Rajesh Vasita

A Review on Extracellular Matrix Mimicking Strategies for an Artificial Stem Cell Niche

In adults, stem cells live in a distinctive microenvironment known as “stem cell niche”. Within such a niche, biochemical and biophysical attributes of extracellular matrix (ECM) executes a major role in regulating stem cell self-renewal and differentiation. However, in conventional in vitro culture system, stem cells are deprived of ECM and hence it is difficult to maintain, expand and differentiate them into a particular lineage. Therefore, different two- and three-dimensional substrates have been designed to mimic the ECM features in vitro. The present review focuses mainly on different scaffolds/substrates made up of or functionalized with proteins, polysaccharides and peptides (biochemical) in form of fibers, gels, gratings, pillars, pits (geometrical) of varying stiffness, and mechanical pressure (mechanical) for replicating stem cell microenvironment that not only helps in understanding stem cell-ECM interactions but also in the development of systems for regenerative medicine applications.

Polycaprolactone–chitosan nanofibers influence cell morphology to induce early osteogenic differentiation

Osteogenic differentiation is highly correlated with cell morphology. Morphological changes are a stimulus as well as a consequence of the differentiation process. Besides, geometrical, biochemical and mechanical properties of a substrate can modulate cell adhesion and morphology. Therefore, in the current study, nanofibrous substrate properties were used to implement necessary changes in cell morphology which induced osteogenic differentiation without biological supplements. A polycaprolactone-chitosan nanofiber substrate had been fabricated with an average diameter of ∼75 nm and an appropriate ratio of polymers that balances surface biocompatibility as well as mechanical strength. DSC and wide-angle XRD analysis revealed miscibility between polymers; whereas a degradation study confirmed the structural integrity of nanofibers. Nanofibers did not cause any cytotoxicity to MC3T3-E1 cells as confirmed by Live/Dead® staining. Morphological studies by SEM and confocal microscopy showed significant changes in terms of cell shape, area, compactness, aspect ratio and nucleus area in cells grown on nanofibers which indicated the osteogenic differentiation inducing potential of nanofibers. This was further confirmed by enhanced mineral deposition and alkaline phosphatase activity up to three weeks. In summary, polycaprolactone-chitosan nanofibers could induce early osteogenic differentiation in MC3T3-E1 pre-osteoblasts without any biological supplements by modulating cell morphology. Moreover, cell morphological features can be used as a predictive and informative approach at the early stages of differentiation experiments.

Antioxidative study of Cerium Oxide nanoparticle functionalised PCL-Gelatin electrospun fibers for wound healing application

Skin wound healing involves a coordinated cellular response to achieve complete reepithelialisation. Elevated levels of reactive oxygen species (ROS) in the wound environment often pose a hindrance in wound healing resulting in impaired wound healing process. Cerium oxide nanoparticles (CeNPs) have the ability to protect the cells from oxidative damage by actively scavenging the ROS. Furthermore, matrices like nanofibers have also been explored for enhancing wound healing. In the current study CeNP functionalised polycaprolactone (PCL)-gelatin nanofiber (PGNPNF) mesh was fabricated by electrospinning and evaluated for its antioxidative potential. Wide angle XRD analysis of randomly oriented nanofibers revealed ∼2.6 times reduced crystallinity than pristine PCL which aided in rapid degradation of nanofibers and release of CeNP. However, bioactive composite made between nanoparticles and PCL-gelatin maintained the fibrous morphology of PGNPNF upto 14 days. The PGNPNF mesh exhibited a superoxide dismutase (SOD) mimetic activity due to the incorporated CeNPs. The PGNPNF mesh enhanced proliferation of 3T3-L1 cells by ∼48% as confirmed by alamar blue assay and SEM micrographs of cells grown on the nanofibrous mesh. Furthermore, the PGNPNF mesh scavenged ROS, which was measured by relative DCF intensity and fluorescence microscopy; and subsequently increased the viability and proliferation of cells by three folds as it alleviated the oxidative stress. Overall, the results of this study suggest the potential of CeNP functionalised PCL-gelatin nanofibrous mesh for wound healing applications.

