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.

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 [...].

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.

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.

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.

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.

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.

Systematic Review: Allogenic Use of Stromal Vascular Fraction (SVF) and Decellularized Extracellular Matrices (ECM) as Advanced Therapy Medicinal Products (ATMP) in Tissue Regeneration.

Stromal vascular fraction (SVF) containing adipose stem cells (ASCs) has been used for many years in regenerative plastic surgery for autologous applications, without any focus on their potential allogenic role. Allogenic SVF transplants could be based on the possibility to use decellularized extracellular matrix (ECM) as a scaffold from a donor then re-cellularized by ASCs of the recipient, in order to develop the advanced therapy medicinal products (ATMP) in fully personalized clinical approaches. A systematic review of this field has been realized in accordance with the Preferred Reporting for Items for Systematic Reviews and Meta-Analyses-Protocols (PRISMA-P) guidelines. Multistep research of the PubMed, Embase, MEDLINE, Pre-MEDLINE, PsycINFO, CINAHL, Clinicaltrials.gov, Scopus database, and Cochrane databases has been conducted to identify articles and investigations on human allogenic ASCs transplant for clinical use. Of the 341 articles identified, 313 were initially assessed for eligibility on the basis of the abstract. Of these, only 29 met all the predetermined criteria for inclusion according to the PICOS (patients, intervention, comparator, outcomes, and study design) approach, and 19 have been included in quantitative synthesis (meta-analysis). Ninety-one percent of the studies previously screened (284 papers) were focused on the in vitro results and pre-clinical experiments. The allogenic use regarded the treatment of perianal fistulas, diabetic foot ulcers, knee osteoarthritis, acute respiratory distress syndrome, refractory rheumatoid arthritis, pediatrics disease, fecal incontinence, ischemic heart disease, autoimmune encephalomyelitis, lateral epicondylitis, and soft tissue defects. The information analyzed suggested the safety and efficacy of allogenic ASCs and ECM transplants without major side effects.

Impact of the Different Preparation Methods to Obtain Autologous Non-Activated Platelet-Rich Plasma (A-PRP) and Activated Platelet-Rich Plasma (AA-PRP) in Plastic Surgery: Wound Healing and Hair Regrowth Evaluation.

Autologous therapies using platelet-rich plasma (PRP) need meticulous preparation-currently, no standardised preparation technique exists. Processing Quantitative Standards (PQSs) define manufacturing quantitative variables (such as time, volume and pressure). Processing Qualitative Standards (PQLSs) define the quality of the materials and methods of manufacturing. The aim of this review is to use existing PQSs and PQLs to report the in vivo/in vitro results obtained by using different Kits, that utilise different procedures (classified as Closed-Technique and Opened-Technique) to isolate autologous human activated (AA-PRP) or non-activated PRP (A-PRP). PQSs included the volumes of blood collected as well as the reagents used, the time/gravity of centrifugation, and the duration, temperature and tilt level/speed of centrifugation. PQLSs included the use of Calcium Chloride CaCl2, Kit weight, transparency of Kit components, the maintenance of a closed sterile processing environment and the use of a small centrifuge. Eight CE marked devices for PRP extraction were evaluated: Angel®, Biomed®, Cascade® and Selphyl®, Mag-18®, i-Stem®, MyCells® and Regenlab®. Using a Kit with the PQSs and PQLSs described in this study enables the isolation of A-PRP, thereby meeting consensus quality criteria. As our understanding of Critical Quality Attributes (CQAs) of A-PRP continues to evolve, especially with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully help isolate A-PRP of desired CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed.

Impact of the Different Preparation Methods to Obtain Human Adipose-Derived Stromal Vascular Fraction Cells (AD-SVFs) and Human Adipose-Derived Mesenchymal Stem Cells (AD-MSCs): Enzymatic Digestion Versus Mechanical Centrifugation.

