOXD1 plays a critical role in the EMT process induced by WPSC, as evidenced by changes in the expression of EMT-related genes and proteins. Clinical analysis revealed that FOXD1 was significantly associated with more aggressive tumor features and poorer prognosis in cancer patients. CONCLUSION: The study highlights FOXD1 as a key player in OSCC pathogenesis and a potential prognostic marker and therapeutic target, particularly when influenced by WPSC exposure. Further research is needed to explore FOXD1 molecular mechanisms and clinical implications to enhance OSCC treatment strategies.

Guidance on the assessment of biocompatibility of biomaterials: Fundamentals and testing considerations.

BACKGROUND: Assessing the biocompatibility of materials is crucial for ensuring the safety and well-being of patients by preventing undesirable, toxic, immune, or allergic reactions, and ensuring that materials remain functional over time without triggering adverse reactions. To ensure a comprehensive assessment, planning tests that carefully consider the intended application and potential exposure scenarios for selecting relevant assays, cell types, and testing parameters is essential. Moreover, characterizing the composition and properties of biomaterials allows for a more accurate understanding of test outcomes and the identification of factors contributing to cytotoxicity. Precise reporting of methodology and results facilitates research reproducibility and understanding of the findings by the scientific community, regulatory agencies, healthcare providers, and the general public. AIMS: This article aims to provide an overview of the key concepts associated with evaluating the biocompatibility of biomaterials while also offering practical guidance on cellular principles, testing methodologies, and biological assays that can support in the planning, execution, and reporting of biocompatibility testing.

Modeling periodontal host-microbe interactions using vascularized gingival connective tissue equivalents.

Gingival connective tissue and its vasculature play a crucial role in the host's immune response against the periodontal microbiome and serve as a bridge between the oral and systemic environments. However, there is a lack of representative models that mimic the complex features of vascularized gingival connective tissue and its interaction with the periodontal microbiome, hindering our understanding of periodontal health and disease. Towards this pursuit, we present the characterization of vascularized gingival connective tissue equivalents (CTEs) as a model to study the interactions between oral biofilm colonizers and gingival tissues in healthy and diseased states. Whole-mount immunolabeling and label-free confocal reflectance microscopy of human fibrin-based matrix embedded with gingival fibroblasts and microvascular endothelial cells demonstrated the generation of bi-cellular vascularized gingival CTEs. Next, we investigated the response of the vascularized gingival CTEs to early, intermediate, and late oral biofilm colonizers. Despite colonization, the early colonizers did not elicit any significant change in the production of the cytokines and chemokines by the CTEs representative of the commensal and homeostatic state. In contrast, intermediate and late colonizers representing a transition to a diseased state exhibited connective tissue and vascular invasion, and elicited a differential immune response accompanied by increased monocyte migration. The culture supernatants produced by the vascularized gingival CTEs in response to early and intermediate colonizers polarized macrophages towards an immunomodulatory M2-like phenotype which activates and protects the host, while the late colonizers polarized towards a pro-inflammatory M1-like phenotype. Lastly,in silicoanalysis showed a high strength of associations between the proteins and transcripts investigated with periodontitis and vascular diseases. In conclusion, the vascularized gingival CTEs provide a biomimeticin vitroplatform to study host-microbiome interactions and innate immune response in periodontal health and diseased states, which potentially paves the way toward the development and assessment of novel periodontal therapeutics.

Modeling Crevicular Fluid Flow and Host-Oral Microbiome Interactions in a Gingival Crevice-on-Chip.

Gingival crevice and gingival crevicular fluid (GCF) flow play a crucial role at the gingiva-oral microbiome interface which contributes toward maintaining the balance between gingival health and periodontal disease. Interstitial flow of GCF strongly impacts the host-microbiome interactions and tissue responses. However, currently available in vitro preclinical models largely disregard the dynamic nature of gingival crevicular microenvironment, thus limiting the progress in the development of periodontal therapeutics. Here, a proof-of-principle "gingival crevice-on-chip" microfluidic platform to culture gingival connective tissue equivalent (CTE) under dynamic interstitial fluid flow mimicking the GCF is described. On-chip co-culture using oral symbiont (Streptococcus oralis) shows the potential to recapitulate microbial colonization, formation of biofilm-like structures at the tissue-microbiome interface, long-term co-culture, and bacterial clearance secondary to simulated GCF (s-GCF) flow. Further, on-chip exposure of the gingival CTEs to the toll-like receptor-2 (TLR-2) agonist or periodontal pathogen Fusobacterium nucleatum demonstrates the potential to mimic early gingival inflammation. In contrast to direct exposure, the induction of s-GCF flow toward the bacterial front attenuates the secretion of inflammatory mediators demonstrating the protective effect of GCF flow. This proposed in vitro platform offers the potential to study complex host-microbe interactions in periodontal disease and the development of periodontal therapeutics under near-microphysiological conditions.

