Biological Effects of PMMA and Composite Resins on Human Gingival Fibroblasts: An In Vitro Comparative Study.

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-1ß 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-1ß 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.

Anti-TLR4 biological response to titanium nitride-coated dental implants: anti-inflammatory response and extracellular matrix synthesis.

Osteointegration is a key process during dental implant placement and is related to titanium surface topography. Implant coating and surface modification methods ameliorate the bone production and the osteogenic process. The current work aimed at evaluating the biological outcomes of two different surfaces of dental implants, machined and titanium nitride (TiN) coated, at an inflammation level using an in vitro model of human periodontal ligament stem cells. The TLR4/MyD88/NF-κB p65/NLRP3 pathway induced by the Porphyromonas gingivalis lipopolysaccharide was studied by means of gene- and protein-level expression. Moreover, the expression of vimentin, vinculin, and fibronectin was evaluated to investigate their effects on the cell adhesion and extracellular matrix deposition. The results of the present study suggest that TiN-coated titanium disks may modulate inflammation by the suppression of the TLR4/MyD88/NF-κB p65/NLRP3 pathway and accelerate extracellular matrix apposition.

Extracellular vesicles (EVs): A promising therapeutic tool in the heart tissue regeneration.

Mesenchymal stem cells (MSCs) treatment has been widely explored as a therapy for myocardial infarction, peripheral ischemic vascular diseases, dilated cardiomyopathy, and pulmonary hypertension. Latest in vitro studies suggest that MSCs can differentiate into contractile cardiomyocytes. One of the best-characterized MSCs products are MSCs-derived extracellular vesicles (EVs). EVs are crucial paracrine effectors of MSCs. Based on previous works, paracrine effects of MSCs play a primary role in the regenerative ability. Hence, in the current paper, we focused our attention on an alternative approach, exploiting products derived from human dental pulp stem cells (hDPSCs) rather than MSCs themselves, which may denote a cost-effective and safer approach. The focus has been on EVs and the bioactive molecules they contain to evaluate their ability to influence the differentiation process toward cardiomyogenic lineage. The expression of GATA4, ACTC1, CX43, and Nkx2.5 was evaluated using Immunofluorescence, real time-PCR, and Western blotting analyses. Furthermore, the expression profiling analysis of the microRNA hsa-miR-200c-3p, targeting the GATA4 gene, was studied. The hsa-miR-200c-3p was found significantly down-regulated in both c-hDPSCs + EVs-hDPSCs and c-hDPSCs + EVs-HL-1 compared to untreated c-hDPSCs underlying a possible epigenetic mechanism behind the prevalent up-regulation of its targeted GATA4 gene. The aim of the present work was to develop an in vitro model of hDPSCs able to differentiate into cardiomyocytes in order to investigate the role of EVs derived from hDPSCs and derived from HL-1 cardiomyocyte cell line in modulating the differentiation process toward cardiomyogenic lineage.

Effects of GHRH Deficiency and GHRH Antagonism on Emotional Disorders in Mice.

Growth hormone (GH)-releasing hormone (GHRH) has been suggested to play a crucial role in brain function. We aimed to further investigate the effects of a novel GHRH antagonist of the Miami (MIA) series, MIA-602, on emotional disorders and explore the relationships between the endocrine system and mood disorders. In this context, the effects induced by MIA-602 were also analyzed in comparison to vehicle-treated mice with GH deficiency due to generalized ablation of the GHRH gene (GHRH knock out (GHRHKO)). We show that the chronic subcutaneous administration of MIA-602 to wild type (+/+) mice, as well as generalized ablation of the GHRH gene, is associated with anxiolytic and antidepressant behavior. Moreover, immunohistochemical and Western blot analyses suggested an evident activation of Nrf2, HO1, and NQO1 in the prefrontal cortex of both +/+ mice treated with MIA-602 (+/+ MIA-602) and homozygous GHRHKO (-/- control) animals. Finally, we also found significantly decreased COX-2, iNOS, NFkB, and TNF-α gene expressions, as well as increased P-AKT and AKT levels in +/+ MIA-602 and -/- control animals compared to +/+ mice treated with vehicle (+/+ control). We hypothesize that the generalized ablation of the GHRH gene leads to a dysregulation of neural pathways, which is mimicked by GHRH antagonist treatment.

Emerging Role of Oral Mesenchymal Stem/Stromal Cells and Their Derivates.

