Differential Migration and Proliferation Potential of the Hydrogel Aided 3D Tumoroid.

For substantial in vitro cancer biology research, the 3D cell culture method has now been regarded as more suitable model expected to be recapitulating maximum in vivo tumor mass relevance. Despite of available techniques to develop in vitro 3D models, a system availing a physiologically relevant in vitro 3D model of primary lung adenocarcinoma with extracellular matrix (ECM) mimicry and similar tumorigenic properties still remains a quest. Thus, in the present study, chemically modified Dextran-Chitosan (MDC) hydrogel has been developed as a 3D tumoroid aiding scaffold. The 3D A549 tumoroids aided by the MDC scaffold have physiologically relevant proliferation, migration, invasive potential, and Gefitinib [targeting epidermal growth factor receptor (EGFR)] efficacy as compared to the 2D cultured cells. The surface topography and wettability of hydrogel availed in vivo micro tumor mass mimicking Lung adenocarcinoma 3D in vitro model. Thus, opening an innovative avenue for elucidating the disease mechanism and drug efficacy on relevant 3D cancer models in vitro.

3D Tumor Models for Breast Cancer: Whither We Are and What We Need.

Three-dimensional (3D) models have led to a paradigm shift in disease modeling in vitro, particularly for cancer. The past decade has seen a phenomenal increase in the development of 3D models for various types of cancers with a focus on studying stemness, invasive behavior, angiogenesis, and chemoresistance of cancer cells, as well as contributions of its stroma, which has expanded our understanding of these processes. Cancer biology is moving into exploring the emerging hallmarks of cancer, such as inflammation, immune evasion, and reprogramming of energy metabolism. Studies into these emerging concepts have provided novel targets and treatment options such as antitumor immunotherapy. However, 3D models that can investigate the emerging hallmarks are few and underexplored. As commonly used immunocompromised mice and syngenic mice cannot accurately mimic human immunology, stromal interactions, and metabolism and require the use of prohibitively expensive humanized mice, there is tremendous scope to develop authentic 3D tumor models in these areas. Taking the specific case of breast cancer, we discuss the currently available 3D models, their applications to mimic signaling in cancer, tumor-stroma interactions, drug responses, and assessment of drug delivery systems and therapies. We discuss the lacunae in the development of 3D tumor models for the emerging hallmarks of cancer, for lesser-explored forms of breast cancer, and provide insights to develop such models. We discuss how the next generation of 3D models can provide a better mimic of human cancer modeling compared to xenograft models and the scope toward preclinical models and precision medicine.

Enhanced biomechanical performance of additively manufactured Ti-6Al-4V bone plates.

As the global trauma fixation devices market expands rapidly, it is imperative to improve the production of fixation devices through enhanced design accuracy and fit for best performance and maximum patient comfort. Selective laser melting (SLM) is one of the mature additive manufacturing methods, which provides a viable route for the rapid production of such devices. In this work, the ability of SLM to produce near-net-shape parts, as desired for medical implants, was utilized for the fabrication of bone plates from Ti-6Al-4V alloy powder. Martensitic microstructure obtained after the printing of alloy resulted in poor ductility, limiting its application in the field of orthopedics. A specially designed repeated cyclic heating and cooling close to but below the ß-transus was used to transform from acicular to a bimodal microstructure without the need for plastic deformation prior to heat treatment for improving the ductility. Bone plates subjected to this heat treatment were mechanically tested by means of tensile and 3-point bend tests and demonstrated large improvement in ductility, and the values were comparable to those similar plates prepared from wrought alloy. Other important properties required for implants were assessed, such as corrosion resistance in simulated body fluid and cytocompatibility in vitro using MC3T3-E1 cells. These results for the bone plate after heat treatment were excellent and similar to those of the additively manufactured and wrought plates. Taken together, the performance of the additively manufactured bone plates after subjecting to heat treatment was similar to those of bone plate manufactured using wrought alloy. These results have important implications for the fabrication of patient-specific metallic orthopedic devices using SLM without compromising their biomechanical performance by subjecting them to a tailored heat treatment.

A designer cell culture insert with a nanofibrous membrane toward engineering an epithelial tissue model validated by cellular nanomechanics.

