Polymicrobial oral biofilm models: simplifying the complex

Over the past century, numerous studies have used oral biofilm models to investigate growth kinetics, biofilm formation, structure and composition, antimicrobial susceptibility and host–pathogen interactions. In vivo animal models provide useful models of some oral diseases; however, these are expensive and carry vast ethical implications. Oral biofilms grown or maintained in vitro offer a useful platform for certain studies and have the advantages of being inexpensive to establish and easy to reproduce and manipulate. In addition, a wide range of variables can be monitored and adjusted to mimic the dynamic environmental changes at different sites in the oral cavity, such as pH, temperature, salivary and gingival crevicular fluid flow rates, or microbial composition. This review provides a detailed insight for early-career oral science researchers into how the biofilm models used in oral research have progressed and improved over the years, their advantages and disadvantages, and how such systems have contributed to our current understanding of oral disease pathogenesis and aetiology

Oral plaque from Type 2 diabetic patients reduces the clonogenic capacity of dental pulp-derived mesenchymal stem cells

Type 2 diabetes (T2D) is a major metabolic disease and a key epigenetic risk factor for the development of additional clinical complications. Among them, periodontitis (PD), a severe inflammatory disease ascribable to a dysregulated physiology and composition of the oral microbiota, represents one of the most relevant complications. Periodontitis can impact the structure of the tooth and likely the stem and progenitor cell pool, which actively contributes to the periodontal microenvironment and homeostasis. Modifications of the oral plaque play a key role in the etiopathogenesis of PD caused by T2D. However, to what extent the biology of the progenitor pool is affected has still to be elucidated. In this short report, we aimed to explore the biological effects of oral plaque derived from T2D patients with PD in comparison to non-diabetic patients with PD. Oral plaque samples were isolated from T2D and non-diabetic subjects with PD. Dental pulp stem cells (DPSCs), derived from the premolar tooth, were conditioned for 21 days with oral plaque samples and tested for their clonogenic ability. Cultures were also induced to differentiate towards the osteogenic lineage, and ALP and osteocalcin gene expression levels were evaluated by real-time qPCR. Results have shown that the number of clones generated by DPSCs exposed to T2D oral plaque was significantly lower compared to controls (ctl). The multivariate analysis confirmed that the decreased clonogenesis was significantly correlated only with T2D diagnosis. Moreover, the effect of T2D oral plaque was specific to DPSCs. Indicators of osteogenic differentiation were not significantly affected. This study provides a new biological insight into the effects ascribable to T2D in PD

Use of bioinformatic strategies as a predictive tool in implant-supported oral rehabilitation: a scoping review

Statement of problem: The use of bioinformatic strategies is growing in dental implant protocols. The current expansion of Omics sciences and artificial intelligence (AI) algorithms in implant dentistry applications have not been documented and analyzed as a predictive tool for the success of dental implants. Purpose: The purpose of this scoping review was to analyze how artificial intelligence algorithms and Omics technologies are being applied in the field of oral implantology as a predictive tool for dental implant success. Material and methods: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews checklist was followed. A search strategy was created at PubMed and Web of Science to answer the question “How is bioinformatics being applied in the area of oral implantology as a predictive tool for implant success?” Results: Thirteen articles were included in this review. Only 3 applied bioinformatic models combining AI algorithms and Omics technologies. These studies highlighted 2 key points for the creation of precision medicine: deep population phenotyping and the integration of Omics sciences in clinical protocols. Most of the studies identified applied AI only in the identification and classification of implant systems, quantification of peri-implant bone loss, and 3-dimensional bone analysis, planning implant placement. Conclusions: The conventional criteria currently used as a technique for the diagnosis and monitoring of dental implants are insufficient and have low accuracy. Models that apply AI algorithms combined with precision methodologies—biomarkers—are extremely useful in the creation of precision medicine, allowing medical dentists to forecast the success of the implant. Tools that integrate the different types of data, including imaging, molecular, risk factor, and implant characteristics, are needed to make a more accurate and personalized prediction of implant success.info:eu-repo/semantics/acceptedVersio

Biofilm-stimulated epithelium modulates the inflammatory responses in co-cultured immune cells

