Recent advances and challenges in graphene-based nanocomposite scaffolds for tissue engineering application.

Graphene-based nanocomposites have recently attracted increasing attention in tissue engineering because of their extraordinary features. These biocompatible substances, in the presence of an apt microenvironment, can stimulate and sustain the growth and differentiation of stem cells into different lineages. This review discusses the characteristics of graphene and its derivatives, such as their excellent electrical signal transduction, carrier mobility, outstanding mechanical strength with improving surface characteristics, self-lubrication, antiwear properties, enormous specific surface area, and ease of functional group modification. Moreover, safety issues in the application of graphene and its derivatives in terms of biocompatibility, toxicity, and interaction with immune cells are discussed. We also describe the applicability of graphene-based nanocomposites in tissue healing and organ regeneration, particularly in the bone, cartilage, teeth, neurons, heart, skeletal muscle, and skin. The impacts of special textural and structural characteristics of graphene-based nanomaterials on the regeneration of various tissues are highlighted. Finally, the present review gives some hints on future research for the transformation of these exciting materials in clinical studies.

Nanotechnology-based products for cancer immunotherapy.

Currently, nanoscale materials and scaffolds carrying antitumor agents to the tumor target site are practical approaches for cancer treatment. Immunotherapy is a modern approach to cancer treatment in which the body's immune system adjusts to deal with cancer cells. Immuno-engineering is a new branch of regenerative medicine-based therapies that uses engineering principles by using biological tools to stimulate the immune system. Therefore, this branch's final aim is to regulate distribution, release, and simultaneous placement of several immune factors at the tumor site, so then upgrade the current treatment methods and subsequently improve the immune system's handling. In this paper, recent research and prospects of nanotechnology-based cancer immunotherapy have been presented and discussed. Furthermore, different encouraging nanotechnology-based plans for targeting various innate and adaptive immune systems will also be discussed. Due to novel views in nanotechnology strategies, this field can address some biological obstacles, although studies are ongoing.

Molecular pathways involved in COVID-19 and potential pathway-based therapeutic targets.

Deciphering the molecular downstream consequences of severe acute respiratory syndrome coronavirus (SARS-CoV)- 2 infection is important for a greater understanding of the disease and treatment planning. Furthermore, greater understanding of the underlying mechanisms of diagnostic and therapeutic strategies can help in the development of vaccines and drugs against COVID-19. At present, the molecular mechanisms of SARS-CoV-2 in the host cells are not sufficiently comprehended. Some of the mechanisms are proposed considering the existing similarities between SARS-CoV-2 and the other members of the ß-CoVs, and others are explained based on studies advanced in the structure and function of SARS-CoV-2. In this review, we endeavored to map the possible mechanisms of the host response following SARS-CoV-2 infection and surveyed current research conducted by in vitro, in vivo and human observations, as well as existing suggestions. We addressed the specific signaling events that can cause cytokine storm and demonstrated three forms of cell death signaling following virus infection, including apoptosis, pyroptosis, and necroptosis. Given the elicited signaling pathways, we introduced possible pathway-based therapeutic targets; ADAM17 was especially highlighted as one of the most important elements of several signaling pathways involved in the immunopathogenesis of COVID-19. We also provided the possible drug candidates against these targets. Moreover, the cytokine-cytokine receptor interaction pathway was found as one of the important cross-talk pathways through a pathway-pathway interaction analysis for SARS-CoV-2 infection.

Effects of in vitro low oxygen tension preconditioning of buccal fat pad stem cells on in Vivo articular cartilage tissue repair.

