Evaluation of cell adhesion molecules (LFA-1 and L-selectin) in ankylosing spondylitis patients after treatment with ß-D-mannuronic acid (M2000).

Background & objectives: To examine ß-D-mannuronic acid (M2000) effects on L-selectin shedding and leucocyte function-associated antigen-1 (LFA-1) expression as mechanisms of action of this drug in patients with ankylosing spondylitis (AS). Methods: To investigate the molecular consequences of ß-D-mannuronic acid on L-selectin shedding, flow cytometry method was used. Furthermore, the effect of it on LFA-1 gene expression was analyzed by using quantitative real time (qRT)-PCR technique. Results: The LFA-1 expression in patients with AS was higher than controls (P=0.046). The LFA-1 expression after 12 wk therapy with ß-D-mannuronic acid was meaningfully decreased (P=0.01). After 12 wk treatment with ß-D-mannuronic acid, the frequency of CD62L-expressing CD4+ T cells in patients with AS, was not considerably altered, compared to the patients before therapy (P=0.5). Furthermore, after 12 wk therapy with ß-D-mannuronic acid, L-selectin expression levels on CD4+ T-cells in patients with AS, were not remarkably changed, compared to the expression levels of these in patients before treatment (P=0.2). Interpretation & conclusions: The results of this study for the first time showed that ß-D-mannuronic acid can affect events of adhesion cascade in patients with AS. Moreover, ß-D-mannuronic acid presented as an acceptable benefit to AS patients and could aid in the process of disease management.

The Open, Randomized, Positive Control Clinical Trial of Guluronic Acid (G2013) on SARS-CoV-2 Patients.

INTRODUCTION: Recently, the coronavirus disease 2019 (COVID-19) infection, with a vast spectrum of clinical and paraclinical symptoms has been a major health concern worldwide. Therapeutical management of COVID-19 includes antiviral and anti-inflammatory drugs. NSAIDs, as the second-line therapy, are often prescribed to relieve the symptoms of COVID-19. The α-L-guluronic acid (G2013) is a non-steroidal patented (PCT/EP2017/067920) agent with immunomodulatory properties. This study investigated the effect of G2013 on the outcome of COVID-19 in moderate to severe patients. METHODS: The disease's symptoms were followed up during hospitalization and for 4 weeks postdischarge in G2013 and control groups. Paraclinical indices were tested at the time of admission and discharge. Statistical analysis was performed on clinical and paraclinical parameters and ICU admission and death rate. RESULTS: The primary and secondary outcomes indicated the efficiency of G2013 on COVID-19 patients' management. There were significant differences in the duration of improvement of fever, coughing, fatigue/malaise. Also, a comparison of paraclinical indices at the time of admission and discharge showed significant change in prothrombin, D-dimer, and platelet. As the main findings of this study, G2013 significantly decreased the percentage of ICU admission (control:17 patients, G2013:1 patient) and death (control: 7 cases, G2013:0). CONCLUSION: These results conclude that G2013 has sufficient potential to be considered for moderate to severe COVID-19 patients, can significantly reduce the clinical and physical complications of this disease, has a positive effect on modulating the coagulopathy process, and aids in saving lives.

Evaluation of therapeutic effects of nanofibrous mat containing mycophenolate mofetil on oral lichen planus: In vitro and clinical trial study.

Objectives: Recently, topical drug delivery system has gained increasing interest in the treatment of oral lesions. Lichen planus is a chronic inflammatory disease affecting mucous membranes and skin. The current study aimed to fabricate a drug delivery system containing mycophenolate mofetil for the treatment of oral lichen planus lesions. Methods: Firstly, a nanofibrous mat containing mycophenolate mofetil, zinc oxide nanoparticles, and aloe vera was designed and fabricated. The antimicrobial, cytocompatibility, anti-inflammatory, and antioxidative characteristics of fabricated scaffolds were evaluated. Then, this nanofibrous mat was applied to 12 patients suffering from bilateral erythematous/erosive Oral Lichen planus (OLP) lesions for 2 weeks. The treatment outcomes, including oral symptoms and lesion size, were compared with the routine topical treatment of these lesions; Triamcinolone ointment. Results: The characterization of nanofibrous mat approved the successful fabrication of scaffolds. The fabricated nanofibers showed notable antimicrobial activity. The amounts of TNF 𝛼, IL6, and reactive oxygen species (ROS) of stimulated human gingival fibroblasts were decreased after exposure to NFs/Myco/Alv/ZnO scaffolds. The clinical trial results demonstrated the same therapeutic effects compared to the commercial ointment, while the symptoms of patients were significantly improved in the mats group.Significance. Considering the successful results of this study, the application of nanofibrous mat can be a promising product for improving treatment outcomes of OLP.

