Pluripotent stem cells: An in vitro model for nanotoxicity assessments.

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd.

Emerging Trends in Biodegradable Microcarriers for Therapeutic Applications.

Microcarriers (MCs) are adaptable therapeutic instruments that may be adjusted to specific therapeutic uses, making them an appealing alternative for regenerative medicine and drug delivery. MCs can be employed to expand therapeutic cells. MCs can be used as scaffolds for tissue engineering, as well as providing a 3D milieu that replicates the original extracellular matrix, facilitating cell proliferation and differentiation. Drugs, peptides, and other therapeutic compounds can be carried by MCs. The surface of the MCs can be altered, to improve medication loading and release, and to target specific tissues or cells. Allogeneic cell therapies in clinical trials require enormous volumes of stem cells, to assure adequate coverage for several recruitment locations, eliminate batch to batch variability, and reduce production costs. Commercially available microcarriers necessitate additional harvesting steps to extract cells and dissociation reagents, which reduces cell yield and quality. To circumvent such production challenges, biodegradable microcarriers have been developed. In this review, we have compiled key information relating to biodegradable MC platforms, for generating clinical-grade cells, that permit cell delivery at the target site without compromising quality or cell yields. Biodegradable MCs could also be employed as injectable scaffolds for defect filling, supplying biochemical signals for tissue repair and regeneration. Bioinks, coupled with biodegradable microcarriers with controlled rheological properties, might improve bioactive profiles, while also providing mechanical stability to 3D bioprinted tissue structures. Biodegradable materials used for microcarriers have the ability to solve in vitro disease modeling, and are advantageous to the biopharmaceutical drug industries, because they widen the spectrum of controllable biodegradation and may be employed in a variety of applications.

Cell-derived nanovesicles from mesenchymal stem cells as extracellular vesicle-mimetics in wound healing.

Wound healing is a dynamic process that involves a series of molecular and cellular events aimed at replacing devitalized and missing cellular components and/or tissue layers. Recently, extracellular vesicles (EVs), naturally cell-secreted lipid membrane-bound vesicles laden with biological cargos including proteins, lipids, and nucleic acids, have drawn wide attention due to their ability to promote wound healing and tissue regeneration. However, current exploitation of EVs as therapeutic agents is limited by their low isolation yields and tedious isolation processes. To circumvent these challenges, bioinspired cell-derived nanovesicles (CDNs) that mimic EVs were obtained by shearing mesenchymal stem cells (MSCs) through membranes with different pore sizes. Physical characterisations and high-throughput proteomics confirmed that MSC-CDNs mimicked MSC-EVs. Moreover, these MSC-CDNs were efficiently uptaken by human dermal fibroblasts and demonstrated a dose-dependent activation of MAPK signalling pathway, resulting in enhancement of cell proliferation, cell migration, secretion of growth factors and extracellular matrix proteins, which all promoted tissue regeneration. Of note, MSC-CDNs enhanced angiogenesis in human dermal microvascular endothelial cells in a 3D PEG-fibrin scaffold and animal model, accelerating wound healing in vitro and in vivo. These findings suggest that MSC-CDNs could replace both whole cells and EVs in promoting wound healing and tissue regeneration.

Black cardamom (Amomum subulatum Roxb.) fruit extracts exhibit apoptotic activity against lung cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried fruits of Amomum subulatum Roxb. (A. subulatum) are widely used as a spice. It is a part of official ayurvedic formulations used in folklore medicine to treat cancer.A. subulatum has been used in ayurvedic formulations to treat various lung conditions such as cough, lung congestion, pulmonary tuberculosis. The present traditional knowledge highlights the effectiveness of A. subulatum in treating cancer and its lung-specific efficacy. AIM OF THE STUDY: This study aims to investigate the cytotoxic potential of A. subulatum on the phenomenal and mechanistic level of lung cancer cells and identify the presence of A. subulatum actives. MATERIALS AND METHODS: The bioactivity of the extracts was tested using MTT assay, apoptotic assay, cell cycle analysis, superoxide production assay, reactive oxygen species (ROS) assay, and western blot analysis. Firstly, five different extracts were prepared using sequential extraction, and then screening of cell lines was performed using MTT assay. RESULTS: Lung cancer cells were selected as the most sensitive target, and dichloromethane extract (DE) was the most active extract. Annexin assay confirmed the mode of cell death as apoptosis. SubG1 peak found in cell cycle analysis substantiated this finding. ROS generation and superoxide showed association with apoptotic death. The upregulation and overexpression of cleaved poly(ADP-ribose)polymerase-1 (PARP-1) showed the failure of DNA repairing machinery contributes to apoptosis. LC-MS findings show the presence of cytotoxic actives cardamonin and alpinetin. CONCLUSIONS: In summary, this study shows the apoptosis-inducing potential of A. subulatum fruit extracts and confirms DNA damage as one of the causes of cell death. Further explorations using bio-fractionation and in-vivo studies are required to determine the most active constituents in A. subulatum.

Enhancement of Skin Delivery of Drugs Using Proposome Depends on Drug Lipophilicity.