AIMS: Progenitor cells-based regenerative strategy has shown promise to repair cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to improve the early chondrogenic differentiation of stem cells. Therefore, this study aimed to determine whether hypoxia preconditioning could be used to enhance the regenerative potential of the combination of buccal fat pad stem cells (BFPSCs) and bilayer chitosan-based hydrogel scaffold for articular cartilage repair. MATERIALS AND METHODS: Human BFPSCs were seeded on the bilayer chitosan-based hydrogel scaffolds in the culture medium. The viability and proliferation of cells on the scaffolds were monitored using scanning electron microscopy (SEM), MTT assay, and DAPI staining. Hypoxia preconditioned BFPSCs-seeded scaffolds were transplanted into rabbit articular cartilage knee defects for 12 weeks. The newly formed tissue was evaluated by cartilage-specific immunohistological analysis and histological staining. KEY FINDINGS: It was found that the chondrogenic differentiation and osteochondral conjunction in articular cartilage defect via BFPSCs-seeded bilayer scaffolds was enhanced by hypoxic preconditioning compared to a normoxic environment. SIGNIFICANCE: Based on our study, the integrity with subchondral bone in osteochondral defect was enhanced by BFPSCs on bilayer scaffold. Thus, this study provides evidence on the design of preconditioned cell-seeded bilayer hydrogels for articular cartilage regeneration.

Impact of Lipid/Magnesium Hydroxide Hybrid Nanoparticles on the Stability of Vascular Endothelial Growth Factor-Loaded PLGA Microspheres.

The purpose of the present study is to characterize poly(d,l-lactide-co-glycolide) (PLGA) composite microcarriers for vascular endothelial growth factor (VEGF) delivery. To reduce the initial burst release and protect the bioactivity, VEGF is encapsulated in soybean l-α-phosphatidylethanolamine (PE) and l-α-phosphatidylcholine (PC) anhydrous reverse micelle (VEGF-RM) nanoparticles. Also, mesoporous nano-hexagonal Mg(OH)2 nanostructure (MNS)-loaded PE/PC anhydrous reverse micelle (MNS-RM) nanoparticles are synthesized to suppress the induced inflammation of PLGA acidic byproducts and regulate the release profile. The flow-focusing microfluidic geometry platforms are used to fabricate different combinations of PLGA composite microspheres (PLGA-CMPs) with MNSs, MNS-RM, VEGF-RM, and native VEGF. The essential parameters of each formulation, such as release profiles, encapsulation efficacy, bioactivity, inflammatory response, and cytotoxicity, are investigated by in vitro and in vivo studies. The results indicate that generated acidic byproducts during the hydrolytic degradation process of PLGA can be buffered, and pH values inside and outside microspheres can remain steady during degradation by MNSs. Furthermore, the significant improvement in the stability of the encapsulated VEGF is confirmed by the bioactivity assay. In vitro release study shows that the VEGF initial burst release is well minimized in the present microcarriers. The present monodisperse PLGA-CMPs can be widely used in various tissue engineering and therapeutic applications.

Comparison of osteogenic differentiation potential of induced pluripotent stem cells and buccal fat pad stem cells on 3D-printed HA/ß-TCP collagen-coated scaffolds.

Production of a 3D bone construct with high-yield differentiated cells using an appropriate cell source provides a reliable strategy for different purposes such as therapeutic screening of the drugs. Although adult stem cells can be a good source, their application is limited due to invasive procedure of their isolation and low yield of differentiation. Patient-specific human-induced pluripotent stem cells (hiPSCs) can be an alternative due to their long-term self-renewal capacity and pluripotency after several passages, resolving the requirement of a large number of progenitor cells. In this study, a new biphasic 3D-printed collagen-coated HA/ß-TCP scaffold was fabricated to provide a 3D environment for the cells. The fabricated scaffolds were characterized by the 3D laser scanning digital microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and mechanical test. Then, the osteogenesis potential of the hiPSC-seeded scaffolds was investigated compared to the buccal fat pad stem cell (BFPSC)-seeded scaffolds through in vitro and in vivo studies. In vitro results demonstrated up-regulated expressions of osteogenesis-related genes of RUNX2, ALP, BMP2, and COL1 compared to the BFPSC-seeded scaffolds. In vivo results on calvarial defects in the rats confirmed a higher bone formation in the hiPSC-seeded scaffolds compared to the BFPSC-seeded groups. The immunofluorescence assay also showed higher expression levels of collagen I and osteocalcin proteins in the hiPSC-seeded scaffolds. It can be concluded that using the hiPSC-seeded scaffolds can lead to a high yield of osteogenesis, and the hiPSCs can be used as a superior stem cell source compared to BFPSCs for bone-like construct bioengineering.

