Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Hybrid extracellular vesicles-liposome incorporated advanced bioink to deliver microRNA.

In additive manufacturing, bioink formulations govern strategies to engineer 3D living tissues that mimic the complex architectures and functions of native tissues for successful tissue regeneration. Conventional 3D-printed tissues are limited in their ability to alter the fate of laden cells. Specifically, the efficient delivery of gene expression regulators (i.e. microRNAs (miRNAs)) to cells in bioprinted tissues has remained largely elusive. In this study, we explored the inclusion of extracellular vesicles (EVs), naturally occurring nanovesicles (NVs), into bioinks to resolve this challenge. EVs show excellent biocompatibility, rapid endocytosis, and low immunogenicity, which lead to the efficient delivery of miRNAs without measurable cytotoxicity. EVs were fused with liposomes to prolong and control their release by altering their physical interaction with the bioink. Hybrid EVs-liposome (hEL) NVs were embedded in gelatin-based hydrogels to create bioinks that could efficiently encapsulate and deliver miRNAs at the target site in a controlled and sustained manner. The regulation of cells' gene expression in a 3D bioprinted matrix was achieved using the hELs-laden bioink as a precursor for excellent shape fidelity and high cell viability constructs. Novel regulatory factors-loaded bioinks will expedite the translation of new bioprinting applications in the tissue engineering field.

Nanoparticle-Based Hybrid Scaffolds for Deciphering the Role of Multimodal Cues in Cardiac Tissue Engineering.

Myocardial microenvironment plays a decisive role in guiding the function and fate of cardiomyocytes, and engineering this extracellular niche holds great promise for cardiac tissue regeneration. Platforms utilizing hybrid hydrogels containing various types of conductive nanoparticles have been a critical tool for constructing engineered cardiac tissues with outstanding mechanical integrity and improved electrophysiological properties. However, there has been no attempt to directly compare the efficacy of these hybrid hydrogels and decipher the mechanisms behind how these platforms differentially regulate cardiomyocyte behavior. Here, we employed gelatin methacryloyl (GelMA) hydrogels containing three different types of carbon-based nanoparticles: carbon nanotubes (CNTs), graphene oxide (GO), and reduced GO (rGO), to investigate the influence of these hybrid scaffolds on the structural organization and functionality of cardiomyocytes. Using immunofluorescent staining for assessing cellular organization and proliferation, we showed that electrically conductive scaffolds (CNT- and rGO-GelMA compared to relatively nonconductive GO-GelMA) played a significant role in promoting desirable morphology of cardiomyocytes and elevated the expression of functional cardiac markers, while maintaining their viability. Electrophysiological analysis revealed that these engineered cardiac tissues showed distinct cardiomyocyte phenotypes and different levels of maturity based on the substrate (CNT-GelMA: ventricular-like, GO-GelMA: atrial-like, and rGO-GelMA: ventricular/atrial mixed phenotypes). Through analysis of gene-expression patterns, we uncovered that the engineered cardiac tissues matured on CNT-GelMA and native cardiac tissues showed comparable expression levels of maturation markers. Furthermore, we demonstrated that engineered cardiac tissues matured on CNT-GelMA have increased functionality through integrin-mediated mechanotransduction (via YAP/TAZ) in contrast to cardiomyocytes cultured on rGO-GelMA.

Flexible and Stretchable PEDOT-Embedded Hybrid Substrates for Bioengineering and Sensory Applications.

Herein, we introduce a flexible, biocompatible, robust and conductive electrospun fiber mat as a substrate for flexible and stretchable electronic devices for various biomedical applications. To impart the electrospun fiber mats with electrical conductivity, poly(3,4-ethylenedioxythiophene) (PEDOT), a conductive polymer, was interpenetrated into nitrile butadiene rubber (NBR) and poly(ethylene glycol) dimethacrylate (PEGDM) crosslinked electrospun fiber mats. The mats were fabricated with tunable fiber orientation, random and aligned, and displayed elastomeric mechanical properties and high conductivity. In addition, bending the mats caused a reversible change in their resistance. The cytotoxicity studies confirmed that the elastomeric and conductive electrospun fiber mats support cardiac cell growth, and thus are adaptable to a wide range of applications, including tissue engineering, implantable sensors and wearable bioelectronics.