Microphysiological Modeling of Gingival Tissues and Host-Material Interactions Using Gingiva-on-Chip.

Gingiva plays a crucial barrier role at the interface of teeth, tooth-supporting structures, microbiome, and external agents. To mimic this complex microenvironment, an in vitro microphysiological platform and biofabricated full-thickness gingival equivalents (gingiva-on-chip) within a vertically stacked microfluidic device is developed. This design allowed long-term and air-liquid interface culture, and host-material interactions under flow conditions. Compared to static cultures, dynamic cultures on-chip enabled the biofabrication of gingival equivalents with stable mucosal matrix, improved epithelial morphogenesis, and barrier features. Additionally, a diseased state with disrupted barrier function representative of gingival/oral mucosal ulcers is modeled. The apical flow feature is utilized to emulate the mechanical action of mouth rinse and integrate the assessment of host-material interactions and transmucosal permeation of oral-care formulations in both healthy and diseased states. Although the gingiva-on-chip cultures have thicker and more mature epithelium, the flow of oral-care formulations induced increased tissue disruption and cytotoxic features compared to static conditions. The realistic emulation of mouth rinsing action facilitated a more physiological assessment of mucosal irritation potential. Overall, this microphysiological system enables biofabrication of human gingiva equivalents in intact and ulcerated states, providing a miniaturized and integrated platform for downstream host-material and host-microbiome applications in gingival and oral mucosa research.

Fluid flow-induced modulation of viability and osteodifferentiation of periodontal ligament stem cell spheroids-on-chip.

Developing physiologically relevant in vitro models for studying periodontitis is crucial for understanding its pathogenesis and developing effective therapeutic strategies. In this study, we aimed to integrate the spheroid culture of periodontal ligament stem cells (PDLSCs) within a spheroid-on-chip microfluidic perfusion platform and to investigate the influence of interstitial fluid flow on morphogenesis, cellular viability, and osteogenic differentiation of PDLSC spheroids. PDLSC spheroids were seeded onto the spheroid-on-chip microfluidic device and cultured under static and flow conditions. Computational analysis demonstrated the translation of fluid flow rates of 1.2 µl min-1 (low-flow) and 7.2 µl min-1 (high-flow) to maximum fluid shear stress of 59 µPa and 360 µPa for low and high-flow conditions, respectively. The spheroid-on-chip microfluidic perfusion platform allowed for modulation of flow conditions leading to larger PDLSC spheroids with improved cellular viability under flow compared to static conditions. Modulation of fluid flow enhanced the osteodifferentiation potential of PDLSC spheroids, demonstrated by significantly enhanced alizarin red staining and alkaline phosphatase expression. Additionally, flow conditions, especially high-flow conditions, exhibited extensive calcium staining across both peripheral and central regions of the spheroids, in contrast to the predominantly peripheral staining observed under static conditions. These findings highlight the importance of fluid flow in shaping the morphological and functional properties of PDLSC spheroids. This work paves the way for future investigations exploring the interactions between PDLSC spheroids, microbial pathogens, and biomaterials within a controlled fluidic environment, offering insights for the development of innovative periodontal therapies, tissue engineering strategies, and regenerative approaches.

Cell-derived nanovesicles from mesenchymal stem cells as extracellular vesicle-mimetics in wound healing.

Wound healing is a dynamic process that involves a series of molecular and cellular events aimed at replacing devitalized and missing cellular components and/or tissue layers. Recently, extracellular vesicles (EVs), naturally cell-secreted lipid membrane-bound vesicles laden with biological cargos including proteins, lipids, and nucleic acids, have drawn wide attention due to their ability to promote wound healing and tissue regeneration. However, current exploitation of EVs as therapeutic agents is limited by their low isolation yields and tedious isolation processes. To circumvent these challenges, bioinspired cell-derived nanovesicles (CDNs) that mimic EVs were obtained by shearing mesenchymal stem cells (MSCs) through membranes with different pore sizes. Physical characterisations and high-throughput proteomics confirmed that MSC-CDNs mimicked MSC-EVs. Moreover, these MSC-CDNs were efficiently uptaken by human dermal fibroblasts and demonstrated a dose-dependent activation of MAPK signalling pathway, resulting in enhancement of cell proliferation, cell migration, secretion of growth factors and extracellular matrix proteins, which all promoted tissue regeneration. Of note, MSC-CDNs enhanced angiogenesis in human dermal microvascular endothelial cells in a 3D PEG-fibrin scaffold and animal model, accelerating wound healing in vitro and in vivo. These findings suggest that MSC-CDNs could replace both whole cells and EVs in promoting wound healing and tissue regeneration.