Enhancing the function of PLGA-collagen scaffold by incorporating TGF-ß1-loaded PLGA-PEG-PLGA nanoparticles for cartilage tissue engineering using human dental pulp stem cells.

Since cartilage has a limited capacity for self-regeneration, treating cartilage degenerative disorders is a long-standing difficulty in orthopedic medicine. Researchers have scrutinized cartilage tissue regeneration to handle the deficiency of cartilage restoration capacity. This investigation proposed to compose an innovative nanocomposite biomaterial that enhances growth factor delivery to the injured cartilage site. Here, we describe the design and development of the biocompatible poly(lactide-co-glycolide) acid-collagen/poly(lactide-co-glycolide)-poly(ethylene glycol)-poly(lactide-co-glycolide) (PLGA-collagen/PLGA-PEG-PLGA) nanocomposite hydrogel containing transforming growth factor-ß1 (TGF-ß1). PLGA-PEG-PLGA nanoparticles were employed as a delivery system embedding TGF-ß1 as an articular cartilage repair therapeutic agent. This study evaluates various physicochemical aspects of fabricated scaffolds by 1HNMR, FT-IR, SEM, BET, and DLS methods. The physicochemical features of the developed scaffolds, including porosity, density, degradation, swelling ratio, mechanical properties, morphologies, BET, ELISA, and cytotoxicity were assessed. The cell viability was investigated with the MTT test. Chondrogenic differentiation was assessed via Alcian blue staining and RT-PCR. In real-time PCR testing, the expression of Sox-9, collagen type II, and aggrecan genes was monitored. According to the results, human dental pulp stem cells (hDPSCs) exhibited high adhesion, proliferation, and differentiation on PLGA-collagen/PLGA-PEG-PLGA-TGFß1 nanocomposite scaffolds compared to the control groups. SEM images displayed suitable cell adhesion and distribution of hDPSCs throughout the scaffolds. RT-PCR assay data displayed that TGF-ß1 loaded PLGA-PEG-PLGA nanoparticles puts forward chondroblast differentiation in hDPSCs through the expression of chondrogenic genes. The findings revealed that PLGA-collagen/PLGA-PEG-PLGA-TGF-ß1 nanocomposite hydrogel can be utilized as a supportive platform to support hDPSCs differentiation by implementing specific physio-chemical features.

Evaluating Mannuronic Acid Effect on Gene Expression Profile of Inflammatory Mediators in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is a multisystem disorder. Various studies have shown the important role of inflammatory factors tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-22, MYD88, and toll-like receptor 2 (TLR2) in this disease. In this study, we investigated the anti-inflammatory effects of B-D-Mannuronic acid (M2000), as a new immunosuppressive drug, on the expression of these inflammatory markers in peripheral blood mononuclear cells (PBMCs) of RA patients. The blood samples of active RA patients and healthy volunteers were used for PBMCsl separation. The cells were cultured with LPS (1 µg/mL), low (5 µg/mL), moderate (25 µg/mL), and high (50 µg/mL) doses of M2000 and a single dose of diclofenac (1 µg/mL) to evaluate TNF-α, IL-6, IL-22, MYD88, and TLR2 genes expression by quantitative real-time (qRT-PCR). Cell surface expression and MFI of TLR2 were assessed; using flow cytometry. Our findings exhibited a significant reduction of TNF-α, IL-6, and MYD88 gene expressions after treatment with three doses of M2000 and an optimum dose of diclofenac. TLR2 gene expression was significantly diminished by moderate and high doses of M2000 and a single dose of diclofenac. Moreoversurface expression of TLR2 was significantly downregulated by moderate and high doses of M2000, while MFI of this receptor was significantly reduced by three doses of M2000. The results of this research showed that M2000 was able to significantly reduce the gene expression of inflammatory molecules  TNF-α, IL-6, MYD88, and TLR2 in patients PBMCs. factor-alpha; Rheumatoid arthritis. These data revealed a part of the molecular mechanisms of M2000 in the treatment process.