Encapsulation of 5-Flurouracil into PLGA Nanofibers and Enhanced Anticancer Effect in Combination with Ajwa-Dates-Extract (Phoenix dactylifera L.).

Side effects connected with chemotherapeutic agents used in cancer treatment has led to alternative modalities of combinatorial therapies in an attempt to reduce the drug dosage and associated risks. In the current study we evaluated the potential use of Ajwa Dates Extract (ADE), reported to have anti-cancer effects, as an adjuvant therapy in combination with 5-flurouracil (5FU) against the human-breast-adenocarcinoma cell line (MFC-7) in vitro. The effects of ADE alone and in combination with 5-FU were evaluated in terms of cell viability and cytotoxicity. For drug delivery purpose, we successfully encapsulated 5FU in both presence and absence of ADE through electrospinning together with poly lactic-co-glycolic acid (PLGA) in different combinations. Physicochemical properties of 5FU and ADE incorporated into PLGA nanofibers remained unaltered as confirmed by Fourier-Transform-Infrared (FTIR), Raman-spectroscopies and X-ray Diffraction (XRD) techniques. The morphological characterization of nanofibers was done using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The surface roughness of PLGA and PLGA + ADE nanofibers increased by incorporation of 5FU. PLGA + ADE nanofibers were in hydrophilic range (<90°) while nanofibers prepared from both PLGA + 5FU and PLGA + 5FU + ADE combinations were in hydrophobic range (∼112°). The percentage inhibition of MCF-7 proliferation at 72 hrs showed an enhanced combinatorial anti-cancer effect of 5FU and ADE on the cells seeded on PLGA + 5FU + ADE mat (47% decrease) while PLGA + 5FU and PLGA + ADE demonstrated only 23% and 16% decrease respectively as compared to controls. The hydrophobicity induced by 5FU can further be investigated to get improved cellular adherence and efficient controlled-drug-release.

Size controlled ultrafine CeO2 nanoparticles produced by the microwave assisted route and their antimicrobial activity.

Cerium oxide (CeO2) nanoparticles (NPs) have a wide range of biological and biomedical applications. This work describes a new methodology for producing ultrafine, highly uniform NPs with controlled sizes using the chemical microwave assisted route. The size of CeO2-NPs decreased from 10 to 5 nm by increasing the molar ratio of cerium nitrate Ce(NO3)3.(6H2O) to that of hexamethylenetetramine (C6H12N) from 1:20 to 20:20. Detailed information about their structural characterization was obtained from the XRD, UV-visible, photoluminescence, Raman spectroscopy, SEM, TEM and AFM. These CeO2-NPs were tested as antimicrobial agent against Gram-negative (Escherichia.coli), Gram-positive (Bacillus.subtilis) bacteria and yeast (Saccharomyces cerevisiae). The obtained results showed significant inhibition of these strain even at low concentration of CeO2-NPs. The CeO2-NPs with the molar ratio 5:20 had the most effective inhibition against E.coli (~70%) at a concentration of 20 µL. The CeO2-NPs with the ratio 12:20 were found to be the most effective against B.subtilis (inhibition ~68%). On the other hand, CeO2-NPs synthesized with the 20:20 molar ratio caused the highest inhibition for S. cerevisiae (~60%). It is observed that at higher NPs concentration (i.e., >20 µL) the inhibition of these strains decreased. The antimicrobial activity may be attributed to the penetrating power of CeO2-NPs size beside the generated oxygen species radicals that caused inhibition of bacterial growth.

Label-Free and Regenerative Electrochemical Microfluidic Biosensors for Continual Monitoring of Cell Secretomes.

Development of an efficient sensing platform capable of continual monitoring of biomarkers is needed to assess the functionality of the in vitro organoids and to evaluate their biological responses toward pharmaceutical compounds or chemical species over extended periods of time. Here, a novel label-free microfluidic electrochemical (EC) biosensor with a unique built-in on-chip regeneration capability for continual measurement of cell-secreted soluble biomarkers from an organoid culture in a fully automated manner without attenuating the sensor sensitivity is reported. The microfluidic EC biosensors are integrated with a human liver-on-a-chip platform for continual monitoring of the metabolic activity of the organoids by measuring the levels of secreted biomarkers for up to 7 d, where the metabolic activity of the organoids is altered by a systemically applied drug. The variations in the biomarker levels are successfully measured by the microfluidic regenerative EC biosensors and agree well with cellular viability and enzyme-linked immunosorbent assay analyses, validating the accuracy of the unique sensing platform. It is believed that this versatile and robust microfluidic EC biosensor that is capable of automated and continual detection of soluble biomarkers will find widespread use for long-term monitoring of human organoids during drug toxicity studies or efficacy assessments of in vitro platforms.

Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs.

Bioprinting is the most convenient microfabrication method to create biomimetic three-dimensional (3D) cardiac tissue constructs, which can be used to regenerate damaged tissue and provide platforms for drug screening. However, existing bioinks, which are usually composed of polymeric biomaterials, are poorly conductive and delay efficient electrical coupling between adjacent cardiac cells. To solve this problem, we developed a gold nanorod (GNR) incorporated gelatin methacryloyl (GelMA)-based bioink for printing 3D functional cardiac tissue constructs. The GNR concentration was adjusted to create a proper microenvironment for the spreading and organization of cardiac cells. At optimized concentration of GNR, the nanocomposite bioink had a low viscosity, similar to pristine inks, which allowed for the easy integration of cells at high densities. As a result, rapid deposition of cell-laden fibers at a high resolution was possible, while reducing shear stress on the encapsulated cells. In the printed GNR constructs, cardiac cells showed improved cell adhesion and organization when compared to the constructs without GNRs. Furthermore, the incorporated GNRs bridged the electrically resistant pore walls of polymers, improved the cell-to-cell coupling, and promoted synchronized contraction of the bioprinted constructs. Given its advantageous properties, this gold nanocomposite bioink may find wide application in cardiac tissue engineering.

Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments.

The ability to modulate stem cell differentiation in a three dimensional (3D) microenvironment for bone tissue engineering in absence of exogenous pharmaceutical agents such as bone morphogenic protein (BMP-2) remains a challenge. In this study, we introduce extracellular matrix (ECM)-mimicking nanocomposite hydrogels to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) for bone regeneration in absence of any osteoinducting factors. In particular, we have reinforced photocrosslinkable collagen-based matrix (gelatin methacryloyl, GelMA) used disk-shaped nanosilicates (nSi), a new class of two-dimensional (2D) nanomaterials. We show that nanoengineered hydrogels supported migration and proliferation of encapsulated hMSCs, with no signs of cell apoptosis or inflammatory cytokine responses. The addition of nSi significantly enhances osteogenic differentiation of encapsulated hMSCs as evident by the increase in alkaline phosphates (ALP) activity and deposition of biomineralized matrix compared to GelMA without nSi. We also show that microfabricated nanoengineered microgels can be used to pattern and control cellular behaviour. Furthermore, we also show that nanoengineered hydrogel have high biocompatibility as determined by in vivo experiments using immunocompetent rat model. Specifically, the hydrogels showed minimum localized immune responses, indicating it ability for tissue engineering applications. Overall, we showed the ability of nanoengineered hydrogels loaded with 2D nanosilicates for osteogenic differentiation of stem cells in vitro, in absence of any growth factors such as BMP-2. Our in vivo studies show high biocompatibility of nanocomposites and show the potential for growth factor free bone regeneration.

Elastomeric nanocomposite scaffolds made from poly(glycerol sebacate) chemically crosslinked with carbon nanotubes.

Carbon nanotube (CNT)-based nanocomposites often possess properties such as high stiffness, electrical conductivity, and thermal stability and have been studied for various biomedical and biotechnological applications. However, the current design approaches utilize CNTs as physical fillers, and thus, the true potential of CNT-based nanocomposites has not been realized. Here, we introduce a general approach to fabricating stiff, elastomeric nanocomposites from poly(glycerol sebacate) (PGS) and CNTs. The covalent crosslinking between the nanotubes and polymer chains resulted in novel property combinations that are not observed in conventional nanocomposites. The addition of 1% CNTs resulted in a five-fold increase in the tensile modulus and a six-fold increase in compression modulus compared with PGS alone, which is far superior to the previously reported studies for CNT-based nanocomposites. Despite a significant increase in mechanical stiffness, the elasticity of the network was not compromised and the resulting nanocomposites showed more than 94% recovery. This study demonstrates that the chemical conjugation of CNTs to a PGS backbone results in stiff and elastomeric nanocomposites. Additionally, in vitro studies using human mesenchymal stem cells (hMSCs) indicated that the incorporation of CNTs into the PGS network significantly enhanced the differentiation potential of the seeded hMSCs, rendering them potentially suitable for applications ranging from scaffolding in musculoskeletal tissue engineering to biosensors in biomedical devices.

