Oriana Trubiani

This in vitro study aimed to analyze the activation of the NFκB p65/MyD88/NALP3 inflammatory pathway and the expression of the BCL-2/BAX/Caspase-3 apoptosis pathway on human gingival fibroblasts (hGFs) after 24 h and 1-week exposure to the eluates of three light-cured commercial composites (GR: GrandioSO, VOCO; BF: Enamel Plus HRi Biofunction, Micerium; FU: Filtek universal restorative, 3M) and the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). A total of 50 light-cured disk-shaped specimens for each composite group (GR, BF and FU) were produced. The tested materials differed in resin matrix composition; notably, GR contained Bis-GMA, whereas BF and FU contained urethane-based dimethacrylates. The hGFs were exposed for 24 h and 1 week to eluates of each group. Pathway activation was evaluated by cell metabolic activity, immunofluorescence analysis and real-time PCR. Inflammatory effects were assessed in hGF culture by measuring proinflammatory cytokine production (ELISA). GR, BF and FU showed a reduction in metabolic activity in a time-dependent manner. GR showed a significant decrease in terms of cell viability after 1 week of exposure and a greater expression of proapoptotic markers than BF and FU. High levels of proinflammatory cytokines were measured after 1 week of culture for GR. The inflammatory pathway was activated in a time-dependent manner. Pathway activation was material-dependent; among the tested composites, GR was found to be more cytotoxic than BF and FU. Despite the limitations of an in vitro study, these findings suggest that dental composite eluates cause time- and material-dependent cytotoxicity, inflammation, and apoptosis in hGFs. Exposure significantly impairs cellular metabolic activity from 24 h up to 1 week. Among the evaluated materials, the GR composite triggered a markedly severe inflammatory and apoptotic effect compared to BF or FU. Ultimately, these findings underscore the potential clinical importance of selecting materials with optimal biocompatibility to minimize adverse tissue reactions, although their direct translation into clinical practice would require further research.

Daily, polluting substances are scattered in the atmosphere by industries, which can potentially damage human health. Particular concern derives from those substances defined as endocrine disruptors Chemicals, which mimic the actions of natural hormones and cause adverse effects on the normal functioning of developmental, metabolic, and reproductive processes like embryonic development, gonad formation, sexual differentiation, and growth, that may lead to the loss of homeostasis. Moreover, perinatal exposure to substances with estrogenic activity may be able to cross the placenta and produce long-lasting consequences in the fetus. Among the potentially contaminating substances involved in endocrine disruption, it is necessary to highlight Bisphenol A (BPA) due to its adverse effects on human health. We used an in vitro model of human periodontal ligament stem cells (hPDLSCs) differentiated into endothelial cells (e-hPDLSCs) to evaluate the angiogenesis effects of BPA. Our data showed that BPA (10 μM) presented a high expression of angiogenesis markers, such as PECAM-1, VEGF, VEGFR, and vWF in hPDLSCs in e-hPDLSCs. The epigenetic mechanism explains these adverse effects caused by BPA, as the vesicles obtained from BPA-treated e-hPDLSCs presented a lower expression of the miRNAs correlated with the angiogenic genes analyzed than the vesicles obtained from untreated e-hPDLSCs.

The use of dental composite resins has significantly increased over recent years, thanks to their esthetics and mechanical features, despite some doubts being raised about their biocompatibility. Residual methacrylate can be eluted from bulk composites, and its amount may significantly increase over time, leading to cytotoxic effects that can involve several inflammatory patterns. The aim of this in vitro study was to evaluate the activation of the inflammatory pathway NFκB p65/MyD88/NALP3 and the apoptosis pathway of BCL-2/BAX/Caspase-3 (CASP-3) expression on human gingival fibroblasts (hGFs) after 24 h and 1-week exposure to the eluates of three heat-cured dental composite resins: GrandioSO, VOCO (GR); Enamel Plus HRi Biofunction, Micerium (BF); and Filtek universal restorative, 3M (FU). The results highlighted that NFκB p65/MyD88/NALP3 was activated after resin exposure in a time-dependent manner. Moreover, immunofluorescence and gene expression analyses showed that pro-apoptotic markers CASP-3 and BAX were elevated, while anti-apoptotic protein BCL-2 was suppressed in hGFs after dental resin exposure. The present in vitro study analyzed mechanisms related to cytotoxicity and apoptosis, suggesting ways to limit composite cytotoxicity through advancements in material technology. The activation of inflammation and apoptotic pathways appeared to be material-dependent, and was less pronounced with BF and FU, which could hypothetically indicate them being a safer clinical choice to preserve periodontal health in daily dental practice.