The gingival epithelium is a physical and immunological barrier to the microbiota of the oral cavity, which interact through soluble mediators with the immune cells that patrol the tissue at the gingival epithelium. We sought to develop a three-dimensional gingivae-biofilm interface model using a commercially available gingival epithelium to study the tissue inflammatory response to oral biofilms associated with “health”, “gingivitis” and “periodontitis”. These biofilms were developed by sequential addition of microorganisms to mimic the formation of supra- and sub-gingival plaque in vivo. Secondly, to mimic the interactions between gingival epithelium and immune cells in vivo, we integrated peripheral blood mononuclear cells and CD14+ monocytes into our three-dimensional model and were able to assess the inflammatory response in the immune cells cultured with and without gingival epithelium. We describe a differential inflammatory response in immune cells cultured with epithelial tissue, and more so following incubation with epithelium stimulated by “gingivitis-associated” biofilm. These results suggest that gingival epithelium-derived soluble mediators may control the inflammatory status of immune cells in vitro, and therefore targeting of the epithelial response may offer novel therapies. This multi-cellular interface model, both of microbial and host origin, offers a robust in vitro platform to investigate host-pathogens at the epithelial surface

Assessing the association between oral hygiene and preterm birth by quantitative light-induced fluorescence

The aim of this study was to investigate the purported link between oral hygiene and preterm birth by using image analysis tools to quantify dental plaque biofilm. Volunteers (η = 91) attending an antenatal clinic were identified as those considered to be “at high risk” of preterm delivery (i.e., a previous history of idiopathic preterm delivery, case group) or those who were not considered to be at risk (control group). The women had images of their anterior teeth captured using quantitative light-induced fluorescence (QLF). These images were analysed to calculate the amount of red fluorescent plaque (ΔR%) and percentage of plaque coverage. QLF showed little difference in ΔR% between the two groups, 65.00% case versus 68.70% control, whereas there was 19.29% difference with regard to the mean plaque coverage, 25.50% case versus 20.58% control. A logistic regression model showed a significant association between plaque coverage and case/control status (Ρ = 0.031), controlling for other potential predictor variables, namely, smoking status, maternal age, and body mass index (BMI)

Pluronic F-127 hydrogel as a promising scaffold for encapsulation of dental-derived mesenchymal stem cells.

Dental-derived mesenchymal stem cells (MSCs) provide an advantageous therapeutic option for tissue engineering due to their high accessibility and bioavailability. However, delivering MSCs to defect sites while maintaining a high MSC survival rate is still a critical challenge in MSC-mediated tissue regeneration. Here, we tested the osteogenic and adipogenic differentiation capacity of dental pulp stem cells (DPSCs) in a thermoreversible Pluronic F127 hydrogel scaffold encapsulation system in vitro. DPSCs were encapsulated in Pluronic (®) F-127 hydrogel and stem cell viability, proliferation and differentiation into adipogenic and osteogenic tissues were evaluated. The degradation profile and swelling kinetics of the hydrogel were also analyzed. Our results confirmed that Pluronic F-127 is a promising and non-toxic scaffold for encapsulation of DPSCs as well as control human bone marrow MSCs (hBMMSCs), yielding high stem cell viability and proliferation. Moreover, after 2 weeks of differentiation in vitro, DPSCs as well as hBMMSCs exhibited high levels of mRNA expression for osteogenic and adipogenic gene markers via PCR analysis. Our histochemical staining further confirmed the ability of Pluronic F-127 to direct the differentiation of these stem cells into osteogenic and adipogenic tissues. Furthermore, our results revealed that Pluronic F-127 has a dense tubular and reticular network morphology, which contributes to its high permeability and solubility, consistent with its high degradability in the tested conditions. Altogether, our findings demonstrate that Pluronic F-127 is a promising scaffold for encapsulation of DPSCs and can be considered for cell delivery purposes in tissue engineering

Functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy

Bio-hybrid artificial organs are an attractive concept to restore organ function through precise biological cooperation with surrounding tissues in vivo. However, in bio-hybrid artificial organs, an artificial organ with fibrous connective tissues, including muscles, tendons and ligaments, has not been developed. Here, we have enveloped with embryonic dental follicle tissue around a HA-coated dental implant, and transplanted into the lower first molar region of a murine tooth-loss model. We successfully developed a novel fibrous connected tooth implant using a HA-coated dental implant and dental follicle stem cells as a bio-hybrid organ. This bio-hybrid implant restored physiological functions, including bone remodelling, regeneration of severe bone-defect and responsiveness to noxious stimuli, through regeneration with periodontal tissues, such as periodontal ligament and cementum. Thus, this study represents the potential for a next-generation bio-hybrid implant for tooth loss as a future bio-hybrid artificial organ replacement therapy

