Enriched Graphene Oxide-Polypropylene Suture Threads Buttons Modulate the Inflammatory Pathway Induced by Escherichia coli Lipopolysaccharide.

Graphene oxide (GO), derived from graphene, has remarkable chemical-physical properties such as stability, strength, and thermal or electric conductivity and additionally shows antibacterial and anti-inflammatory properties. The present study aimed to evaluate the anti-inflammatory effects of polypropylene suture threads buttons (PPSTBs), enriched with two different concentrations of GO, in the modulation of the inflammatory pathway TLR4/MyD 88/NFκB p65/NLRP3 induced by the Escherichia coli (E. coli) lipopolysaccharide (LPS-E). The gene and the protein expression of inflammatory markers were evaluated in an in vitro model of primary human gingival fibroblasts (hGFs) by real-time PCR, western blotting, and immunofluorescence analysis. Both GO concentrations used in the polypropylene suture threads buttons-GO constructs (PPSTBs-GO) decreased the expression of inflammatory markers in hGFs treated with LPS-E. The hGFs morphology and adhesion on the PPSTBs-GO constructs were also visualized by inverted light microscopy, scanning electron microscopy (SEM), and real-time PCR. Together, these results suggest that enriched PPSTBs-GO modulates the inflammatory process through TLR4/MyD 88/NFκB p65/NLRP3 pathway.

Current Evidence on Bisphenol A Exposure and the Molecular Mechanism Involved in Related Pathological Conditions.

Bisphenol A (BPA) is one of the so-called endocrine disrupting chemicals (EDCs) and is thought to be involved in the pathogenesis of different morbid conditions: immune-mediated disorders, type-2 diabetes mellitus, cardiovascular diseases, and cancer. The purpose of this review is to analyze the mechanism of action of bisphenol A, with a special focus on mesenchymal stromal/stem cells (MSCs) and adipogenesis. Its uses will be assessed in various fields: dental, orthopedic, and industrial. The different pathological or physiological conditions altered by BPA and the related molecular pathways will be taken into consideration.

Correction: Marconi et al. Human Periodontal Ligament Stem Cells Response to Titanium Implant Surface: Extracellular Matrix Deposition. Biology 2021, 10, 931.

In the original publication [...].

Hypoxia: molecular pathophysiological mechanisms in human diseases

Hypoxia, a low O2 tension, is a fundamental feature that occurs in physiological events as well as pathophysiological conditions, especially mentioned for its role in the mechanism of angiogenesis, glucose metabolism, and cell proliferation/survival. The hypoxic state through the activation of specific mechanisms is an aggravating circumstance commonly noticed in multiple sclerosis, cancer, heart disease, kidney disease, liver disease, lung disease, and in inflammatory bowel disease. On the other hand, hypoxia could play a key role in tissue regeneration and repair of damaged tissues, especially by acting on specific tissue stem cells, but their features may result as a disadvantage when it is concerned for neoplastic stem cells. Furthermore, hypoxia could also have a potential role in tissue engineering and regenerative medicine due to its capacity to improve the performance of biomaterials. The current review aims to highlight the hypoxic molecular mechanisms reported in different pathological conditions to provide an overview of hypoxia as a therapeutic agent in regenerative and molecular therapy. (AU)

Hypoxia: molecular pathophysiological mechanisms in human diseases.

Hypoxia, a low O2 tension, is a fundamental feature that occurs in physiological events as well as pathophysiological conditions, especially mentioned for its role in the mechanism of angiogenesis, glucose metabolism, and cell proliferation/survival. The hypoxic state through the activation of specific mechanisms is an aggravating circumstance commonly noticed in multiple sclerosis, cancer, heart disease, kidney disease, liver disease, lung disease, and in inflammatory bowel disease. On the other hand, hypoxia could play a key role in tissue regeneration and repair of damaged tissues, especially by acting on specific tissue stem cells, but their features may result as a disadvantage when it is concerned for neoplastic stem cells. Furthermore, hypoxia could also have a potential role in tissue engineering and regenerative medicine due to its capacity to improve the performance of biomaterials. The current review aims to highlight the hypoxic molecular mechanisms reported in different pathological conditions to provide an overview of hypoxia as a therapeutic agent in regenerative and molecular therapy.