MTA-Enriched Polymeric Scaffolds Enhanced the Expression of Angiogenic Markers in Human Dental Pulp Stem Cells.

Revascularization of the pulp tissue is one of the fundamental processes and challenges in regenerative endodontic procedures (REPs). In this regard, the current study is aimed at synthesizing the mineral trioxide aggregate- (MTA-) based scaffolds as a biomaterial for REPs. Poly (ε-caprolactone) (PCL)/chitosan (CS)/MTA scaffolds were constructed and evaluated by FTIR, SEM, XRD, and TGA analyses. Proliferation and adhesion of human dental pulp stem cells (hDPSCs) were assessed on these scaffolds by scanning electron microscopy (SEM) and MTT assays, respectively. The expression of angiogenic markers was investigated in gene and protein levels by real-time PCR and western blotting tests. Our results indicated that the obtained appropriate physicochemical characteristics of scaffolds could be suitable for REPs. The adhesion and proliferation level of hDPSCs were significantly increased after seeding on PCL/CS/MTA scaffolds. The expression levels of VEGFR-2, Tie2, and Angiopoietin-1 genes were statistically increased on the PCL/CS/MTA scaffold. In support of these findings, western blotting results showed the upregulation of these markers at protein levels in PCL/CS/MTA scaffold (P < 0.05). The current study results suggested that PCL/CS/MTA scaffolds provide appropriate structures for the adhesion and proliferation of hDPSCs besides induction of the angiogenesis process in these cells.

Towards Induction of Angiogenesis in Dental Pulp Stem Cells Using Chitosan-Based Hydrogels Releasing Basic Fibroblast Growth Factor.

INTRODUCTION: Chitosan is a natural biopolymer that attracted enormous attention in biomedical fields. The main components of regenerative endodontic procedures (REPs), as well as tissue engineering, are scaffolds, stem cells, and growth factors. As one of the basic factors in the REPs is maintaining vascularization, this study was aimed at developing basic fibroblast growth factor- (bFGF-) loaded scaffolds and investigating their effects on the angiogenic induction in human dental pulp stem cells (hDPSCs). METHODS: Poly (ε-caprolactone) (PCL)/chitosan- (CS-) based highly porous scaffold (PCL/CS) was prepared and evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analyses. The adhesion and survival potency of seeded cells were assessed by SEM and MTT assays, respectively. The amount of angiogenic markers was investigated in gene and protein levels by real-time PCR and western blotting assays, respectively. RESULTS: Based on our findings, the SEM and FTIR tests confirmed the appropriate structure of synthesized scaffolds. Besides, the adhesion and survival rate of cells and the levels of VEGFR-2, Tie2, and Angiopoietin-1 genes were increased significantly in the PCL/CS/bFGF group. Also, the western blotting results showed the upregulation of these markers at protein levels, which were considerably higher at the PCL/CS/bFGF group (P < 0.05). CONCLUSIONS: On a more general note, this study demonstrates that the bFGF-loaded PCL/CS scaffolds have the potential to promote angiogenesis of hDPSCs, which could provide vitality of dentin-pulp complex as the initial required factor for regenerative endodontic procedures.

Novel nanocomposite scaffold based on gelatin/PLGA-PEG-PLGA hydrogels embedded with TGF-ß1 for chondrogenic differentiation of human dental pulp stem cells in vitro.

