Silk ProteinsEnriched Nanocomposite Hydrogels Based on Modified MMT Clay and Poly(2-hydroxyethyl methacrylate-co-2-acrylamido-2-methylpropane Sulfonic Acid) Display Favorable Properties for Soft Tissue Engineering.

Due to their remarkable structures and properties, three-dimensional hydrogels and nanostructured clay particles have been extensively studied and have shown a high potential for tissue engineering as solutions for tissue defects. In this study, four types of 2-hydroxyethyl methacrylate/2-acrylamido-2-methylpropane sulfonic acid/montmorillonite (HEMA/AMPSA/MMT) hydrogels enriched with sericin, and fibroin were prepared and studied in the context of regenerative medicine for soft tissue regenerative medicine. Our aim was to obtain crosslinked hydrogel structures using modified montmorillonite clay as a crosslinking agent. In order to improve the in vitro and in vivo biocompatibility, silk proteins were further incorporated within the hydrogel matrix. Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) were performed to prove the chemical structures of the modified MMT and nanocomposite hydrogels. Swelling and rheological measurements showed the good elastic behavior of the hydrogels due to this unique network structure in which modified MMT acts as a crosslinking agent. Hydrogel biocompatibility was assessed by MTT, LDH and LIVE/DEAD assays. The hydrogels were evaluated for their potential to support adipogenesis in vitro and human stem cells isolated from adipose tissue were seeded in them and induced to differentiate. The progress was assessed by evaluation of expression of adipogenic markers (ppar-γ2, perilipin) evaluated by qPCR. The potential of the materials to support tissue regeneration was further evaluated on animal models in vivo. All materials proved to be biocompatible, with better results on the 95% HEMA 5% AMPSA enriched with sericin and fibroin material. This composition promoted a better development of adipogenesis compared to the other compositions studied, due the addition of sericin and fibroin. The results were confirmed in vivo as well, with a better progress of soft tissue regeneration after implantation in mice. Therefore, hydrogel 95% HEMA 5% AMPSA enriched with sericin as well as fibroin showed the best results that recommend it for future soft tissue engineering application.

Regenerative Potential of Mesenchymal Stem Cells' (MSCs) Secretome for Liver Fibrosis Therapies.

Chronic liver injuries lead to liver fibrosis and then to end-stage liver cirrhosis. Liver transplantation is often needed as a course of treatment for patients in critical conditions, but limitations associated with transplantation prompted the continuous search for alternative therapeutic strategies. Cell therapy with stem cells has emerged as an attractive option in order to stimulate tissue regeneration and liver repair. Transplanted mesenchymal stem cells (MSCs) could trans-differentiate into hepatocyte-like cells and, moreover, show anti-fibrotic and immunomodulatory effects. However, cell transplantation may lead to some uncontrolled side effects, risks associated with tumorigenesis, and cell rejection. MSCs' secretome includes a large number of soluble factors and extracellular vesicles (EVs), through which they exert their therapeutic role. This could represent a cell-free strategy, which is safer and more effective than MSC transplantation. In this review, we focus on cell therapies based on MSCs and how the MSCs' secretome impacts the mechanisms associated with liver diseases. Moreover, we discuss the important therapeutic role of EVs and how their properties could be further used in liver regeneration.

Complexation with Random Methyl-ß-Cyclodextrin and (2-Hidroxypropyl)-ß-Cyclodextrin Enhances In Vivo Anti-Fibrotic and Anti-Inflammatory Effects of Chrysin via the Inhibition of NF-κB and TGF-ß1/Smad Signaling Pathways and Modulation of Hepatic Pro/Anti-Fibrotic miRNA.