Regenerative medicine is the branch of medicine that aims to repair and regenerate damaged tissues and presents promising avenues for addressing a wide range of currently incurable diseases. Regenerative medicine is based on the use of cell therapy with stem cells that can differentiate into differentiated cells of specific tissues. There are various types of stem cells, which are different in potential and derivation. The aim of this review is to summarize the types of stem cells most studied and recently discovered, from adult stem cells to innovative induced pluripotent stem cells (iPSCs), for regenerative medicine purposes. The stem cells involved in the identification of new regenerative therapeutic approaches are analyzed here through a classification based on the tissues’ embryonic derivation: stem cells from ectodermal derivation tissues, stem cells from mesodermal derivation tissues, stem cells from endodermal derivation tissues, and iPSCs.

Two-dimensional culture systems have been used for a long time in the research field but their disadvantages make it difficult to reproduce the in vivo environment. Three-dimensional culture systems overcome these limitations, simulating the physiological context of an organism, from the molecular level to the cellular, tissue, and organ complexity levels. This review focuses on 3D cellular models, such as spheroids and tumoroids, which reproduce tumor heterogeneity and microenvironments. It also includes 3D cultures of mesenchymal stem cells (MSCs), particularly those derived from teeth. In conclusion, 3D models are profoundly impacting the biomedical field by offering more accurate in vitro platforms for drug development and disease modeling, thereby significantly reducing the reliance on animal testing and leading to the advancement of personalized and regenerative medicine.

Barrier function regulation, angiogenic potential, and immune response modulation are only a few of the many roles of the vascular system that nowadays represent one of the main targets for environmental pollutants, in particular, pesticides. We have used human umbilical vein endothelial cells (HUVECs) as an in vitro model to investigate the effects of pesticides on the activation of the NALP3-CASP1-IL-1β inflammatory pathway using real time PCR (RT-PCR) and immunofluorescence investigations, reactive oxygen species (ROS) generation, and morphological alterations with scanning electron microscopy (SEM) analysis. Our findings offer a comprehensive evaluation of the cellular and molecular damage induced by pesticide exposure and show strong inflammasome activation. They indicate that these chemicals may initiate necroptosis and drive prolonged inflammation in endothelial cells. This study provides crucial insights into how pesticides contribute to endothelial dysfunction, highlighting the need for further investigation into their inflammatory and immune-modulatory effects on vascular health.

Background: The aim of this study was to evaluate blood pressure (BP) difference between cuff inflation and deflation and to investigate whether hypertension grade and patients’ characteristics could be influential. Methods: We selected 328 asymptomatic patients, 219 with mild systolic/diastolic hypertension, 75 with moderate systolic/diastolic hypertension and 34 with severe systolic/diastolic hypertension. Results: Hypertension grade was a determinant of the difference between systolic/diastolic BP during cuff inflation and deflation; the difference was progressively and significantly higher from mild to moderate to severe hypertension (1.80 ± 1.03/1.21 ± 0.56 vs. 5.32 ± 1.09/3.04 ± 0.81 vs. 9.74 ± 1.46/4.88 ± 0.73 mmHg, respectively (all differences were significant). Age, gender, body mass index, smoking habits and laboratory parameters were not associated with BP differences. The observed differences led to a reclassification of 24% of patients with moderate and 32% of patients with severe hypertension to a lower grade, but all were classified as hypertensive patients during cuff inflation. Conclusions: Hypertension grade influences the difference in systolic/diastolic BP during cuff inflation and deflation. This difference leads to a reclassification of hypertension grade during cuff inflation within the hypertensive range but does not influence the definition of hypertensive status. Future studies are needed to confirm whether the differences in systolic/diastolic BP between cuff inflation and deflation are due to BP increases induced by sympathetic activation, as well as a potential different behavior of the brachial artery during closing or opening, or both.