Comparison of digital and conventional radiographic techniques

This study aimed to evaluate the correlation between conventional and digital radiographic methods in the measurement of periapical lesions in primary molars and compares the time used to obtain the radiographic images betweenboth methods. Material and methods: This crossover study included children between 4 to 8-year-old with periapical lesion in primary mandibular molars. Fifteen molars were randomly assigned firstly to receive conventional or digital periapical radiograph during the steps of endodontic treatment. The time to obtain the radiographic image was evaluated in seconds and compared by the Mann-Whitney test. The periapical lesions measurement (mm2) were performed by the Image J software and the degree of correlation of measurement between both techniques was evaluated by the Spearman correlation test. Data was analyzed using the GraphPad Prism software (α = 0.05). Results: A strong positive correlation between the measurement of lesions occurred on conventional methods in comparison with the measurement on digital methods (r2=0.778; p<0.0006); however, the time to obtain the radiographic images was shorter in the digital method (p<0.0001). Conclusion: The digital method had a shorter amount of time to obtain the images and strong correlation for the lesions measurement in comparison to the conventional method. Therefore, the digital radiograph method is preferable for use in children

Bioengineering and Cell-derived Strategies for Salivary Gland Regeneration

Xerostomia (dry mouth symptoms) is a group of incurable debilitating conditions of salivary glands caused by aging, radiation/chemical exposure, or aberrant inflammation in the salivary glands. During this PhD thesis, we aimed to evaluate whether cell-derived strategies (e.g., extracellular vesicles, EVs) could be a potential new therapy to ameliorate salivary gland injury and restore function after radiotherapy or in autoimmune diseases. In addition, we aimed to develop new imaging techniques for both 2D and 3D analysis of larger samples which allows for quantification of disease and regenerative features. Firstly, we constructed an in vivo murine model of 25 Gy irradiation-induced salivary gland damage to evaluate the potential of human dental pulp stem cell (hDPSCs)-derived EVs. EVs were injected 3x weekly via tail vein, beginning immediately after irradiation. Salivary gland function was evaluated 18 days after irradiation using salivary gland flow rate (SFR), gene expression (by qRT-PCR) and histopathology. Next, we tested different methods to generate PCSS using a vibratome and evaluated the slices in terms of viability (by WST-1), gene expression (by qRT-PCR), secreted α-amylase activity (by α-amylase assay kit) and histological/light sheet fluorescence microscopy (LSFM) three-dimensional imaging. Following irradiation, SFR decreased while senescence-associated β-galactosidase-positive cells (via immunofluorescences) and senescence-related genes and secretory-phenotypes (e.g., p21 and MMP3 in qRT-PCR) increased. SFR was unchanged following EVs treatment, but senescence-associated genes and secretory-phenotypes decreased. We also demonstrated that in an animal model of Sjögren’s syndrome, which exhibit dry mouth symptoms, that hDPSCs-EVs could inhibit the acquisition of the senescent phenotype in salivary gland epithelial cells (SGECs) and alleviate the loss of glandular function. EVs were also found to perform these effects through an underlying immunomodulatory mechanism. For PCSS, we developed protocols to produce viable slices of controled thicknesses which retained the ability to secrete functional α-amylase for at least two days in ex vivo culture. Phenotypic salivary gland cell epithelial markers (e.g., Keratin 5 and Aquaporin 5) increased over time in PCSS (by qRT-PCR), indicating the retention of cells that are necessary for salivary glands’ function. We developed workflows to perform LSFM 3D visualization in whole salivary glands as well as the PCSS model. In conclusion, hDPSCs-EVs reduced senescence of salivary gland epithelial cells in both murine irradiation and Sjögren’s syndrome models and may become a promising future for xerostomia patients. For the murine PCSS, we successfully established an executable operating procedure at the methodological level to reliably generate viable and functional murine PCSS and developed new state-of-the-art analytical methods (such as LFSM 3D imaging and qRT-PCR) to increase the diversity of objective tools to evaluate PCSS. Therefore, this work laid the foundation for the future application of other therapies (such as irradiation therapy or EVs therapy) to the PCSS model. Those future applications could include drug screening or mechanism of injury study. At the same time, we developed a sustainable histology process to reduce xylene utilization in histological processing for salivary gland tissue processing. Therefore, this work has developed a set of in vitro and in vivo experiments with state-of-the-art methods to better understand disease mechanisms and to evaluate new therapies for salivary glands