C-Myc Expression in Oral Squamous Cell Carcinoma: Molecular Mechanisms in Cell Survival and Cancer Progression.

Oral squamous cell carcinoma (OSCC) represents 90% of malignant epithelial cancer that occurs in the oral cavity. The c-Myc factor is expressed in multiple types of cancer, comprising head and neck squamous cell carcinoma (HNSCC), where it plays a fundamental role in tumor prognosis and in the self-renewal of tumor stem cells. However, the role of c-Myc in controlling OSCC cells is not well-known. The aim of the present study is the evaluation of the biological roles and regulatory mechanism of c-Myc in the pathogenesis of OSCC. Results indicated that c-Myc, c-Jun, Bcl-2, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), ERK 1/2 and pERK1/2 were overexpressed in a cellular model of squamous cell carcinoma, Cal-27. Doxorubicin (Doxo), a common chemotherapeutic agent, inhibited cell invasion, hypoxia, angiogenesis and inflammation in a cellular model of Cal-27 cells as indicated by downregulation of MMP-9, VEGF, ERK 1/2 and pERK 1/2 as well as promoted apoptosis as evidenced by the downregulation of Bcl-2 protein. This work aimed at underlying the functional relevance of c-Myc in OSCC and the HIF-Myc collaboration by integrating the knowledge on this molecular link in an OSCC tumor microenvironment. The results obtained showed for the first time the vital role of c-Myc in Cal-27 in cell survival/proliferation and tumor growth as well as the negative regulatory effect of Doxo against c-Myc signaling pathway.

Improved osteogenic differentiation by extremely low electromagnetic field exposure: possible application for bone engineering.

Human periodontal ligament mesenchymal stem cells (hPDLSCs) are a promising cell type model for regenerative medicine applications due to their anti-inflammatory, immunomodulatory and non-tumorigenic potentials. Extremely low-frequency electromagnetic fields (ELF-EMF) are reported to affect biological properties such as cell proliferation and differentiation and modulate gene expression profile. In this study, we investigated the effects of an intermittent ELF-EMF exposure (6 h/day) for the standard differentiation period (28 days) and for 10 days in hPDLSCs in the presence or not of osteogenic differentiation medium (OM). We evaluated cell proliferation, de novo calcium deposition and osteogenic differentiation marker expression in sham and ELF-EMF-exposed cells. After ELF-EMF exposure, compared with sham-exposed, an increase in cell proliferation rate (p < 0.001) and de novo calcium deposition (p < 0.001) was observed after 10 days of exposure. Real-time PCR and Western blot results showed that COL1A1 and RUNX-2 gene expression and COL1A1, RUNX-2 and OPN protein expression were upregulated respectively in the cells exposed to ELF-EMF exposure along with or without OM for 10 days. Altogether, these results suggested that the promotion of osteogenic differentiation is more efficient in ELF-EMF-exposed hPDLSCs. Moreover, our analyses indicated that there is an early induction of hPDLSC differentiation after ELF-EMF application.

A Narrative Review: Gingival Stem Cells as a Limitless Reservoir for Regenerative Medicine.

The gingival tissue can be collected in an easy way and represent an accessible source to isolate gingival-derived mesenchymal stem cells (GMSCs). GMSCs are a subpopulation of dental-derived mesenchymal stem cells that show the mesenchymal stem cells (MSCs) features, such as differentiation abilities and immunomodulatory properties. Dental-derived stem cells are also expandable in vitro with genomic stability and the possibility to maintain the stemness properties over a prolonged period of passages. Moreover, several preclinical studies have documented that the extracellular vesicles (EVs) released from GMSCs possess similar biological functions and therapeutic effects. The EVs may represent a promising tool in the cell-free regenerative therapy approach. The present review paper summarized the GMSCs, their multi-lineage differentiation capacities, immunomodulatory features, and the potential use in the treatment of several diseases in order to stimulate tissue regeneration. GMSCs should be considered a good stem cell source for potential applications in tissue engineering and regenerative dentistry.

Decellularized Dental Pulp, Extracellular Vesicles, and 5-Azacytidine: A New Tool for Endodontic Regeneration.

