Chor Yong Tay

Nanoparticles strengthen intracellular tension and retard cellular migration.

Nanoparticles can have profound effects on cell biology. Here, we show that after TiO2, SiO2, and hydroxyapatite nanoparticles treatment, TR146 epithelial cell sheet displayed slower migration. Cells after exposure to the nanoparticles showed increased cell contractility with significantly impaired wound healing capability however without any apparent cytotoxicity. We showed the mechanism is through nanoparticle-mediated massive disruption of the intracellular microtubule assembly, thereby triggering a positive feedback that promoted stronger substrate adhesions thus leading to limited cell motility.

Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage.

Stem cell response can be influenced by a multitude of chemical, topological and mechanical physiochemical cues. While extensive studies have been focused on the use of soluble factors to direct stem cell differentiation, there are growing evidences illustrating the potential to modulate stem cell differentiation via precise engineering of cell shape. Fibronectin were printed on poly(lactic-co-glycolic acid) (PLGA) thin film forming spatially defined geometries as a means to control the morphology of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs that were cultured on unpatterned substrata adhered and flattened extensively (approximately 10,000 microm(2)) while cells grown on 20 microm micropatterend wide adhesive strips were highly elongated with much smaller area coverage of approximately 2000 microm(2). Gene expression analysis revealed up-regulation of several hallmark markers associated to neurogenesis and myogenesis for cells that were highly elongated while osteogenic markers were specifically down-regulated or remained at its nominal level. Even though there is clearly upregulated levels of both neuronal and myogenic lineages but at the functionally relevant level of protein expression, the myogenic lineage is dominant within the time scale studied as determined by the exclusive expression of cardiac myosin heavy chain for the micropatterned cells. Enforced cell shape distortion resulting in large scale rearrangement of cytoskeletal network and altered nucleus shape has been proposed as a physical impetus by which mechanical deformation is translated into biochemical response. These results demonstrated for the first time that cellular shape modulation in the absence of any induction factors may be a viable strategy to coax lineage-specific differentiation of stem cells.

Micro‐/Nano‐engineered Cellular Responses for Soft Tissue Engineering and Biomedical Applications

The development of biomedical devices and reconstruction of functional ex vivo tissues often requires the need to fabricate biomimetic surfaces with features of sub-micrometer precision. This can be achieved with the advancements in micro-/nano-engineering techniques, allowing researchers to manipulate a plethora of cellular behaviors at the cell-biomaterial interface. Systematic studies conducted on these 2D engineered surfaces have unraveled numerous novel findings that can potentially be integrated as part of the design consideration for future 2D and 3D biomaterials and will no doubt greatly benefit tissue engineering. In this review, recent developments detailing the use of micro-/nano-engineering techniques to direct cellular orientation and function pertinent to soft tissue engineering will be highlighted. Particularly, this article aims to provide valuable insights into distinctive cell interactions and reactions to controlled surfaces, which can be exploited to understand the mechanisms of cell growth on micro-/nano-engineered interfaces, and to harness this knowledge to optimize the performance of 3D artificial soft tissue grafts and biomedical applications.

Effect of zinc oxide nanomaterials-induced oxidative stress on the p53 pathway

Excessive production of reactive oxygen species (ROS) is a hallmark feature in nanomaterials (NMs) induced cellular toxicity. However, the inter-relationship between NMs induced ROS generation and the cells innate ability to regulate intracellular ROS level in effecting a particular cellular outcome is currently underexplored. Here, using a BJ fibroblast p53 knockdown system, we showed that p53 may be implicated in playing a dual regulatory role to determine cell survivability in response to oxidative stress induced by ZnO NMs. At low level of ZnO NMs induced ROS, p53 triggers expression of antioxidant genes such as SOD2, GPX1, SESN1, SESN2 and ALDH4A1 to restore oxidative homeostasis while at high concentration of ZnO NMs, the elevated level of intracellular ROS activated the apoptotic pathway through p53. The implication of our finding that p53 can function as an important regulator in determining ZnO induced cytotoxicity is highlighted by the differential action of ZnO on p53 deficient and proficient colorectal cell lines. p53 deficient cells cancer cells such as DLD-1 and SW480 are more susceptible to ZnO induced cell death compared to p53 proficient cells such as colon epithelial cells NCM460 and HCT116 cells in a ROS dependent manner. Collectively, our findings showcased a role p53 plays in the context of nanotoxicity and highlights the need to consider the interplay of physicochemical properties of NMs and cell biology.