Fabrication of Decellularized Engineered Extracellular Matrix through Bioreactor-Based Environment for Bone Tissue Engineering.

Extracellular matrix (ECM)-contained grafts can be achieved by decellularization of native bones or synthetic scaffolds. Limitations associated with harvesting the native bone has raised interest in preparing in vitro ECM bioscaffold for bone tissue engineering. Here, we intend to develop an ECM-contained construct via decellularizing an engineered gelatin-coated ß-tricalcium phosphate (gTCP) scaffold. In order to find an optimal protocol for decellularization of cell-loaded gTCP scaffolds, they were seeded with buccal fat pad-derived stem cells. Then, four decellularization protocols including sodium dodecyl sulfate, trypsin, Triton X-100, and combined solution methods were compared for the amounts of residual cells and remnant collagen and alteration of scaffold structure. Then, the efficacy of the selected protocol in removing cells from gTCP scaffolds incubated in a rotating and perfusion bioreactor for 24 days was evaluated and compared with static condition using histological analysis. Finally, decellularized scaffolds, reloaded with cells, and their cytotoxicity and osteoinductive capability were evaluated. Complete removal of cells from gTCP scaffolds was achieved from all protocols. However, treatment with Triton X-100 showed significantly higher amount of remnant ECM. Bioreactor-incubated scaffolds possessed greater magnitude of ECM proteins including collagen and glycosaminoglycans. Reseeding the decellularized scaffolds also represented higher osteoinductivity of bioreactor-based scaffolds. Application of Triton X-100 as decellularization protocol and usage of bioreactors are suggested as a suitable technique for designing ECM-contained grafts for bone tissue engineering.

Mesenchymal Stem Cell Spheroids Embedded in an Injectable Thermosensitive Hydrogel: An In Situ Drug Formation Platform for Accelerated Wound Healing.

The ability of mesenchymal stem cells (MSCs) to enhance cutaneous wound healing has been well established. Extensive expansion of cells to reach sufficient cell numbers for regenerating tissues has always limited cell-based therapies. An ingenious solution to address this challenge is to develop a strategy to increase the immunomodulatory effects of MSCs without expanding them. In this study, we employed a simple characteristic of cells. It was observed that an optimized three-dimensional (3D) MSC culture in spheroid forms significantly improved their paracrine effects. An electrospray (ES) encapsulation apparatus was used to encapsulate individual or 3D spheroid MSCs into microscale alginate beads (microbeads). Furthermore, alginate microbeads were embedded in an injectable thermosensitive hydrogel matrix, which gels at skin temperature. The hydrogel fills and seals the wounds cavities, maintains high humidity at the wound area, absorbs exudate, and fixes microbeads, protecting them from direct contact with the harsh wound environment. In vitro investigations revealed that secretion of interleukin 10 (IL-0) and transforming growth factor ß1 (TGF-ß1) gene was gradually enhanced, providing a delivery platform for prolonged release of bioactive molecules. In vivo study on full-thickness wounds showed granulation and re-epithelialization, only after 7 days. Moreover, increased expression of α-smooth muscle actin (α-SMA) in the first 14 days after treatment ensured wound contraction. Besides, a gradual decrease in α-SMA secretion resulted in reduced scar formation. Well-organized collagen fibrils and high expression of the angiogenesis biomarker CD31 confirmed the promoting effect of the hydrogel on the wound-healing process. The proposed wound-dressing system would potentially be used in scalable and effective cell-based wound therapies.