Dental pulp is a major component of the dental body that serves to maintain the tooth life and function. The aim of the present work was to develop a system that functions as a growth-permissive microenvironment for dental pulp regeneration using a decellularized dental pulp (DDP) matrix, 5-Aza-2'-deoxycytidine (5-Aza), and Extracellular Vesicles (EVs) derived from human Dental Pulp Stem Cells (hDPSCs). Human dental pulps extracted from healthy teeth, scheduled to be removed for orthodontic purpose, were decellularized and then recellularized with hDPSCs. The hDPSCs were seeded on DDP and maintained under different culture conditions: basal medium (CTRL), EVs, 5-Aza, and EVs+-5-Aza. Immunofluorescence staining and Western blot analyses were performed to evaluate the proteins' expression related to dentinogenesis, such as ALP, RUNX2, COL1A1, Vinculin, DMP1, and DSPP. Protein contents found in the DDP recellularized with hDPSCs were highly expressed in samples co-treated with EVs and 5-Aza compared to other culture conditions. This study developed a DDP matrix loaded by hDPSCs in co-treatment with EVs, which might enhance the dentinogenic differentiation with a high potentiality for endodontic regeneration.

The Beneficial Effect of Carvacrol in HL-1 Cardiomyocytes Treated with LPS-G: Anti-Inflammatory Pathway Investigations.

Carvacrol (CAR), a natural phenolic monoterpene, possesses different biological activities, such as anti-inflammatory and antioxidant activities. The current study aimed to evaluate the response of HL-1 cardiomyocytes to an inflammatory stimulus triggered by lipopolysaccharide from Porphyromonas gingivalis (LPS-G), alone or in co-treatment with CAR, to investigate the potential protective role of CAR in the inflammatory process through modulation of the TLR4/NFκB/NALP3/IL-1ß pathway and ROS production. In an in vitro experiment, HL-1 cardiomyocytes were exposed to LPS-G and incubated with CAR. We evaluated the anti-inflammatory effect of CAR by the reduction in TLR4, NFκB, NALP3, and IL-1ß expression using immunofluorescence staining. Western blot analysis also validated the modulation of the TLR4/NFκB/NALP3/IL-1ß pathway. ROS analyses confirmed the protective effects of CAR. Our results suggest that CAR could provide a significant protection role against inflammatory stimulus generated by LPS-G, involving the suppression of the TLR4/NFκB/NALP3/IL-1ß signaling pathway.

Enhanced Extracellular Matrix Deposition on Titanium Implant Surfaces: Cellular and Molecular Evidences.

The surface structure of the titanium dental implants can modulate the activity of mesenchymal stem cells in order to promote the upregulation of osteoblastic related genes and the release of extracellular matrix (ECM) components. The present work was focused on the in vitro evaluation of the interaction of human periodontal ligament stem cells (hPDLSCs) and two different implant titanium surfaces topography (CTRL and TEST). This study was aimed at analyzing the cytotoxicity of the dental implant surfaces, the cellular adhesion capacity, and the improvement in the release of ECM molecules in an in vitro model. These parameters were carried out by means of the microscopic evaluation, viability assays, immunofluorescence, Western blot and RT-PCR investigations. The knowledge of the cell/implant interaction is essential for implant healing in order to obtain a more performing surfaces that promote the ECM release and provide the starting point to initiate the osseointegration process.

Human Periodontal Ligament Stem Cells Response to Titanium Implant Surface: Extracellular Matrix Deposition.