In the current study, a novel nanocomposite hydrogel scaffold comprising of natural-based gelatin and synthetic-based (poly D, L (lactide-co-glycolide) -b- poly (ethylene glycol)-b- poly D, L (lactide-co-glycolide) (PLGA-PEG-PLGA) triblock copolymer was developed and loaded with transforming growth factor- ß1 (TGF-ß1). Synthesized scaffolds' chemical structure was examined by 1H NMR and ATR-FTIR. Scanning electron microscopy (SEM) confirmed particle size and morphology of the prepared nanoparticles as well as the scaffolds. The morphology analysis revealed a porous interconnected structure throughout the scaffold with a pore size dimension of about 202.05 µm. The swelling behavior, in vitro degradation, mechanical properties, density, and porosity were also evaluated. Phalloidin/DAPI staining was utilized for confirming the extended cytoskeleton of the chondrocytes. Alcian blue staining was conducted to determine cartilaginous matrix sulfated glycosaminoglycan (sGAG) synthesis. Eventually, over a period of 21 days, a real-time RT-PCR analysis was applied to measure the mRNA expression of chondrogenic marker genes, type-II collagen, SOX 9, and aggrecan, in hDPSCs cultured for up to 21 days to study the influence of gelatin/PLGA-PEG-PLGA-TGF-ß1 hydrogels on hDPSCs. The findings of the cell-encapsulating hydrogels analysis suggested that the adhesion, viability, and chondrogenic differentiation of hDPSCs improved by gelatin/PLGA-PEG-PLGA-TGF-ß1 nanocomposite hydrogels. These data supported the conclusion that gelatin/PLGA-PEG-PLGA-TGF-ß1 nanocomposite hydrogels render the features that allow thein vitrofunctionality of encapsulated hDPSCs and hence can contribute the basis for new effective strategies for the treatment of cartilage injuries.

The Effects of Mannuronic Acid on IL-1ß, IL-17A, STAT1, and STAT3 Gene Expressions and TLR2 and TLR4 Molecules in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic neurologic disease defined by inflammation and demyelination of the central nervous system that comes with variable degrees of axonal and neuronal damage. The efficacy of ß-D-mannuronic acid (M2000) as a novel drug with immunosuppressive properties (patented: PCT/EP2017/067920), has been shown in an experimental model of MS. In this study, the effects of M2000 on interleukin (IL)-1ß, IL-17A, signal transducer and activator of transcription (STAT) 1, and STAT3 gene expressions and Toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) molecules in patients with secondary progressive MS were evaluated. In this study, 14 patients with secondary progressive MS and 14 healthy subjects (as control group) were entered from the phase 2 clinical trial (Clinical Trial identifier, IRCT2016111313739N6). The gene expressions of IL-1ß, IL-17A, STAT1, and STAT3 were assessed at the baseline and then measured after 6 months of therapy with M2000 by using the quantitative real-time polymerase chain reaction method. Moreover, the expressions of TLR2 and TLR4 molecules on peripheral blood mononuclear cells were evaluated by the flow cytometry method. The gene expressions of IL-17A, STAT1, and STAT3 in patients with MS decreased after 6 months of therapy with M2000 comparing before treatment. Also, the gene expression of IL-1ß decreased numerically after 6 months. Furthermore, the expressions of TLR2 and TLR4 on PBMCs of the patients declined when compared to baseline. The results of this investigation revealed that M2000 could downregulate IL-17, STAT1, and STAT3 genes in patients with secondary progressive MS and also reduce the expressions of TLR2 and TLR4 on PBMCs. Moreover, M2000 declined numerically IL-ß gene expression.

Adverse Effects of Anti-Covid-19 Drug Candidates and Alcohol on Cellular Stress Responses of Hepatocytes.