Mesenchymal stem/stromal cells (MSCs) have fewer ethical, moral, and safety problems in comparison with embryonic stem cells [...].

Inhibition of LPS-Induced Inflammatory Response of Oral Mesenchymal Stem Cells in the Presence of Galectin-3.

Galectin-3 (GAL-3) is a beta-galactoside binding lectin produced by mesenchymal stem cells (MSCs) and other cell sources under inflammatory conditions. Several studies have reported that GAL-3 exerts an anti-inflammatory action, regulated by its natural ligand GAL-3 BP. In the present study, we aimed to assess the GAL-3 mediated regulation of the MSC function in an LPS-induced inflammation setting. Human gingival mesenchymal stem cells (hGMSCs) were stimulated in vitro with LPSs; the expression of TLR4, NFκB p65, MyD88 and NALP3 were assessed in the hGMSCs via immunofluorescence imaging using confocal microscopy, Western blot assay, and RT-PCR before and after the addition of GAL-3, both alone and with the addition of its inhibitors. LPSs stimulated the expression of TLR4, NFκB p65, MyD88 and NALP3 in hGMSCs, which was inhibited by GAL-3. The addition of either GAL3-BP or the antibody to GAL-3 were able to revert the GAL-3-mediated effects, restoring the expression of TLR4, NFκB p65, MyD88 and NALP3. GAL-3 induces the downregulation of the LPS-induced inflammatory program in MSCs.

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.

Obesity and Wound Healing: Focus on Mesenchymal Stem Cells.

Chronic wounds represent nowadays a major challenge for both clinicians and researchers in the regenerative setting. Obesity represents one of the major comorbidities in patients affected by chronic ulcers and therefore diverse studies aimed at assessing possible links between these two morbid conditions are currently ongoing. In particular, adipose tissue has recently been described as having metabolic and endocrine functions rather than serving as a mere fat storage deposit. In this setting, adipose-derived stem cells, a peculiar subset of mesenchymal stromal/stem cells (MSCs) located in adipose tissue, have been demonstrated to possess regenerative and immunological functions with a key role in regulating both adipocyte function and skin regeneration. The aim of the present review is to give an overview of the most recent findings on wound healing, with a special focus on adipose tissue biology and obesity.

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 [...].

Protective effect of oral stem cells extracellular vesicles on cardiomyocytes in hypoxia-reperfusion.

Hypoxia signaling plays an important role in physiological and pathological conditions. Hypoxia in the heart tissue can produce different consequences depending on the duration of exposure to the hypoxic state. While acute hypoxic exposure leads to a reversible acclimatization in heart tissue with normal systemic oxygen supply, chronic hypoxia exacerbates cardiac dysfunction, leads to a destruction of the tissue. Extracellular vesicles (EVs) are small membrane vesicles that act as mediators of intercellular communication. EVs are secreted by different cell types and those produced by oral cavity-derived mesenchymal stem cells (MSCs), including human gingival MSCs (hGMSCs), have pro-angiogenic and anti-inflammatory effects and showed therapeutic role in tissue regeneration. The aim of the present work was to evaluate the potential protective and regenerative role of EVs produced by hGMSCs, in an in vitro model of hypoxia-conditioned HL-1 cardiomyocytes through the expression analysis of following inflammatory, oxidative stress, angiogenesis, cell survival and apoptotic markers: HIF-1α, P300, NFkB, CCL2, IL1B, IL6, NRF2, CASP-3, BAX and VEGF. Results showed that hGMSCs-derived EVs exerted protection HL-1 cardiomyocytes exposed to both pre and post hypoxic conditions. Moreover, modulation of CASP3 and BAX expression demonstrated that EVs reduced the apoptosis. The analysis of microRNAs in EVs derived from hGMSCs was performed to assess the epigenetic regulation of the presented markers. The following microRNAs: hsa-miR-138-5p, hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-21-5p, hsa-miR-324-5p, hsa-miR-133a-3p, hsa-miR-150-5p, hsa-miR-199a-5p, hsa-miR-128-3p and hsa-miR-221-3p can directly or indirectly target the studied genes by determining their modulation obtained in our study. The data from this study suggested that EVs obtained from hGMSCs may be considered for the cell free treatment option in hypoxia-driven cardiac tissue dysfunction.