Engineered platforms for culturing cells of the skin and other epithelial tissues are useful for the regeneration and development of in vitro tissue models used in drug screening. Recapitulating the biomechanical behavior of the cells is one of the important hallmarks of successful tissue generation on these platforms. The biomechanical behavior of cells profoundly affects the physiological functions of the generated tissue. In this work, a designer nanofibrous cell culture insert (NCCI) device was developed, consisting of a free-hanging polymeric nanofibrous membrane. The free-hanging nanofibrous membrane has a well-tailored architecture, stiffness, and topography to better mimic the extracellular matrix of any soft tissue than conventional, flat tissue culture polystyrene (TCPS) surfaces. Human keratinocytes (HaCaT cells) cultured on the designer NCCIs exhibited a 3D tissue-like phenotype compared to the cells cultured on TCPS. Furthermore, the biomechanical characterization by bio-atomic force microscopy (Bio-AFM) revealed a markedly altered cellular morphology and stiffness of the cellular cytoplasm, nucleus, and cell-cell junctions. The nuclear and cytoplasmic moduli were reduced, while the stiffness of the cellular junctions was enhanced on the NCCI compared to cells on TCPS, which are indicative of the fluidic state and migratory phenotype on the NCCI. These observations were corroborated by immunostaining, which revealed enhanced cell-cell contact along with a higher expression of junction proteins and enhanced migration in a wound-healing assay. Taken together, these results underscore the role of the novel designer NCCI device as an in vitro platform for epithelial cells with several potential applications, including drug testing, disease modeling, and tissue regeneration.

Biomimetic polycaprolactone-chitosan nanofibrous substrate influenced cell cycle and ECM secretion affect cellular uptake of nanoclusters.

Biomimetic cell culture substrates are developed as an alternative to the conventional substrates. They provide necessary biochemical and biophysical cues to the cells from their surrounding environment for their optimal growth, behaviour and physiology. Changes in physiology of cells growing on biomimetic substrate can essentially affect results of in vitro biological experiments such as drug cytotoxicity, nanoparticle internalization or signalling pathways. As majority of ECM proteins are fibrous in nature, nanofibrous scaffolds have more biomimicking properties. Therefore, in this study, we developed ECM mimicking polycaprolactone-chitosan nanofiber substrate and evaluated its effect on cell morphology, proliferation, cell cycle and ECM production. Further, cellular uptake of BSA-AuNCs has been assessed on conventional and biomimetic substrate in order to demonstrate the effect of these events on cellular properties. It was observed that the cells that were grown for 15 days on the nanofibers, had majority of cells in the proliferative phase of cell cycle compared to TCPS. Moreover, these cells showed extensive collagen and fibronectin production. Due to these conditions C3H10T1/2 cells displayed higher cell internalization of BSA-AuNCs. Overall, this study indicates that the nano-topographical and biochemical environment could alter the cell proliferative behaviour and ECM production, which affects the cell internalization of BSA-AuNCs. Also, PCL-chitosan nanofibrous substrate could be a better alternative to TCPS for cell culture studies.

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 Schiff's 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.

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.

Design and synthesis of BODIPY-clickate based Hg(2+) sensors: the effect of triazole binding mode with Hg(2+) on signal transduction.

BODIPY-clickates, F1 and F2, for the detection of Hg(2+) have been designed, synthesized and characterized. Both F1 and F2 showed hyperchromic shifts in the UV-visible spectra in response to increasing Hg(2+) concentrations. Hg(2+) ion binding caused perturbation of the emission quenching process and chelation induced enhanced bathochromic emission of F1 and F2 to 620 nm and 660 nm, respectively. Job's plot clearly indicated that the binding ratio of F1 and F2 with Hg(2+) was 1 : 1. The NMR titration of BODIPY-clickates with Hg(2+) confirmed that aromatic amines and triazoles were involved in the binding event. Furthermore, HRMS data of F1-Hg(2+) and F2-Hg(2+) supported the formation of mercury complexes of BODIPY-clickates. The dissociation constant for the interaction between fluorescent probes F1 and F2 with Hg(2+) was found to be 24.4 ± 5.1 µM and 22.0 ± 3.9 µM, respectively. The Hg(2+) ion induced fluorescence enhancement was almost stable in a pH range of 5 to 8. Having less toxicity to live cells, both the probes were successfully used to map the Hg(2+) ions in live A549 cells.