During the pandemic, dexamethasone (DEX), remdesivir (RDV), hydroxychloroquine (HCQ), thapsigargin (TG), camostat mesylate (CaM), and pralatrexate were repurposed drugs for coronavirus disease 2019 (COVID-19). However, the side effects on the liver associated with the anti-COVID therapies are unknown. Cellular stresses by these drugs at 0-30 µM were studied using HepG2, Huh7, and/or primary human hepatocytes. DEX or RDV induced endoplasmic reticulum stress with increased X-box binding protein 1 and autophagic response with increased accumulation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II). DEX and RDV had additive effects on the stress responses in the liver cells, which further increased expression of activating transcription factor 4 and C/EBP homology protein 1 (CHOP), and cell death. Alcohol pretreatment (50 mM) and DEX induced greater cellular stress responses than DEX and RDV. Pralatrexate induced Golgi fragmentation, cell cycle arrest at G0/G1 phase, activations of poly (ADP-ribose) polymerase-1 (PARP) and caspases, and cell death. Pralatrexate and alcohol had synergistic effects on the cell death mediators of Bim, caspase3, and PARP. The protease inhibitor CaM and TG induced autophagic response and mitochondrial stress with altered mitochondrial membrane potential, B-cell lymphoma 2, and cytochrome C. TG and HCQ induced autophagic response markers of Unc-51 like autophagy activating kinase, LC3-II, Beclin1, and Atg5, and severe ER stress marker CHOP. Conclusion: These results suggest that the anti-COVID-19 drugs, especially with drug-drug or alcohol-drug combinations, cause cellular stress responses and injuries in the liver cells.

Evaluation of the success rate of pit and fissure sealants on first molars: 12 months follow-up study.

INTRODUCTION: Dental caries by far is the most prevalent concern of the preadolescents and adolescents in dental clinics. Despite the provision of local fluoride, the occlusal surfaces of teeth are susceptible to dental caries. Pit and fissure sealant therapy is a preventive method to decrease dental caries in permanent teeth. The present study aimed to evaluate the success of fissure sealant treatments of first molar teeth, at 3, 6 and 12 months follow-ups. MATERIALS AND METHODS: Sixty-five children were randomly selected. The subjects had already received fissure sealants in the department of public health dentistry. Demographic data, including age and gender, sealant failure and the type of failure were recorded in the relevant checklists. Feigal criteria were used to evaluate the success or failure of fissure sealant treatments. RESULTS: Overall success rate was 74.3% for 1 year. Evaluation of the failure rate showed that at the 3-month interval, 20.6% of the sealants exhibited failure (57.1% due to margin discoloration and 42.9% due to lack of margin adaptation). 28.6% of the sealants failed at the 6-month (75% due to marginal discoloration and 25% due to anatomical form) and 41.2% failed at the 12-month interval (57.1% due to marginal discoloration and 42.9% due to the lack of margin adaptation). CONCLUSION: The total failure rate of fissure sealant failures after 1 year was 27.7%. The most frequent reason for the failure of fissure sealants was marginal discoloration.

ß-D-mannuronic Acid (M2000) and Inflammatory Cytokines in COVID-19; An In vitro Study.

coronavirus disease of 2019 (COVID-19) can be complicated by acute respiratory distress syndrome (ARDS) and may be associated with cytokine storm and multiorgan failure. Anti-inflammatory agents, such as systemic corticosteroids, monoclonal antibodies, and nonsteroidal anti-inflammatory drugs (NSAIDs) can be used for this purpose. In this study, we evaluated the immunomodulatory effect of mannuronic acid (M2000), which is a novel NSAID, on COVID-19-related cytokine storms. This study was conducted in vitro on blood samples of 30 COVID-19 patients who presented with ARDS to a referral center. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and incubated with phorbol myristate acetate for 24 hours. M2000 was administered with the dosages of 25 µg/well and 50 µg/well after 4 hours of incubation at 37°C. The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess mRNA gene expression. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the supernatant PBMC levels of interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ. Both mRNA expression and the supernatant PBMC levels of IL-17, TNF-α, IL­6, and IFN­Î³ were decreased in PBMCs of COVID-19 patients treated with M2000 compared with the control  group. For the first time, it was observed that M2000 could be effective in alleviating the inflammatory cascade of COVID-19 patients based on an in vitro model. After further studies in vitro and in animal models, M2000 could be considered a novel NSAID drug in COVID-19 patients.

The Antimicrobial, Antioxidative, and Anti-Inflammatory Effects of Polycaprolactone/Gelatin Scaffolds Containing Chrysin for Regenerative Endodontic Purposes.