Chrysin (CHR) is a natural flavonoid with a wide range of pharmacological activities, including hepatoprotection, but poor water solubility. By including water-soluble hydroxypropyl (HPBCD) and randomly methylated (RAMEB) ß-cyclodextrin, we aimed to increase its biodisponibility and the effectiveness of the antifibrotic effects of chrysin at oral administration. Liver fibrosis in mice was induced in 7 weeks by CCl4 i.p. administration, and afterwards treated with 50 mg/kg of CHR-HPBCD, CHR-RAMEB, and free chrysin. CCl4 administration increased hepatic inflammation (which was augmented by the upregulation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB), tumor necrosis factor (TNF)-α, and interleukin 6 (IL-6) and induced fibrosis, as determined using histopathology and electron microscopy. These results were also confirmed by the upregulation of Collagen I (Col I) and matrix metalloproteinase (MMP) expression, which led to extracellular fibrotic matrix proliferation. Moreover, the immunopositivity of alpha-smooth muscle actin (a-SMA) in the CCl4 group was evidence of hepatic stellate cell (HSC) activation. The main profibrotic pathway was activated, as confirmed by an increase in the transforming growth factor- ß1 (TGF-ß1) and Smad 2/3 expression, while Smad 7 expression was decreased. Treatment with CHR-HPBCD and CHR-RAMEB considerably reduced liver injury, attenuated inflammation, and decreased extracellular liver collagen deposits. CHR-RAMEB was determined to be the most active antifibrotic complex. We conclude that both nanocomplexes exert anti-inflammatory effects and antifibrotic effects in a considerably stronger manner than for free chrysin administration.

A novel experimental approach to evaluate guided bone regeneration (GBR) in the rat femur using a 3D-printed CAD/CAM zirconia space-maintaining barrier.

INTRODUCTION: Obtaining a certain bone volume is an important goal in implantology or orthopedics. Thus, after tooth extraction, quite a lot of horizontal and vertical alveolar bone is lost in time and can be detrimental to the implant treatment outcome, while the treatment of critical bone defects is a considerable challenge for surgery. OBJECTIVES: In this study we designed a new in vivo model as an useful experimental tool to assess guided bone regeneration (GBR) using a computer-aided design/manufacturing (CAD-CAM) space-maintaining barrier. METHODS: The barrier was 3D printed with three progressive heights, surgically placed on rat femur, and GBR results were analyzed at 2, 4, and 8 weeks by X-ray and bone mineral density analysis, histology/morphometry and by immunofluorescence and immunohistochemistry for osteogenesis and angiogenesis evaluation. RESULTS: The obtained results show that the proposed experimental model provides a real-time useful information on progressive bone tissue formation, which depends on the volume of isolated space created for GBR and on molecular events that lead to satisfactory vertical and horizontal bone augmentation and osteointegration. CONCLUSION: In conclusion, the proposed customized three-dome space-maintaining barrier is suitable as an experimental tool to assess the potential of using the designed barriers in dentistry and orthopedics to promote the formation of new bone and determine their space- and time-dependent limitations. Meanwhile, guided bone augmentation for dentistry requires subsequent evaluation on an alveolar bone preclinical model followed by clinical implementation.

Complexation with Random Methyl-ß-Cyclodextrin and (2-Hydroxypropyl)-ß-Cyclodextrin Promotes Chrysin Effect and Potential for Liver Fibrosis Therapy.

Liver fibrosis results from chronic liver injury and is characterized by the accumulation of extracellular matrix in excess driven by hepatic stellate cells (HSCs) activation. Chrysin (CHR) is a natural flavonoid that is limited by its low solubility to exert its anti-inflammatory, antioxidant and anti-fibrotic properties. The aim of this study was to investigate the biocompatibility of CHR complexes with two cyclodextrins (CDs)-(2-hydroxypropyl)-ß-cyclodextrin (HPBCD) and random methyl-ß-cyclodextrin (RAMEB), and their potential to induce anti-inflammatory, antioxidant and anti-fibrotic effects. Biocompatibility of the complexes was evaluated on Huh7 and LX2 cell lines: MTT and Live/Dead tests indicated the cell viability and an LDH test showed the cytotoxicity. Immunohistochemical staining of Nuclear Factor Kappa B (NF-κB) nuclear translocation was performed to evaluate the anti-inflammatory effect of the complexes. Oxygen Radical Absorbance assay, Superoxide Dismutase activity and Glutathione Peroxidase (GPx) assays indicated the antioxidant properties of the chrysin complexes. Finally, the complexes' anti-fibrotic potential was evaluated at the protein and gene level of α-sma. In HSCs, CDs induced higher cytotoxicity correlated with lower cell viability than CHR-CD. The 1:1 CHR-RAMEB pretreatment avoided p65 translocation. The 1:2 CHR-RAMEB complex increased ORAC values, improved SOD activity and produced the highest stimulation of GPx activity. CHR-RAMEB reduced α-sma expression at lower concentration than CHR-HPBCD, proving to be more efficient. In conclusion, both CHR-CD complexes proved to be biocompatible, but CHR-RAMEB showed improved anti-inflammatory, antioxidant and anti-fibrotic effects that could recommend its further use in liver fibrosis treatment.