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)

The Aftermath of Long-Term Cigarette Smoking on Telomere Length and Mitochondrial DNA Copy Number in Human Cumulus Cells Prior to In Vitro Fertilization-A Pilot Study.

Cigarette smoking among women of reproductive age is known to take a toll on systemic health and fertility potential by severely impacting ovarian tissues and cells, such as granulosa and cumulus cells (CCs). The purpose of this study was to determine the potential damage caused by tobacco smoke at a molecular level in the CCs of females who had undergone in vitro fertilization. The level of intracellular damage was determined by estimating the average telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN), as well as the expression profile of telomere maintenance genes TERF1, TERF2, POT1 and microRNAs miR-155, miR-23a and miR-185. Western blotting analysis was performed to detect consequent protein levels of TERF1, TERF2 and POT1. Our results evidenced significantly lower relative TL and mtDNA-CN and a down-regulation pattern for all three described genes and corresponding proteins in the CCs of smokers compared with controls (p < 0.05). No significant differences were found in the miRNAs' modulation. Combined, our data add another piece to the puzzle of the complex regulatory molecular networks controlling the general effects of tobacco smoke in CCs. This pilot study extends the until now modest number of studies simultaneously investigating the mtDNA-CN and TL pathways in the human CCs of smoking women.

The Role of Hypoxia in Improving the Therapeutic Potential of Mesenchymal Stromal Cells. A Comparative Study From Healthy Lung and Congenital Pulmonary Airway Malformations in Infants.

Mesenchymal stromal cells (MSCs) play an important role in the field of regenerative medicine thanks to their immunomodulatory properties and their ability to secrete paracrine factors. The use of MSCs has also been tested in children with congenital lung diseases inducing fibrosis and a decrease in lung function. Congenital malformations of the pulmonary airways (CPAM) are the most frequently encountered lung lesion that results from defects in early development of airways. Despite the beneficial properties of MSCs, interventions aimed at improving the outcome of cell therapy are needed. Hypoxia may be an approach aimed to ameliorate the therapeutic potential of MSCs. In this regard, we evaluated the transcriptomic profile of MSCs collected from pediatric patients with CPAM, analyzing similarities and differences between healthy tissue (MSCs-lung) and cystic tissue (MSCs-CPAM) both in normoxia and in cells preconditioned with hypoxia (0.2%) for 24 h. Study results showed that hypoxia induces cell cycle activation, increasing in such a way the cell proliferation ability, and enhancing cell anaerobic metabolism in both MSCs-lung and MSCs-CPAM-lung. Additionally, hypoxia downregulated several pro-apoptotic genes preserving MSCs from apoptosis and, at the same time, improving their viability in both comparisons. Finally, data obtained indicates that hypoxia leads to a greater expression of genes involved in the regulation of the cytoskeleton in MSCs-lung than MSCs-CPAM.

Microplastics Affect the Inflammation Pathway in Human Gingival Fibroblasts: A Study in the Adriatic Sea.

The level of environmental microplastics in the sea is constantly increasing. They can enter the human body with food, be absorbed through the gut and have negative effects on the organism's health after its digestion. To date, microplastics (MPs) are considered new environmental pollutants in the air sea and they are attracting wide attention. The possible toxic effects of MPs isolated at different sea depths of 1, 24 and 78 m were explored in an in vitro model of human gingival fibroblasts (hGFs). MPs isolated from the sea showed different size and were then divided into different sample groups: 1, 24 and 78 m. The results obtained revealed that MPs are able to activate the inflammatory pathway NFkB/MyD88/NLRP3. In detail, the exposure to MPs from 1 and 78 m led to increased levels of inflammatory markers NFkB, MyD88 and NLRP3 in terms of proteins and gene expression. Moreover, cells exposed to MPs showed a lower metabolic activity rate compared to unexposed cells. In conclusion, these findings demonstrate that the inflammation process is stimulated by MPs exposure, providing a new perspective to better understand the intracellular mechanism.

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.

Exosomes as Carriers for Notch Molecules.

Exosomes are extracellular vesicles involved in cell-to-cell communication as well as extrusion of biological material. Using dental pulp stem cells culture as a model, we hereby describe a method for the packaging of Delta-like 4 (DLL4), a representative Notch ligand, into newly generated exosomes. We then provide methods of analysis to confirm the presence of Notch proteins and transcripts internalization and transport via exosomes.

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.