Mechanical behavior of human mesenchymal stem cells during adipogenic and osteogenic differentiation

Human mesenchymal stem cells (hMSCs) have gained widespread attention in the field of tissue engineering but not much is known about the changes of mechanical properties during the process of cell lineage commitment and the mechanisms of these behaviors. It is believed that exploring the inter-relations between stem cells mechanical properties and lineage commitment will shed light on the mechanobiology aspect of differentiation. hMSCs were cultured in adipogenic and osteogenic mediums and the elastic moduli were monitored using micropipette aspiration. It was found that hMSCs undergoing osteogenesis have an instantaneous Youngs modulus of 890 +/- 219 Pa and an equilibrium Youngs modulus of 224 +/- 40 Pa, each is about 2-fold higher than the control group. Interestingly, cells cultured in adipogenic medium exhibited a slight increase in the cellular modulus followed by a decrease relative to that of the control group. Gene expression study was employed to gain insights into this phenomenon. Concomitant up regulation of actin binding filamin A (FLNa) and gamma-Tubulin with the cellular elastic modulus indicated their important role in mechanical regulation during hMSCs differentiation. Statistical results showed that cell shape and cell area changed with cellular mechanical properties, which means that cell morphology has a close relation with cell elastic modulus in the initial stage of differentiation. Collectively, these results provide a quantitative description of hMSCs mechanical behavior during the process of differentiation as well as the possible accompanying mechanism at the biomolecular level.

Ultrabright organic dots with aggregation-induced emission characteristics for cell tracking.

Noninvasive fluorescence cell tracking provides critical information on the physiological displacement and translocation of actively migrating cells, which deepens our understanding of biomedical engineering, oncological research, stem cell transplantation and therapies. Non-viral fluorescent protein transfection based cell tracing has been widely used but with issues related to cell type-dependent expression, lagged readout, immunogenicity and mutagenesis. Alternative cell tracking methods are therefore desired to attain reliable, stable, and efficient labeling over a long time. In this work, we have successfully developed ultra-bright organic dots with aggregation-induced emission (AIE dots) and demonstrated their capabilities for cellular imaging and cell tracking. The AIE dots possess high fluorescence, super photostability, and excellent cellular retention and biocompatibility. As compared to commonly used pMAX-GFP plasmid labeling approach, the organic AIE dots showed excellent cell labeling on all tested human cell lines and superior tracing performance, which opens up new opportunities in the cell-based immunotherapies and other related biological researches.

Mechanistic Investigation of the Biological Effects of SiO2, TiO2, and ZnO Nanoparticles on Intestinal Cells

Silicon dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) are currently among the most widely used nanoparticles (NPs) in the food industry. This could potentially lead to unintended exposure of the gastrointestinal tract to these NPs. This study aims to investigate the potential side-effects of these food-borne NPs on intestinal cells and to mechanistically understand the observed biological responses. Among the panel of tested NPs, ZnO NPs are the most toxic. Consistently in all three tested intestinal cell models, ZnO NPs invoke the most inflammatory responses from the cells and induce the highest intracellular production of reactive oxygen species (ROS). The elevated ROS levels induce significant damage to the DNA of the cells, resulting in cell-cycle arrest and subsequently cell death. In contrast, both SiO2 and TiO2 NPs elicit minimum biological responses from the intestinal cells. Overall, the study showcases the varying capability of the food-borne NPs to induce a cellular response in the intestinal cells. In addition to physicochemical differences in the NPs, the genetic landscape of the intestinal cell models governs the toxicology profile of these food-borne NPs.