The study aims to investigate the propylene glycol-based liposomes named 'proposomes' in enhancing skin permeation of drugs with different physicochemical properties. Ibuprofen, tofacitinib citrate, rhodamine B, and lidocaine were loaded into proposomes. These drug formulations were analyzed for particle size, zeta potential, polydispersity index, entrapment efficiency, and in vitro skin permeation. The confocal laser scanning microscopy was performed on skin treated with calcein and rhodamine B laden proposomes. The transdermal delivery relative to physicochemical properties of drugs such as logP, melting point, molecular weight, solubility, etc., were analyzed. We tested the safety of the proposomes using reconstructed human skin tissue equivalents, which were fabricated in-house. We also used human cadaver skin samples as a control. The proposomes had an average diameter of 128 to 148 nm. The drug's entrapment efficiencies were in the range of 42.9-52.7%, translating into the significant enhancement of drug permeation through the skin. The enhancement ratio was 1.4 to 4.0, and linearly correlated to logP, molecular weight, and melting point. Confocal imaging also showed higher skin permeation of calcein and rhodamine B in proposome than in solution. The proposome was found safe for skin application. The enhancement of skin delivery of drugs through proposomes was dependent on the lipophilicity of the drug. The entrapment efficiency was positively correlated with logP of the drug, which led to high drug absorption.

Fabrication of vascularized tissue constructs under chemically defined culture conditions.

Three-dimensional (3D) biofabrication techniques that enable the production of multicellular tissue equivalents for applications in basic biology, drug screening and regenerative medicne. Fabrication of these tissue constructs with in-built microvasculature enables recapitulation of the biological environment of the native tissues. Here, we present the fabrication of 3D vascularized tissue constructs containing microvascular networks using human embryonic stem cell (hESC)-derived endothelial cells (ECs) and pericytes encapsulated within a fibrin-based matrix and cultured under chemically defined conditions. Firstly, by manipulating the developmental signaling pathways under chemically defined culture conditions, hESCs were efficiently differentiated to hESC-ECs and hESC-pericytes through intermediate stages of lateral plate and paraxial mesoderm respectively. Next, encapsulation of these hESC-derived vascular cells within fibrin-based matrix and culture under chemically defined conditions, result in self-assembly of hESC-ECs into a network of microvessels within a period of 6-9 d. With the supporting influence of hESC-pericytes, the microvascular network with lumen was stable for at least 3 weeks. Quantification of the fractal dimensions of the microvascular networks demonstrate the increasing complexity of the vascular network with increasing endothelial cell densities. Dextran permeation studies in the presence or absence of vasodilating agent (histamine) showed the presence of hollow lumen, modulation of barrier properties of the microvasculature and its functional response to histamine. Hence, this versatile in vitro 3D model of vascularized constructs generated under chemically defined conditions is well suited to study early angiogenesis for in vitro drug testing applications and provide a clinically amenable, fundamental step towards fabrication of complex and functional tissues for regenerative applications in the future.

Proposome for transdermal delivery of tofacitinib.

Tofacitinib citrate (TC) has recently gained interest in treating skin disorders such as psoriasis, atopic dermatitis and baldness. Unfortunately, the oral administration shows side effects, such as decreased neutrophil counts. To this end, the topical delivery of TC can be used to reduce the risk associated with systemic exposure. However, TC shows minimal absorption via skin. Hence, the objective of this study is to enhance the skin delivery of TC using a non-invasive approach. The liposomes based on propylene glycol, named as proposomes, carrying TC, were studied. The vesicle characteristics and in vitro skin permeation were assessed. The proposomes enhanced the skin permeability of TC by 4-11 folds. The composition of proposomes was found to affect the skin permeation and deposition of TC. The proposomes were stable for at least 6 months. Overall, proposomes were effective for targeted topical drug delivery.

PLGA nanoparticles as chlorhexidine-delivery carrier to resin-dentin adhesive interface.

OBJECTIVE: To characterize and deliver fabricated CHX-loaded PLGA-nanoparticles inside micron-sized dentinal-tubules of demineralized dentin-substrates and resin-dentin interface. METHODS: Nanoparticles fabricated by emulsion evaporation were assessed in-vitro by different techniques. Delivery of drug-loaded nanoparticles to demineralized dentin substrates, interaction with collagen matrix, and ex-vivo CHX-release profiles using extracted teeth connected to experimental setup simulating pulpal hydrostatic pressure were investigated. Furthermore, nanoparticles association/interaction with a commercial dentin-adhesive applied to demineralized dentin substrates were examined. RESULTS: The results showed that the formulated nanoparticles demonstrated attractive physicochemical properties, low cytotoxicity, potent antibacterial efficacy, and slow degradation and gradual CHX release profiles. Nanoparticles delivered efficiently inside dentinal-tubules structure to sufficient depth (>10µm) against the simulated upward pulpal hydrostatic-pressure, even after bonding-resins infiltration and were attached/retained on collagen-fibrils. These results verified the potential significance of this newly introduced drug-delivery therapeutic strategy for future clinical applications and promote for a new era of future dental research. SIGNIFICANCE: This innovative drug-delivery strategy has proven to be a reliable method for delivering treatments that could be elaborated for other clinical applications in adhesive and restorative dentistry.

In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors.

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.