The appropriate endodontic material should eliminate the infection and inflammation to provide a situation for regeneration and healing of pulp tissue besides biomineralization. Chrysin is one of the active ingredients of plant flavonoids, which has significant anti-inflammatory and antimicrobial properties. In the present study, this natural substance was evaluated for antioxidant, anti-inflammatory, and mineralization properties on dental pulp stem cells (DPSCs). SEM, FTIR, and TGA tests were used to determine the successful synthesize of chrysin-loaded scaffolds. The antimicrobial effects of the synthesized scaffold against Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis were assessed by the agar diffusion test and live/dead assay. The proliferation of DPSCs on these scaffolds was determined by the MTT assay, DAPI staining, and DNA extraction. Moreover, the antioxidant and anti-inflammation activity of chrysin-loaded scaffolds on inflamed DPSCs was evaluated. Alkaline phosphatase activity and Alizarin Red S Stain tests were done to evaluate the mineralization of DPSCs seeded on these scaffolds. The chrysin-loaded scaffolds reported antimicrobial effects against evaluated bacterial strains. The proliferation of DPSCs seeded on these scaffolds was increased significantly (p < 0.05). The TNFα and DCF levels in inflamed DPSCs showed a significant decrease in the presence of chrysin-loaded scaffolds (p < 0.05). The ALP activity and formation of mineralized nodules of DPSCs on these scaffolds were significantly increased compared with the control group (p < 0.05). These results indicated that chrysin as an ancient therapeutic agent can accelerate the healing and regeneration of damaged pulp tissue, and this active ingredient can be a potential natural substance for regenerative endodontic procedures.

Synthesis, characterization, and evaluation of curcumin-loaded endodontic reparative material.

Curcumin (CUR) is an ancient therapeutic agent with remarkable antimicrobial and anti-inflammatory properties. The purpose of the current study was to synthesize and evaluate a curcumin-based reparative endodontic material to reduce infection and inflammation besides the induction of mineralization during the healing of the dentin-pulp complex. Poly-ɛ-caprolactone (PCL)/gelatin (Gel)/CUR scaffold was synthesized and assessed by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermo-gravimetric analysis (TGA). Agar diffusion test was performed against E. coli, A. baumannii, P. aeruginosa, S. aureus, E. faecalis, and S. mutans. Moreover, proliferative, antioxidative, anti-inflammatory, and calcification properties of these scaffolds on human dental pulp stem cells (hDPSCs) were evaluated. The results showed that PCL/Gel/CUR scaffold had antibacterial effects. Also, these CUR-based scaffolds had significant inhibitory effects on the expression of tumor necrosis factor α and DCF from inflamed hDPSCs (p < 0.05). Moreover, the induction of mineralization in hDPSCs significantly increased after seeding on CUR-based scaffolds (p < 0.05). Based on these findings, the investigated CUR-loaded material was fabricated successfully and provided an appropriate structure for the attachment and proliferation of hDPSCs. It was found that these scaffolds had antimicrobial, antioxidant, and anti-inflammatory characteristics and could induce mineralization in hDPSCs, which is essential for healing and repairing the injured dentin-pulp complex.

Evaluation of the Effect of Mannuronic Acid as a Novel NSAID With Immunosuppressive Properties on Expression of SOCS1, SOCS3, SHIP1, and TRAF6 Genes and Serum Levels of IL-6 and TNF-α in Patients With Multiple Sclerosis.