Study of locust bean gum reinforced cyst-chitosan and oxidized dextran based semi-IPN cryogel dressing for hemostatic application

Severe blood loss due to traumatic injuries remains one of the leading causes of death in emergency settings. Chitosan continues to be the candidate material for hemostatic applications due to its inherent hemostatic properties. However, available chitosan-based dressings have been reported to have an acidic odor at the wound site due to the incorporation of acid based solvents for their fabrication and deformation under compression owing to low mechanical strength limiting its usability. In the present study semi-IPN cryogel was fabricated via Schiffs base cross-linking between the polyaldehyde groups of oxidized dextran and thiolated chitosan in presence of locust bean gum (LBG) known for its hydrophilicity. Polymerization at −12 °C yielded macroporous semi-IPN cryogels with an average pore size of 124.57 ± 20.31 μm and 85.46% porosity. The hydrophobicity index of LBG reinforced semi-IPN cryogel was reduced 2.42 times whereas the swelling ratio was increased by 156.08% compare to control cryogel. The increased hydrophilicity and swelling ratio inflated the compressive modulus from 28.1 kPa to 33.85 for LBG reinforced semi-IPN cryogel. The structural stability and constant degradation medium pH were also recorded over a period of 12 weeks. The cryogels demonstrated lower adsorption affinity towards BSA. The cytotoxicity assays (direct, indirect) with 3T3-L1 fibroblast cells confirmed the cytocompatibility of the cryogels. The hemolysis assay showed <5% hemolysis confirming blood compatibility of the fabricated cryogel, while whole blood clotting and platelet adhesion assays confirmed the hemostatic potential of semi-IPN cryogel.

Nano-delivery of Food-Derived Biomolecules: An Overview

Certain bioactive molecules present in food have therapeutic effects such as antioxidant, anti-carcinogenic, anti-mutagenic, anti-ageing and anti-inflammatory activity. Although these molecules can be consumed via food, their effects are often subordinated due to degradation during digestion and lesser bioavailability at target site. Nanocarriers such as nanoparticles, nanoliposomes and nanopolymersomes have been developed for the food-derived biomolecules that can overcome traditional drug delivery limitations and provide advantages such as increased solubility and stability, protection against degradation, target specificity, increased bioavailability and regulated release. This chapter discusses different types of nanocarriers for delivery of food-derived biomolecules along with their advantages over conventional drug delivery methods. It also addresses the clearance of these nanocarriers from the body, their toxicity and ethics-related concerns. Overall, it provides an overview of nanocarriers used for delivery of food-derived bioactive molecules.

Bi-functional oxidized dextran–based hydrogel inducing microtumors: An in vitro three-dimensional lung tumor model for drug toxicity assays:

Cancer is a serious death causing disease having 8.2 million deaths in 2012. In the last decade, only about 10% of chemotherapeutic compounds showed productivity in drug screening. Two-dimensional culture assays are the most common in vitro drug screening models, which do not precisely model the in vivo condition for reliable preclinical drug screening. Three-dimensional scaffold–based cell cultures perhaps mimic tumor microenvironment and recapitulate physiologically more relevant tumor. This study was carried out to develop bi-functional oxidized dextran–based cell instructive hydrogel that provides three-dimensional environment to cancer cells for inducing microtumor. Oxidized dextran was blended with thiolated chitosan to fabricate an in situ self-gelable hydrogel (modified dextran–chitosan) in a one-step process. The hydrogels characterization revealed cross-linked network structure with highly porous structure and water absorption. The modified dextran–chitosan hydrogel showed reduced hydrophobicity and has reduced protein absorption, which resulted in changing the A549 cell adhesiveness, and encouraged them to form microtumor. The cells were proliferated in clusters having spherical morphology with randomly oriented stress fiber and large nucleus. Further microtumors were studied for hypoxia where reactive oxygen species generation demonstrated 15-fold increase as compared to monolayer culture. Drug-sensitivity results showed that microtumors generated on modified dextran–chitosan hydrogel showed resistance to doxorubicin with having 33%–58% increased growth than two-dimensional monolayer model at concentrations of 25–100 µM. In summary, the modified dextran–chitosan scaffold can provide surface chemistry that induces three-dimensional microtumors with physiologically relevant properties to in vivo tumor including growth, morphology, extracellular matrix production, hypoxic phenotype, and drug response. This model can be potentially utilized for drug toxicity studies and cancer disease modeling to understand tumor phenotype and progression.