Elastomeric nanocomposite scaffolds made from poly (glycerol sebacate) chemically crosslinked with carbon nanotubes.

Carbon nanotube (CNT)-based nanocomposites often possess properties such as high stiffness, electrical conductivity, and thermal stability and have been studied for various biomedical and biotechnological applications. However, the current design approaches utilize CNTs as physical filler, and thus, the true potential of CNT-based nanocomposites has not been achieved. Here, we introduce a general approach of fabricating stiff, elastomeric nanocomposites from poly(glycerol sebacate) (PGS) and CNTs. The covalent crosslinking between the nanotubes and polymer chains resulted in novel property combinations that are not observed in conventional nanocomposites. The addition of 1% CNTs resulted a five-fold increase in the tensile modulus and a six-fold increase in compression modulus compared with PGS alone, which is far superior to the previously reported studies for CNT-based nanocomposites. Despite significant increase in mechanical stiffness, the elasticity of the network was not compromised and the resulting nanocomposites showed more than 94% recovery. This study demonstrates that the chemical conjugation of CNTs to a PGS backbone results in stiff and elastomeric nanocomposites. Additionally, in vitro studies using human mesenchymal stem cells (hMSCs) indicated that the incorporation of CNTs to PGS network significantly enhanced the differentiation potential of the seeded hMSCs rendering them potentially suitable for applications ranging from scaffolding in musculoskeletal tissue engineering to biosensors in biomedical devices.

Candida albicans: A Model Organism for Studying Fungal Pathogens.

Candida albicans is an opportunistic human fungal pathogen that causes candidiasis. As healthcare has been improved worldwide, the number of immunocompromised patients has been increased to a greater extent and they are highly susceptible to various pathogenic microbes and C. albicans has been prominent among the fungal pathogens. The complete genome sequence of this pathogen is now available and has been extremely useful for the identification of repertoire of genes present in this pathogen. The major challenge is now to assign the functions to these genes of which 13% are specific to C. albicans. Due to its close relationship with yeast Saccharomyces cerevisiae, an edge over other fungal pathogens because most of the technologies can be directly transferred to C. albicans from S. cerevisiae and it is amenable to mutation, gene disruption, and transformation. The last two decades have witnessed enormous amount of research activities on this pathogen that leads to the understanding of host-parasite interaction, infections, and disease propagation. Clearly, C. albicans has emerged as a model organism for studying fungal pathogens along with other two fungi Aspergillus fumigatus and Cryptococcus neoformans. Understanding its complete life style of C. albicans will undoubtedly be useful for developing potential antifungal drugs and tackling Candida infections. This will also shed light on the functioning of other fungal pathogens.

Human fungal pathogen Candida albicans in the postgenomic era: an overview.

Candida albicans is an opportunistic human fungal pathogen and is responsible for candidiasis. Owing to the improvement in healthcare, the number of immunocompromised patients in hospitals has increased worldwide and these individuals are susceptible to infections caused by many pathogenic microbes, among which C. albicans is one of the major players. Currently, the complete genome sequence of this pathogen is available and the size of this was estimated to be of 16 Mb. Annotation of C. albicans genome revealed that there are 6114 open reading frames (ORFs), of which 774 are specific to C. albicans. This poses a challenge as well as an opportunity to the Candida community to understand the functions of the unknown genes, especially those specific to C. albicans. Efforts have been made by the Candida community to systematically delete the ORFs and assign the functions. This will, in turn, help in understanding the biology of C. albicans and its interactions with animals as well as humans, and better drugs can be developed to treat Candida infections. In this article, we review updates on the Candida biology in the context of the availability of the genome sequence, its functional analysis and anti-Candida therapy. Finally, in the light of present trends in Candida research and current challenges, various opportunities are identified and suggestions are made.