Autologous therapies using adipose-derived stromal vascular fraction (AD-SVFs) and adult adipose-derived mesenchymal stem cells (AD-MSCs) warrant careful preparation of the harvested adipose tissue. Currently, no standardized technique for this preparation exists. Processing quantitative standards (PQSs) define manufacturing quantitative variables (such as time, volume, and pressure). Processing qualitative standards (PQLSs) define the quality of the materials and methods in manufacturing. The purpose of the review was to use PQSs and PQLSs to report the in vivo and in vitro results obtained by different processing kits that use different procedures (enzymatic vs. non-enzymatic) to isolate human AD-SVFs/AD-MSCs. PQSs included the volume of fat tissue harvested and reagents used, the time/gravity of centrifugation, and the time, temperature, and tilt level/speed of incubation and/or centrifugation. PQLSs included the use of a collagenase, a processing time of 30 min, kit weight, transparency of the kit components, the maintenance of a closed sterile processing environment, and the use of a small centrifuge and incubating rocker. Using a kit with the PQSs and PQLSs described in this study enables the isolation of AD-MSCs that meet the consensus quality criteria. As the discovery of new critical quality attributes (CQAs) of AD-MSCs evolve with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully isolate AD-MSCs of various CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed.

Human Oral Stem Cells, Biomaterials and Extracellular Vesicles: A Promising Tool in Bone Tissue Repair.

Tissue engineering and/or regenerative medicine are fields of life science exploiting both engineering and biological fundamentals to originate new tissues and organs and to induce the regeneration of damaged or diseased tissues and organs. In particular, de novo bone tissue regeneration requires a mechanically competent osteo-conductive/inductive 3D biomaterial scaffold that guarantees the cell adhesion, proliferation, angiogenesis and differentiation into osteogenic lineage. Cellular components represent a key factor in tissue engineering and bone growth strategies take advantage from employment of mesenchymal stem cells (MSCs), an ideal cell source for tissue repair. Recently, the application of extracellular vesicles (EVs), isolated from stem cells, as cell-free therapy has emerged as a promising therapeutic strategy. This review aims at summarizing the recent and representative research on the bone tissue engineering field using a 3D scaffold enriched with human oral stem cells and their derivatives, EVs, as a promising therapeutic potential in the reconstructing of bone tissue defects.

Transcriptomic Analysis of Stem Cells Treated with Moringin or Cannabidiol: Analogies and Differences in Inflammation Pathways.

Inflammation is a common feature of many neurodegenerative diseases. The treatment of stem cells as a therapeutic approach to repair damage in the central nervous system represents a valid alternative. In this study, using Next-Generation Sequencing (NGS) technology, we analyzed the transcriptomic profile of human Gingival Mesenchymal Stem Cells (hGMSCs) treated with Moringin [4-(α-l-ramanosyloxy)-benzyl isothiocyanate] (hGMSCs-MOR) or with Cannabidiol (hGMSCs-CBD) at dose of 0.5 or 5 µM, respectively. Moreover, we compared their transcriptomic profiles in order to evaluate analogies and differences in pro- and anti-inflammatory pathways. The hGMSCs-MOR selectively downregulate TNF-α signaling from the beginning, reducing the expression of TNF-α receptor while hGMSCs-CBD limit its activity after the process started. The treatment with CBD downregulates the pro-inflammatory pathway mediated by the IL-1 family, including its receptor while MOR is less efficient. Furthermore, both the treatments are efficient in the IL-6 signaling. In particular, CBD reduces the effect of the pro-inflammatory JAK/STAT pathway while MOR enhances the pro-survival PI3K/AKT/mTOR. In addition, both hGMSCs-MOR and hGMSCs-CBD improve the anti-inflammatory activity enhancing the TGF-ß pathway.

3D Printing PLA/Gingival Stem Cells/ EVs Upregulate miR-2861 and -210 during Osteoangiogenesis Commitment.