The major challenge for dentistry is to provide the patient an oral rehabilitation to maintain healthy bone conditions in order to reduce the time for loading protocols. Advancement in implant surface design is necessary to favour and promote the osseointegration process. The surface features of titanium dental implant can promote a relevant influence on the morphology and differentiation ability of mesenchymal stem cells, induction of the osteoblastic genes expression and the release of extracellular matrix (ECM) components. The present study aimed at evaluating the in vitro effects of two different dental implants with titanium surfaces, TEST and CTRL, to culture the human periodontal ligament stem cells (hPDLSCs). Expression of ECM components such as Vimentin, Fibronectin, N-cadherin, Laminin, Focal Adhesion Kinase (FAK) and Integrin beta-1 (ITGB1), and the osteogenic related markers, as runt related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), were investigated. Human PDLSCs cultured on the TEST implant surface demonstrated a better cell adhesion capability as observed by Scanning Electron Microscopy (SEM) and immunofluorescence analysis. Moreover, immunofluorescence and Western blot experiments showed an over expression of Fibronectin, Laminin, N-cadherin and RUNX2 in hPDLSCs seeded on TEST implant surface. The gene expression study by RT-PCR validated the results obtained in protein assays and exhibited the expression of RUNX2, ALP, Vimentin (VIM), Fibronectin (FN1), N-cadherin (CDH2), Laminin (LAMB1), FAK and ITGB1 in hPDLSCs seeded on TEST surface compared to the CTRL dental implant surface. Understanding the mechanisms of ECM components release and its regulation are essential for developing novel strategies in tissue engineering and regenerative medicine. Our results demonstrated that the impact of treated surfaces of titanium dental implants might increase and accelerate the ECM apposition and provide the starting point to initiate the osseointegration process.

Role of ascorbic acid in the regulation of epigenetic processes induced by Porphyromonas gingivalis in endothelial-committed oral stem cells.

Periodontitis is a common inflammatory disease that affects the teeth-supporting tissue and causes bone and tooth loss. Moreover, in a worldwide population, periodontal disease is often associated with cardiovascular diseases. Emerging studies have reported that one of the major pathogens related to periodontitis is Porphyromonas gingivalis (P. gingivalis), which triggers the inflammatory intracellular cascade. Here, we hypothesized a possible protective effect of ascorbic acid (AA) in the restoration of the physiological molecular pathway after exposure to lipopolysaccharide derived from P. gingivalis (LPS-G). In particular, human gingiva-derived mesenchymal stem cells (hGMSCs) and endothelial-differentiated hGMSCs (e-hGMSCs) exposed to LPS-G showed upregulation of p300 and downregulation of DNA methyltransferase 1 (DNMT1), proteins associated with DNA methylation and histone acetylation. The co-treatment of AA and LPS-G showed a physiological expression of p300 and DNMT1 in hGMSCs and e-hGMSCs. Moreover, the inflammatory process triggered by LPS-G was demonstrated by evaluation of reactive oxygen species (ROS) and their intracellular localization. AA exposure re-established the physiological ROS levels. Despite the limitations of in vitro study, these findings collectively expand our knowledge regarding the molecular pathways involved in periodontal disease, and suggest the involvement of epigenetic modifications in the development of periodontitis.

The Effect of Liposomal Curcumin as an Anti-Inflammatory Strategy on Lipopolysaccharide e from Porphyromonas gingivalis Treated Endothelial Committed Neural Crest Derived Stem Cells: Morphological and Molecular Mechanisms.

Curcumin, a yellow polyphenol extracted from the turmeric root is used as a diet supplement. It exhibits anti-inflammatory, antioxidant, and antitumor properties by modulating different intracellular mechanisms. Due to their low solubility in water, the curcumin molecules must be encapsulated into liposomes to improve the bioavailability and biomedical potential. For the periodontal tissue and systemic health, it is essential to regulate the local inflammatory response. In this study, the possible beneficial effect of liposomes loaded with curcumin (CurLIP) in neural crest-derived human periodontal ligament stem cells (hPDLSCs) and in endothelial-differentiated hPDLSCs (e-hPDLSCs) induced with an inflammatory stimulus (lipopolysaccharide obtained from Porphyromonas gingivalis, LPS-G) was evaluated. The CurLIP formulation exhibited a significant anti-inflammatory effect by the downregulation of Toll-like receptor-4 (TLR4)/Myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa light chain enhancer of activated B cells (NFkB)/NLR Family Pyrin Domain Containing 3 (NLRP3)/Caspase-1/Interleukin (IL)-1ß inflammation cascade and reactive oxygen species (ROS) formation. Moreover, the exposure to LPS-G caused significant alterations in the expression of epigenetic modifiers, such as DNA Methyltransferase 1 (DNMT1) and P300, while the CurLIP treatment showed physiological expression. Overall, our in vitro study provides novel mechanistic insights into the intracellular pathway exert by CurLIP in the regulation of inflammation and epigenetic modifications.