Cyclodextrin Complexation Improves the Solubility and Caco-2 Permeability of Chrysin.

Chrysin is a bioflavonoid that can be found in natural products such as honey and propolis, and it possesses several biological effects such as antioxidant, anti-inflammatory, and anti-cancer activity. However, it is poorly soluble in water, and its bioavailability is limited. The aim of this research is to investigate the chrysin solubilization capacity of different ß-cylcodextrin derivatives and compare their biological activities. Chrysin was complexed with ß-cyclodextrin (ßCD), hydroxypropyl-ß-, (HPBCD) sulfobutylether-ß-, (SBECD), and randomly-methylated-ß-cyclodextrin (RAMEB) by the lyophilization method in 1:1 and 1:2 molar ratios. The solubilities of the chrysin-cyclodextrin complexes were tested, and the solubilization abilities of cyclodextrins were studied by phase solubility experiments. The cytotoxicity of the complexes was measured by the MTT method, and the permeability enhancement was tested on Caco-2 monolayers. The solubility study showed that the complexes formed with RAMEB had the highest solubility in water. The phase solubility experiments confirmed the strongest interaction between RAMEB and chrysin. In the viability test, none of the complexes showed cytotoxicity up to 100 µM concentration. The permeability study revealed that both at 1:1 and 1:2 ratios, the RAMEB complexes were the most effective to enhance chrysin permeability through the Caco-2 monolayers. In conclusion, cyclodextrins, especially RAMEB, are suitable for improving chrysin solubility and absorption.

Cellular Interplay as a Consequence of Inflammatory Signals Leading to Liver Fibrosis Development.

Inflammation has been known to be an important driver of fibrogenesis in the liver and onset of hepatic fibrosis. It starts off as a process meant to protect the liver from further damage, but it can become the main promoter of liver fibrosis. There are many inflammation-related pathways activated during liver fibrosis that lead to hepatic stellate cells (HSCs) activation and collagen-deposition in the liver. Such events are mostly modulated upstream of HSCs and involve signals from hepatocytes and innate immune cells. One particular event is represented by cell death during liver injury that generates multiple inflammatory signals that further trigger sterile inflammation and enhancement of inflammatory response. The assembly of inflammasome that responds to danger-associated molecular patterns (DAMPs) stimulates the release of pro-inflammatory cytokines and at the same time, initiates programmed cell death called pyroptosis. This review focuses on cellular and molecular mechanisms responsible for initiation and progress of inflammation in the liver.

Proteomic Technology "Lens" for Epithelial-Mesenchymal Transition Process Identification in Oncology.

The epithelial-mesenchymal transition (EMT) is a complex transformation process that induces local and distant progression of many malignant tumours. Due to its complex array of proteins that are dynamically over-/underexpressed during this process, proteomic technologies gained their place in the EMT research in the last years. Proteomics has identified new molecular pathways of this process and brought important insights to develop new therapy targets. Various proteomic tools and multiple combinations were developed in this area. Out of the proteomic technology armentarium, mass spectrometry and array technologies are the most used approaches. The main characteristics of the proteomic technology used in this domain are high throughput and detection of minute concentration in small samples. We present herein, using various proteomic technologies, the identification in cancer cell lines and in tumour tissue EMT-related proteins, proteins that are involved in the activation of different cellular pathways. Proteomics has brought besides standard EMT markers (e.g., cell-cell adhesion proteins and transcription factors) other future potential markers for improving diagnosis, monitoring evolution, and developing new therapy targets. Future will increase the proteomic role in clinical investigation and validation of EMT-related biomarkers.