Improved antifungal activity and stability of chitosan nanofibers using cellulose nanocrystal on banknote papers.

Microorganisms can spread on the surface of banknotes and cause many infectious diseases. Chitosan nanofibers (CNFs) and cellulose nanocrystals (CNCs) are nanomaterials, which can affect the antimicrobial properties. In this study, the fungal species that grew on the surfaces of collected banknotes from different places were identified. To examine the antifungal effect of the both nanomaterials on the banknotes, the stable coatings using CNFs and CNCs emulsions were prepared by roller coating. The results revealed that the most colonies in the banknotes obtained from the bakeries and butcheries were Aspergillus sp., whereas the colonies in bus terminals and the hospitals were Aspergillus niger and Penicillium, respectively. The results showed that the CNCs had no antifungal effect alone on the aforementioned species, but it could improve the antifungal effect, adhesion, and stability of CNFs on the banknote surfaces. This study suggested a new approach to decrease the infection spreads through banknotes.

Electrical stimulation of somatic human stem cells mediated by composite containing conductive nanofibers for ligament regeneration.

One of the advances in the field of biomedical nanotechnology, is conductive nanofiber fabrication and the discovery of its applications. Biocompatible flexible nanofibers that have a good biocompatibility, mechanical properties and morphology. Poly (3, 4-ethylene dioxythiophene) (PEDOT) is a conductive polymer that has recently been used in medical applications. In this study, the electrospinning technique and vapor phase polymerization combination method with freeze drying was used to produce Silk fibroin/PEDOT/Chitosan nanocomposite scaffold. The aim of our study was to develop a ligament construct of PEDOT/Silk bilayer nanofibrous scaffold, to mimic the aligned collagen fiber bundles and Chitosan sponge coating was done on these fibrous scaffolds, to mimic the glycosaminoglycans of ECM sheath. The developed constructs were characterized. The unrestricted somatic human stem cells (USSC), were cultured on the scaffold. Then, the effect of applying DC electric pulses to cells cultured on polymer was assessed. Cellular function was actively exhibited in scaffold with electrical induction, as evident by the high expression of collagen I, collagen III, decorin, biglycan and aggrecan genes. Novel scaffold plus electrical stimulation shows facilitating cell seeding and promoting cell proliferation, differentiation. This composites can be used in this new field for stem cells differentiation to target tissues.

Enhanced Cardiac Differentiation of Human Cardiovascular Disease Patient-Specific Induced Pluripotent Stem Cells by Applying Unidirectional Electrical Pulses Using Aligned Electroactive Nanofibrous Scaffolds.

In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (ß) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.

In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

Significant changes of 5-Hydroxytriptamine 3A receptor gene expression in peripheral blood mononuclear cells of allergic asthmatic patients.

Asthma is a chronic inflammatory disorder of the airways. The stress is a factor for asthma which indicates a disorder in the function of communicational mediators of nervous and immunological systems such as neurotransmitters. A study indicated that blood serotonin concentration increases in asthmatic patients. Other study indicates that one kind of the serotonin receptors, named 5HT3A, on PBMCs causes secretion of series of pro-inflammatory cytokines which play important roles in allergic asthma disease. Thus, we evaluated the ratio expression level of 5HT3A subtype receptors in asthma. The Peripheral Blood Mononuclear Cells were separated from whole blood of 30 allergic asthmatic patients and 30 normal controls by a gradient density centrifugation technique, then the total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for 5-hydroxytryptamine 3A subtype receptor mRNA and beta-actin as internal control. Results revealed that relative gene expression of 5-hydroxytryptamine 3A subtype receptor increased significantly in Peripheral Blood Mononuclear Cells of patients with asthma in comparison with normal individuals. To conclude, considering 5-hydroxytryptamine 3A subtype receptor role in accomplishment of asthma symptoms, this increase in its expression may exacerbate the seriousness of asthma disease.