Epithelial-Mesenchymal Transition (EMT): The Type-2 EMT in Wound Healing, Tissue Regeneration and Organ Fibrosis.

Theepithelial-mesenchymal transition (EMT) is an essential event during cell development, in which epithelial cells acquire mesenchymal fibroblast-like features including reduced intercellular adhesion and increased motility. EMT also plays a key role in wound healing processes, which are mediated by inflammatory cells and fibroblasts. These cells secrete specific factors that interact with molecules of the extracellular matrix (ECM) such as collagens, laminins, elastin and tenascins. Wound healing follows four distinct and successive phases characterized by haemostasis, inflammation, cell proliferation and finally tissue remodeling. EMT is classified into three diverse subtypes: type-1 EMT, type-2 EMT and type-3 EMT. Type-1 EMT is involved in embryogenesis and organ development. Type-2 EMT is associated with wound healing, tissue regeneration and organ fibrosis. During organ fibrosis, type-2 EMT occurs as a reparative-associated process in response to ongoing inflammation and eventually leads to organ destruction. Type-3 EMT is implicated in cancer progression, which is linked to the occurrence of genetic and epigenetic alterations, in detail the ones promoting clonal outgrowth and the formation of localized tumors. The current review aimed at exploring the role of EMT process with particular focus on type-2 EMT in wound healing, fibrosis and tissue regeneration, as well as some recent progresses in the EMT and tissue regeneration field, including the modulation of EMT by biomaterials.

Transforming Growth Factor-Beta1 and Human Gingival Fibroblast-to-Myofibroblast Differentiation: Molecular and Morphological Modifications.

After oral mucosal injury, the healing response following specific steps that lead to wound closure and to tissue repair. Multiple cell populations are involved in this process; in particular, fibroblasts play a key role in the production of extracellular matrix (ECM). During wound healing the remodeling of ECM is a key stage to restore the tissue functionality through multifunctional fibroblast populations that are placed in the connective tissues of gingiva and periodontal ligament. Notably, a fibroblast sub-type (myofibroblast) is centrally involved in collagen synthesis and fibrillar remodeling. The present work evidenced the role of Transforming Growth Factor-beta1 (TGF-ß1) to mediate human gingival fibroblasts (hGFs) differentiation into myofibroblasts derived from gingival fibroblasts (myo-hGFs). The morphological and functional features were analyzed through Confocal Laser Scanning Microscopy (CLSM), flow cytometry, and western blotting analyses. The specific markers, such as alpha-Smooth Muscle Actin (α-SMA), Vimentin, E-cadherin, ß-catenin, and Smad 2/3, were modulated in myo-hGFs after the induction with TGF-ß1, at different time points (24, 48, and 72 h). After 72 h of treatment TGF-ß1 operates as an inducer of hGFs into myo-hGFs differentiation. We propose that TGF-ß1 may promote in vitro the fibroblasts-to-myofibroblasts transition via the morphological and molecular modifications, as the induction of α-SMA, Vimentin, E-cadherin, ß-catenin, and Smad 2/3.

Oral Bone Tissue Regeneration: Mesenchymal Stem Cells, Secretome, and Biomaterials.

In the last few decades, tissue engineering has become one of the most studied medical fields. Even if bone shows self-remodeling properties, in some cases, due to injuries or anomalies, bone regeneration can be required. In particular, oral bone regeneration is needed in the dentistry field, where the functional restoration of tissues near the tooth represents a limit for many dental implants. In this context, the application of biomaterials and mesenchymal stem cells (MSCs) appears promising for bone regeneration. This review focused on in vivo studies that evaluated bone regeneration using biomaterials with MSCs. Different biocompatible biomaterials were enriched with MSCs from different sources. These constructs showed an enhanced bone regenerative power in in vivo models. However, we discussed also a future perspective in tissue engineering using the MSC secretome, namely the conditioned medium and extracellular vesicles. This new approach has already shown promising results for bone tissue regeneration in experimental models.

Antioxidant Ascorbic Acid Modulates NLRP3 Inflammasome in LPS-G Treated Oral Stem Cells through NFκB/Caspase-1/IL-1ß Pathway.