Introduction: In recent years, advancements in technology and the refinement of engineering techniques have facilitated the development of tissue engineering, placing particular emphasis on the use of 3D-biomaterials with several structural and chemical geometric features. In particular, increasing information on biomaterial geometric surfaces has allowed for a better understanding of tissue regenerative processes. In the present study a comparison between BioRipar^®, bovine pericardium membrane, modified with micrometric roundish regular open pores (BioR-Ps) and BioRipar^® without pores (BioR-NPs) has been investigated.Methods: The expression of adhesion molecules such as: fibronectin, vimentin, focal adhesion kinase (FAK), integrin 1β, integrin α5, E-cadherin, and molecules involved in neovascularization processes such as: vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGF-R) were evaluated in an in vitro model containing primary culture of human periodontal ligament stem cells (hPDLSCs) through multiparametric analysis.Results: The results indicated a markedly significant expression of all the abovementioned molecules in hPDLSCs cultured withBioR-Ps compared to hPDLSCs cultured with BioR-NPs. Scanning electron microscopy analysis indicated a marked interaction between the cells and the substrate, particularly evident in the proximity of open pores in the hPDLSCs cultured on the BioR-P surface compared to hPDLSCs cultured on the BioR-NP surface. Thus, the presence of micrometric open pores on the scaffold stimulates the proliferation potential of cells apart from their adhesion ability on the patch, particularly near the poresDiscussion: Expression of angiogenic molecules strengthened the performance of the modified BioR-Ps. During synthesis, 3D-biomaterial micrometric open-pores enable better bonding between cells and materials, increasing contact area and promoting cellular molecular signals in biomaterial-guided tissue engineering.

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers’ markers’ expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration.

The use of temporary resin for provisional restorations is a fundamental step to maintain the position of prepared teeth, to protect the pulpal vitality and the periodontal health as well as the occlusion. The present study aimed at evaluating the biological effects of two resins used in dentistry for temporary restorations, Coldpac (Yates Motloid) and ProTemp 4™ (3M ESPE ™), and their eluates, in an in vitro model of human gingival fibroblasts (hGFs). The activation of the inflammatory pathway NFκB p65/NLRP3/IL-1β induced by the self-curing resin disks was evaluated by real-time PCR, Western blotting and immunofluorescence analysis. The hGFs adhesion on resin disks was investigated by means of inverted light microscopy and scanning electron microscopy (SEM). Our results suggest that hGF cells cultured in adhesion and with eluate derived from ProTemp 4™ (3M ESPE ™) resin evidenced a downregulation in the expression of the inflammatory mediators such as NFκB p65, NLRP3 and IL-1β compared to the cells cultured with Coldpac (Yates Motloid) after 24 h and 1 week of culture. Furthermore, the cells cultured with ProTemp 4™ (3M ESPE ™) after 24 h and 1 week of culture reported a higher cell viability compared to the cells cultured with Coldpac (Yates Motloid), established by MTS cell analysis. Similar results were obtained when hGFs were placed in culture with the eluate derived from ProTemp 4™ (3M ESPE ™) resin which showed a higher cell viability compared to the cells cultured with eluate derived from Coldpac (Yates Motloid). These results highlighted the lower pro-inflammatory action and improved cell biocompatibility of ProTemp 4™ (3M ESPE ™), suggesting a better performance in terms of cells–material interaction.