Multiple sclerosis (MS) is described as a chronic inflammatory, demyelinating disease of the central nervous system on an autoimmune basis, which is the most frequent reason for nontraumatic disability in youth. The efficacy and safety of ß-D-nannuronic acid (M2000) as a novel immunosuppressive drug (patented PCT/EP2017/067920) has been shown in an experimental model of MS and also in a phase 2 clinical trial. The effects of M2000 on SOCS1, SOCS3, TRAF6, and SHIP1 gene expression and also serum levels of IL-6 and TNF-α in secondary progressive multiple sclerosis patients have been assessed in this study. In this study, 14 secondary progressive multiple sclerosis patients and 14 healthy subjects (as the control group) were recruited from the phase 2 clinical trial (Clinical Trial identifier, IRCT2016111313739N6). Gene expression of SOCS1, SOCS3, TRAF6, and SHIP1 was measured at baseline and after 6 months of therapy with M2000 using a quantitative real-time polymerase chain reaction method. Furthermore, the serum levels of IL-6 and TNF-α were assessed by the enzyme-linked immunosorbent assay method. Our results showed that the gene expression of SOCS1, SOCS3, and SHIP1 was increased after 6 months of therapy with M2000 in MS patients. Moreover, the serum levels of IL-6 and TNF-α of patients declined compared with baseline, but this was not statistically significant. The results of this study demonstrated that M2000, with immunosuppressive properties, could upregulate SOCS1, SOCS3, and SHIP1 genes in patients with secondary progressive multiple sclerosis.

In vivo evaluation of biocompatibility and immune modulation potential of poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone)-gelatin hydrogels enriched with nano-hydroxyapatite in the model of mouse.

Biocompatible, biodegradable, and injectable hydrogels are a novel and promising approach for bone regeneration. In this study, poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone) (PCL-PEG-PCL), PCL-PEG-PCL-gelatin (Gel), PCL-PEG-PCL-Gel/nano-hydroxyapatite (nHA) injectable hydrogels were synthesized and evaluated in a mouse model of subcutaneous transplantation after 14 days. PCL-PEG-PCL-Gel and PCL-PEG-PCL-Gel/nHA hydrogels were fabricated with in situ precipitation method. Structure, intermolecular interaction, and the reaction between the PCL-PEG-PCL, Gel, and nHA were evaluated using a scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (H-NMR), and carbon nuclear magnetic resonance (C-NMR). Fourteen days after subcutaneous injection, the existence of an immune system reaction was investigated using Hematoxylin and Eosin (H&E) staining. Using immunofluorescence imaging, the number of CD68+ cells was determined in the periphery of the hydrogel. The CD8/CD4 lymphocyte ratio was also calculated in blood samples. We monitored the expression of CCL-2, BCL-2, IL-10, and CD31 using real-time PCR assay. The chemical evaluation revealed the successful integration of Gel and nHA to the PCL-PEG-PCL backbone. Histological examination showed the lack of inflammation at the site of injection. No toxicological effects were determined in hepatic and renal tissues. The addition of nHA to the PCL-PEG-PCL-Gel decreased biodegradation time. None of the hydrogels caused statistically significant differences in the number of CD68 cells (p > 0.05). The CD8/CD4 lymphocyte ratio remained unchanged in all groups (p > 0.05). Compared to the PCL-PEG-PCL group, the addition of nHA and Gel increased the expression of CCL-2, BCL-2, IL-10, and CD31 (p < 0.05). In conclusion, the current study showed that PCL-PEG-PCL-Gel/nHA hydrogels could be used in in vivo conditions without prominent toxic effects and inflammatory responses.

The osteogenic differentiation of human dental pulp stem cells in alginate-gelatin/Nano-hydroxyapatite microcapsules.

BACKGROUND: Microcapsule is considered as a promising 3D microenvironment for Bone Tissue Engineering (BTE) applications. Microencapsulation of cells in an appropriate scaffold not only protected the cells against excess stress but also promoted cell proliferation and differentiation. Through the current study, we aimed to microcapsulate the human Dental Pulp Stem Cells (hDPSCs) and evaluated the proliferation and osteogenic differentiation of those cells by using MTT assay, qRT-PCR, Alkaline phosphatase, and Alizarine Red S. RESULTS: The SEM results revealed that Alg/Gel microcapsules containing nHA showed a rough and more compact surface morphology in comparison with the Alg/Gel microcapsules. Moreover, the microencapsulation by Alg/Gel/nHA could improve cell proliferation and induce osteogenic differentiation. The cells cultured in the Alg/Gel and Alg/Gel/nHA microcapsules showed 1.4-fold and 1.7-fold activity of BMP-2 gene expression more in comparison with the control group after 21 days. The mentioned amounts for the BMP-2 gene were 2.5-fold and 4-fold more expression for the Alg/Gel and Alg/Gel/nHA microcapsules after 28 days. The nHA, addition to hDPSCs-laden Alg/Gel microcapsule, could up-regulate the bone-related gene expressions of osteocalcin, osteonectin, and RUNX-2 during the 21 and 28 days through the culturing period, too. Calcium deposition and ALP activities of the cells were observed in accordance with the proliferation results as well as the gene expression analysis. CONCLUSION: The present study demonstrated that microencapsulation of the hDPSCs inside the Alg/Gel/nHA hydrogel could be a potential approach for regenerative dentistry in the near future.