Human gingival mesenchymal stem cells (hGMSCs) and endothelial committed hGMSCs (e-hGMSCs) have considerable potential to serve as an in vitro model to replicate the inflammation sustained by Porphyromonas gingivalis in periodontal and cardiovascular diseases. The present study aimed to investigate the effect of ascorbic acid (AA) on the inflammatory reverting action of lipopolysaccharide (LPS-G) on the cell metabolic activity, inflammation pathway and reactive oxygen species (ROS) generation in hGMSCs and e-hGMSCs. Cells were treated with LPS-G (5 µg mL-1) or AA (50 µg mL-1) and analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, immunofluorescence and Western blot methods. The rate of cell metabolic activity was decreased significantly in LPS-G-treated groups, while groups co-treated with LPS-G and AA showed a logarithmic cell metabolic activity rate similar to untreated cells. AA treatment attenuated the inflammatory effect of LPS-G by reducing the expression of TLR4/MyD88/NFκB/NLRP3/Caspase-1/IL-1ß, as demonstrated by Western blot analysis and immunofluorescence acquisition. LPS-G-induced cells displayed an increase in ROS production, while AA co-treated cells showed a protective effect. In summary, our work suggests that AA attenuated LPS-G-mediated inflammation and ROS generation in hGMSCs and e-hGMSCs via suppressing the NFκB/Caspase-1/IL-1ß pathway. These findings indicate that AA may be considered as a potential factor involved in the modulation of the inflammatory pathway triggered by LPS-G in an vitro cellular model.

Ascorbic Acid: A New Player of Epigenetic Regulation in LPS-gingivalis Treated Human Periodontal Ligament Stem Cells.

Periodontitis is usually sustained from microorganism of oral cavity, like Porphyromonas gingivalis (P. gingivalis). Periodontal disease is an infectious disease that afflicts a large number of people. Researches are investigating on the mesenchymal stem cells (MSCs) response to inflammatory events in combination with antioxidant substances. In particular, ascorbic acid (AA) increased cell proliferation, upregulated the cells pluripotency marker expression, provide a protection from inflammation, and induced the regeneration of periodontal ligament tissue. The purpose of the present research was to investigate the effects of AA in primary culture of human periodontal ligament stem cells (hPDLSCs) exposed to P. gingivalis lipopolysaccharide (LPS-G). The effect of AA on hPDLSCs exposed to LPS-G was determined through the cell proliferation assay. The molecules involved in the inflammatory pathway and epigenetic regulation have been identified using immunofluorescence and Western blot analyses. miR-210 level was quantified by qRT-PCR, and the ROS generation was finally studied. Cells co-treated with LPS-G and AA showed a restoration in terms of cell proliferation. The expression of NFκB, MyD88, and p300 was upregulated in LPS-G exposed cells, while the expression was attenuated in the co-treatment with AA. DNMT1 expression is attenuated in the cells exposed to the inflammatory stimulus. The level of miR-210 was reduced in stimulated cells, while the expression was evident in the hPDLSCs co-treated with LPS-G and AA. In conclusion, the AA could enhance a protective effect in in vitro periodontitis model, downregulating the inflammatory pathway and ROS generation and modulating the miR-210 level.

Stem Cells Secretome from Oral Tissue Could Represent a Promising Therapeutic Approach in COVID-19-Disease?

At present, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has quickly become a health emergency because no specifics vaccines or drugs, at this moment, are available. Recent studies have shown that the transplantation of mesenchymal stem cells (MSCs) into Coronavirus Disease 2019 (COVID-19) patients could represent a promising strategy for the development of new therapeutic methods. We speculate and suggest that the secretome of human Oral Tissue Stem Cells (hOTSCs), for their immunomodulatory and anti-inflammatory specific properties, could exert beneficial effects on the COVID-19 patients through an innovative aerosolisation technique. This non-invasive technique can offer multiple advantages in prophylaxis, as well as the prevention and treatment of severe epidemic respiratory syndrome with minimum risk and optimal therapeutic effects. This has the potential to create a novel pathway towards immunomodulatory therapy for the treatment of COVID-19 positive patients.