Bone tissue regeneration strategies require approaches that provide an osteogenic and angiogenic microenvironment able to drive the bone growth. Recently, the development of 3D printing biomaterials, including poly(lactide) (3D-PLA), enriched with mesenchymal stem cells (MSCs) and/or their derivatives, such as extracellular vesicles (EVs) has been achieving promising results. In this study, in vitro results showed an increased expression of osteogenic and angiogenic markers, as RUNX2, VEGFA, OPN and COL1A1 in the living construct 3D-PLA/human Gingival MSCs (hGMSCs)/EVs. Considering that EVs carry and transfer proteins, mRNA and microRNA into target cells, we evaluated miR-2861 and miR-210 expression related to osteoangiogenesis commitment. Histological examination of rats implanted with 3D-PLA/hGMSCs/EVs evidenced the activation of bone regeneration and of the vascularization process, confirmed also by MicroCT. In synthesis, an upregulation of miR-2861 and -210 other than RUNX2, VEGFA, OPN and COL1A1 was evident in cells cultured in the presence of the biomaterial and EVs. Then, these results evidenced that EVs may enhance bone regeneration in calvaria defects, in association with an enhanced vascularization offering a novel regulatory system in the osteoangiogenesis evolution. The application of new strategies to improve biomaterial engraftment is of great interest in the regenerative medicine and can represent a way to promote bone regeneration.

Moringin Pretreatment Inhibits the Expression of Genes Involved in Mitophagy in the Stem Cell of the Human Periodontal Ligament.

Moringin [4-(α-L-rhamnosyloxy) benzyl isothiocyanate] is an isothiocyanate extracted from Moringa oleifera seeds. It is an antioxidant known for several biological properties useful in the treatment of neurodegenerative diseases. Several neurodegenerative disorders such as Parkinson's and Alzheimer's diseases are linked to dysfunctional mitochondria due to the resulting increase of Reactive Oxygen Species (ROS). Stem cell-based therapeutic treatments in neurodegenerative diseases provide an alternative strategy aimed to replace the impaired tissue. In this study were investigated the deregulated genes involved in mitophagy in the human periodontal ligament stem cells pretreated with moringin. The RNA-seq study reveals the downregulation of PINK1, with a fold change (FC) of -0.56, such as the genes involved in the phagophore formation (MAP1LC3B FC: -0.73, GABARAP FC: -0.52, GABARAPL1 FC: -0.70, GABARAPL2 FC: -0.39). The moringin pretreatment downregulates the pro-apoptotic gene BAX (-0.66) and upregulates the anti-apoptotic genes BCL2L12 (FC: 1.35) and MCL1 (FC: 0.36). The downregulation of the most of the caspases (CASP1 FC: -1.43, CASP4 FC: -0.18, CASP6 FC: -1.34, CASP7 FC: -0.46, CASP8 FC: -0.65) implies the inactivation of the apoptotic process. Our results suggest that mitochondrial dysfunctions induced by oxidative stress can be inhibited by moringin pretreatment in human periodontal ligament stem cells (hPDLSCs).

Endothelial committed oral stem cells as modelling in the relationship between periodontal and cardiovascular disease.

In the present study we have mimicked, in vitro, an inflammatory process using Lipopolysaccharide derived from Porphyromonas Gingivalis (LPS-G) and human Periodontal Ligament Stem Cells induced to endothelial differentiation (e-hPDLSCs). The research project has been organized into the three following steps: i) induction of hPDLSCs toward endothelial differentiation; ii) evaluation of the molecular signaling pathway involved in the response to the LPS-G, and iii) functional response evaluation of the living construct constituted by porcine decellularized valve/e-hPDLSCs treated with LPS-G. Obtained results showed that 5 µg/ml LPS-G stimulus provokes: a slowdown of cell growth starting from 24 hr and the release of IL6, IL8, and MCP1 molecules. Signaling network analyzed showed the activation of TLR4/ NFkB/ERK1/2/p-ERK1/2 signaling mediated by MyD88 in LPS-G stimulated e-hPDLSCs, moreover a time course put in evidence a nuclear traslocation of ERK1/2 and p-ERK1/2 in differentiated samples. Following, the ability of e-hPDLSCs to expand and colonize the decellularized porcine heart valves was appraised at ultrastructural level. Considering that, the Reactive Oxygen Species (ROS) play an important role in the progression and development of cardiovascular disease (CVD), in LPS-G living construct model e-hPDLSCs/decellularized porcine heart valves (dPHV), ROS production was assessed. Time lapse experiments evidenced that LPS-G provokes in e-hPDLSCs a rapid and sustained increase in ROS generation, negligible on undifferentiated cells. From obtained data, by multiparametric analyses, a reasonable conclusion may be that the inflammation process activated by LPS-G can affect endothelial cells and could represent in vivo a possible pathological and predictor state of CVD.