Graphene Oxide Enhances Chitosan-Based 3D Scaffold Properties for Bone Tissue Engineering.

The main goal of bone tissue engineering (BTE) is to refine and repair major bone defects based on bioactive biomaterials with distinct properties that can induce and support bone tissue formation. Graphene and its derivatives, such as graphene oxide (GO), display optimal properties for BTE, being able to support cell growth and proliferation, cell attachment, and cytoskeleton development as well as the activation of osteogenesis and bone development pathways. Conversely, the presence of GO within a polymer matrix produces favorable changes to scaffold morphologies that facilitate cell attachment and migration i.e., more ordered morphologies, greater surface area, and higher total porosity. Therefore, there is a need to explore the potential of GO for tissue engineering applications and regenerative medicine. Here, we aim to promote one novel scaffold based on a natural compound of chitosan, improved with 3 wt.% GO, for BTE approaches, considering its good biocompatibility, remarkable 3D characteristics, and ability to support stem cell differentiation processes towards the bone lineage.

Versatile Biomaterial Platform Enriched with Graphene Oxide and Carbon Nanotubes for Multiple Tissue Engineering Applications.

Carbon-based nanomaterials, such as graphene oxide (GO) or carbon nanotubes (CNTs) are currently used in various medical applications due to their positive influence on biocompatibility, adhesion, proliferation, and differentiation, as well as their contribution to modulating cell behavior in response to nanomaterial substrates. In this context, in this study, novel flexible membranes based on cellulose acetate (CA) enriched with CNT and GO in different percentages were tested for their versatility to be used as substrates for soft or hard tissue engineering (TE), namely, for their ability to support human adipose-derived stem cells (hASCs) adhesion during adipogenic or osteogenic differentiation. For this purpose, differentiation markers were assessed both at gene and protein levels, while histological staining was performed to show the evolution of the processes in response to CA-CNT-GO substrates. Micro-CT analysis indicated porous morphologies with open and interconnected voids. A slightly lower total porosity was obtained for the samples filled with the highest amount of GO and CNTs, but thicker walls, larger and more uniform pores were obtained, providing beneficial effects on cell behavior and increased mechanical stability. The addition of 1 wt% GO and CNT to the biocomposites enhanced hASCs adhesion and cytoskeleton formation. The evolution of both adipogenic and osteogenic differentiation processes was found to be augmented proportionally to the GO-CNT concentration. In conclusion, CA-CNT-GO biomaterials displayed good properties and versatility as platforms for cell differentiation with potential as future implantable materials in TE applications.

Efficiency of Multiparticulate Delivery Systems Loaded with Flufenamic Acid Designed for Burn Wound Healing Applications.

Burns are soft tissue injuries that require particular care for wound healing. Current tissue engineering approaches are aimed at identifying the most efficient treatment combinations to restore the tissue properties and function by using adapted scaffolds or delivery platforms for tissue repair and regeneration by triggering molecules. To reduce the inflammation associated with skin burns, the addition of an anti-inflammatory factor in these scaffolds would greatly increase the quality of the therapy. Therefore, this study is aimed at obtaining and validating a novel multiparticulate system based on a collagen matrix with controlled delivery of flufenamic acid anti-inflammatory drug for burn wound healing applications. In this work, we have characterized the properties and biocompatibility of these multiparticulate drug delivery systems (MDDS) and we have demonstrated their efficiency against burns and soft tissue lesions, particularly when the drug was microencapsulated, and thus with a controlled release. This study contributes to the advancement in therapy of burns and burn wound healing applications.