Influence of ß-D-mannuronic Acid, as a New Member of Non-steroidal Anti- Inflammatory Drugs Family, on the Expression Pattern of Chemokines and their Receptors in Rheumatoid Arthritis.

BACKGROUND: Based on the encouraging results of phase III clinical trial of ß-Dmannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. METHODS: PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 µg/mL) of M2000 and optimum dose (1 µg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. RESULTS: CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression was downregulated significantly followed by the treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after the treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by the treatment of these cells with a high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by the treatment of these cells with a high dose of M2000 and optimum dose of diclofenac. CONCLUSION: According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

Evaluation of the adhesion of human dental pulp stem cells to different endodontic biomaterials before and after setting.

Background. Stem cell-based treatment modalities have been potential strategies for tissue regeneration in many conditions. Several studies have evaluated the biologic properties of DPSCs and their efficacy in the treatment of a variety of diseases. The present study was undertaken to evaluate the adhesion behavior of DPSCs on different endodontic materials before and after setting. Methods. The crowns of the selected teeth were removed, and the root canals were prepared and obturated with gutta-percha and AH26 sealer. A retrograde cavity was prepared at root ends. Different materials were placed in the cavities. Then the samples were attached to the wells with the use of a chemical glue. Dental pulp stem cells were allowed to proliferate to reach a count of 2 million and transferred to -12well plates in association with a culture medium. Finally, the samples attached to the wells were exposed to the stem cells immersed in the culture medium before and after setting. Then adhesion of the stem cells was evaluated using SEM. Results. The SEM results showed cellular adhesion in the samples containing CEM cement both before and after setting. The samples containing MTA Angelus and ProRoot MTA exhibited cellular adhesion before setting, with no cellular adhesion after setting. The samples containing AH26 and MTA Fillapex sealers exhibited cellular adhesion after setting, with no adhesion before setting. The samples containing simvastatin exhibited no cellular adhesion before setting; this material had dissolved in the culture medium after setting evaluation. Conclusion. The results of the present study showed that of all the materials tested, CEM cement had the highest capacity for dental pulp stem cell adhesion.

Pichia fermentans originates apoptosis in human oral squamous cell carcinoma by over-expressing BAX and CASP 9 genes.

Squamous cell carcinoma (SCC) is one of the most common malignant tumors of the oral cavity. Probiotics have often been considered as effective anti-tumoral candidates. This study aimed to investigate the role of Pichia fermentans YSH secretion metabolites on the induction of apoptosis in SCC. Cytotoxicity, apoptotic effects, and visualization DNA damage were evaluated by MTT, flow cytometry, and DAPI staining assays, respectively. Real-time PCR was employed for evaluation of the mechanism of cellular apoptosis. P. fermentans YSH secretions (IC50) showed cellular cytotoxicity in human tongue squamous carcinoma (HSC4, RRID:CVCL_1289) cells (85% apoptosis) similar to the cytotoxicity of cisplatin whereas only 21% apoptosis was observed in human epithelial normal (KDR, RRID:CVCL_9V14) cells. The prophylactic efficacy of reference yeast, which regarded as a reference, was not comparable to P. fermentans YSH illustrating strain-dependent properties of bioactivities on oral disease control and prevention. According to our result, the main cytotoxicity is related to apoptosis mechanisms induced by apoptosis genes inducing BAX and CASP. However, follow-up researches should be performed to recognize the compounds to be utilized as effective anticancer therapeutics.