Characterization of silver diamine fluoride cytotoxicity using microfluidic tooth-on-a-chip and gingival equivalents.

OBJECTIVE: This study aims to characterize the cytotoxicity potential of silver diamine fluoride (SDF) on dental pulp stem cells (DPSC) and gingival equivalents. METHODS: DPSC cultured on 96-well plates was exposed directly to SDF (0.0001-0.01%) and cell viability (IC50) quantified. Effect of SDF on DPSC viability under flow (with dentin barrier) conditions was evaluated using a custom-designed microfluidic "tooth-on-a-chip". Permeability of dentin discs (0.5-1.5 mm thickness) was evaluated using lucifer yellow permeation assay. Dentin discs were treated with 38% SDF (up to 3 h), and cell viability (live/dead assay) of the DPSC cultured in the inlet (unexposed) and outlet (exposed) regions of the pulp channel was evaluated. To assess the mucosal corrosion potential, gingival equivalents were treated with 38% SDF for 3 or 60 min (OECD test guideline 431) and characterized by MTT assay and histomorphometric analysis. RESULTS: DPSC exposed directly to SDF showed a dose-dependent reduction in cell viability (IC50: 0.001%). Inlet channels (internal control) of the tooth-on-a-chip exposed to PBS and SDF-exposed dentin discs showed> 85% DPSC viability. In contrast, the outlet channels of SDF-exposed dentin discs showed a decreased viability of< 31% and 0% (1.5 and ≤1.0 mm thick dentin disc, respectively) (p < 0.01). The gingiva equivalents treated with SDF for 3 and 60 min demonstrated decreased epithelial integrity, loss of intercellular cohesion and corneal layer detachment with significant reduction in intact epithelial thickness (p < 0.05). SIGNIFICANCE: SDF penetrated the dentin (≤1 mm thick) inducing significant death of the pulp cells. SDF also disrupted gingival epithelial integrity resulting in mucosal corrosion.

Differential immune responses of 3D gingival and periodontal connective tissue equivalents to microbial colonization.

Gingival and periodontal ligament fibroblasts are functionally distinct cell types within the dento-gingival unit that participate in host immune response. Their microenvironment influences the behavior and immune response to microbial challenge. We developed three-dimensional gingival and periodontal connective tissue equivalents (CTEs) using human fibrin-based matrix. The CTEs were characterized, and the heterogeneity in their innate immune response was investigated. The CTEs demonstrated no to minimal response to planktonic Streptococcus mitis and Streptococcus oralis, while their biofilms elicited a moderate increase in IL-6 and IL-8 production. In contrast, Fusobacterium nucleatum provoked a substantial increase in IL-6 and IL-8 production. Interestingly, the gingival CTEs secreted significantly higher IL-6, while periodontal counterparts produced higher IL-8. In conclusion, the gingival and periodontal CTEs exhibited differential responses to various bacterial challenges. This gives insights into the contribution of tissue topography and fibroblast heterogeneity in rendering protective and specific immune responses toward early biofilm colonizers.

Two-Photon Fluorescence Microscopy and Applications in Angiogenesis and Related Molecular Events.

The role of angiogenesis in health and disease have gained considerable momentum in recent years. Visualizing angiogenic patterns and associated events of surrounding vascular beds in response to therapeutic and laboratory-grade biomolecules has become a commonplace in regenerative medicine and the biosciences. To achieve high-quality imaging for elucidating the molecular mechanisms of angiogenesis, the two-photon excitation fluorescence (2PEF) microscopy, or multiphoton fluorescence microscopy is increasingly utilized in scientific investigations. The 2PEF microscope confers several distinct imaging advantages over other fluorescence excitation microscopy techniques-for the observation of in-depth, three-dimensional vascularity in a variety of tissue formats, including fixed tissue specimens and in vivo vasculature in live specimens. Understanding morphological and subcellular changes that occur in cells and tissues during angiogenesis will provide insights to behavioral responses in diseased states, advance the engineering of physiologically relevant tissue models, and provide biochemical clues for the design of therapeutic strategies. We review the applicability and limitations of the 2PEF microscope on the biophysical and molecular-level signatures of angiogenesis in various tissue models. Imaging techniques and strategies for best practices in 2PEF microscopy will be reviewed. Impact Statement Deep live tissue imaging provides unique opportunities to study angiogenesis and associated events in real-time. In contrast to cross-sectional data provided by conventional methods, two-photon microscopy enables high-resolution tissue imaging, data acquisition over time, real-time visualization of angiogenic events, and reduces the number of animal models used in scientific research. This review provides insights on different two-photon microscopy methods and its application in live and deep tissue imaging of angiogenesis on in vitro and in vivo tissues. We believe that the current trends in imaging can transform the investigation of angiogenesis, cancer research, and biofabrication of vascularized tissues.