Grading of aortic stenosis severity: a head-to-head comparison between cardiac magnetic resonance imaging and echocardiography.

AIM: To prospectively evaluate the accuracy of cardiac magnetic resonance (cMR) imaging for the assessment of aortic valve effective orifice area (EOA) by continuity equation and anatomical aortic valve area (AVA) by direct planimetry, as compared with transthoracic (TTE) and transesophageal (TEE) two-dimensional (2D) echocardiography, respectively. METHODS AND RESULTS: A total of 31 patients (21 men, 10 women, mean age 69 ± 10 years) with moderate-to-severe aortic stenosis (AS) diagnosed by TTE and scheduled for elective aortic valve replacement, underwent both cMR and TEE. AVA by cMR was obtained from balanced steady-state free-precession cine-images. EOA was computed from phase-contrast MR flow analysis. AVA at cMR (0.93 ± 0.42 cm2) was highly correlated with TEE-derived planimetry (0.92 ± 0.32 cm2) (concordance correlation coefficient, CCC = 0.85). By excluding 11 patients with extensively thickened and heavily calcified cusps, the CCC increased to 0.93. EOA at cMR (0.86 ± 0.30 cm2) showed a strong correlation with TTE-derived EOA (0.78 ± 0.25 cm2) (CCC = 0.82). CONCLUSIONS: cMR imaging is an accurate alternative for the grading of AS severity. Its use may be recommended especially in patients with poor transthoracic acoustic windows and/or in case of discordance between 2D echocardiographic parameters.

MicroRNA 210 Mediates VEGF Upregulation in Human Periodontal Ligament Stem Cells Cultured on 3DHydroxyapatite Ceramic Scaffold.

The aim of the present research was the evaluation of the behavior of human periodontal ligament stem cells (hPDLSCs), cultured in presence of Endobon® Xenograft Granules (G), a fully deproteinated hydroxyapatite ceramic scaffold derived from cancellous bovine bone. hPDLSCs were seeded with and without G for 24 h to 1 week. The cell growth, morphological features, adhesiveness, differentiation ability, modulation of miR-210 and Vascular Endothelial Growth Factor (VEGF) secretion were analyzed by means of MTT assay, Scanning Electron Microscopy (SEM), Confocal Laser Scanning Microscopy (CLSM), Alizarin Red S assay, RT-PCR and ELISA test, respectively. hPDLSCs grown on the biomaterial showed the ability to form focal adhesion on the substrate, as demonstrated by vinculin expression. These data were supported by SEM analysis showing that an adhesiveness process associated to cell growth occurs between cells and biomaterials. The osteogenic differentiation, evaluated by morphological, biochemical, and RT-PCR analysis, was pronounced in the hPDLSCs grown in the three-dimensional inorganic bovine bone substitute in the presence of osteoinductive conditions. In addition, an upregulation of miR-210 and VEGF was evident in cells cultured in presence of the biomaterial. Our results inspire us to consider granules not only an adequate biocompatible three-dimensional biomaterial, but also an effective inductor of miR-210 and VEGF; in fact, the involvement of miR-210 in VEGF secretion could offer a novel regulatory system in the early steps of the bone-regeneration process.

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.