Biomimicry 3D Gastrointestinal Spheroid Platform for the Assessment of Toxicity and Inflammatory Effects of Zinc Oxide Nanoparticles

Our current mechanistic understanding on the effects of engineered nanoparticles (NPs) on cellular physiology is derived mainly from 2D cell culture studies. However, conventional monolayer cell culture may not accurately model the mass transfer gradient that is expected in 3D tissue physiology and thus may lead to artifactual experimental conclusions. Herein, using a micropatterned agarose hydrogel platform, the effects of ZnO NPs (25 nm) on 3D colon cell spheroids of well-defined sizes are examined. The findings show that cell dimensionality plays a critical role in governing the spatiotemporal cellular outcomes like inflammatory response and cytotoxicity in response to ZnO NPs treatment. More importantly, ZnO NPs can induce different modes of cell death in 2D and 3D cell culture systems. Interestingly, the outer few layers of cells in 3D model could only protect the inner core of cells for a limited time and periodically slough off from the spheroids surface. These findings suggest that toxicological conclusions made from 2D cell models might overestimate the toxicity of ZnO NPs. This 3D cell spheroid model can serve as a reproducible platform to better reflect the actual cell response to NPs and to study a more realistic mechanism of nanoparticle-induced toxicity.

Thickness sensing of hMSCs on collagen gel directs stem cell fate

Mechanically compliant substrate provides crucial biomechanical cues for multipotent stem cells to regulate cellular fates such as differentiation, proliferation and maintenance of their phenotype. Effective modulus of which cells sense is not only determined by intrinsic mechanical properties of the substrate, but also the thickness of substrate. From our study, it was found that interference from underlying rigid support at hundreds of microns away could induce significant cellular response. Human mesenchymal stem cells (hMSCs) were cultured on compliant biological gel, collagen type I, of different thickness but identical ECM composition and local stiffness. The cells sensed the thin gel (130 μm) as having a higher effective modulus than the thick gel (1440 μm) and this was reflected in their changes in morphology, actin fibers structure, proliferation and tissue specific gene expression. Commitment into neuronal lineage was observed on the thin gel only. Conversely, the thick gel (1440 μm) was found to act like a substrate with lower effective modulus that inhibited actin fiber polymerization. Stem cells on the thick substrate did not express tissue specific genes and remained at their quiescent state. This study highlighted the need to consider not only the local modulus but also the thickness of biopolymer gel coating during modulation of cellular responses.

Nano-hydroxyapatite and Nano-titanium Dioxide Exhibit Different Subcellular Distribution and Apoptotic Profile in Human Oral Epithelium

Nanomaterials (NMs) such as titanium dioxide (nano-TiO2) and hydroxyapatite (nano-HA) are widely used in food, personal care, and many household products. Due to their extensive usage, the risk of human exposure is increased and may trigger NMs specific biological outcomes as the NMs interface with the cells. However, the interaction of nano-TiO2 and nano-HA with cells, their uptake and subcellular distribution, and the cytotoxic effects are poorly understood. Herein, we characterized and examined the cellular internalization, inflammatory response and cytotoxic effects of nano-TiO2 and nano-HA using TR146 human oral buccal epithelial cells as an in vitro model. We showed both types of NMs were able to bind to the cellular membrane and passage into the cells in a dose dependent manner. Strikingly, both types of NMs exhibited distinct subcellular distribution profile with nano-HA displaying a higher preference to accumulate near the cell membrane compared to nano-TiO2. Exposure to both types of NMs caused an elevated reactive oxygen species (ROS) level and expression of inflammatory transcripts with increasing NMs concentration. Although cells treated with nano-HA induces minimal apoptosis, nano-TiO2 treated samples displayed approximately 28% early apoptosis after 24 h of NMs exposure. We further showed that nano-TiO2 mediated cell death is independent of the classical p53-Bax apoptosis pathway. Our findings provided insights into the potential cellular fates of human oral epithelial cells as they interface with industrial grade nano-HA and nano-TiO2.