Alteration of Young's modulus in mesenchymal stromal cells during osteogenesis measured by atomic force microscopy.

Mechanical properties of biological tissues are increasingly recognized as an important parameter for the indication of disease states as well as tissue homeostasis and regeneration. Multipotent mesenchymal stromal/stem cells (MSCs), which play important roles in bone formation and remodeling, are potential cell sources for regenerative medicine. However, the cellular mechanical properties of differentiating MSCs corresponding to the substrate stiffness has not been sufficiently studied. In this study, we used Atomic Force Microscopy (AFM) to measure changes of stiffness of human MSCs cultured in rigid Petri dish and on polyacrylamide (PA) substrates during osteogenic differentiation. The results showed that the Young's modulus of MSC cytoplasmic outer region increased over time during osteogenesis. There is a strong linear correlation between the osteogenic induction time and the Young's modulus of the cells cultured in rigid Petri dishes in the first 15 days after the induction; the Young's modulus approaches to a plateau after day 15. On the other hand, the Young's moduli of MSCs cultured on PA gels with stiffness of 7 kPa and 42 kPa also increase over time during osteogenic differentiation, but the inclination of such increase is much smaller than that of MSCs differentiating in rigid dishes. Herein, we established a protocol of AFM measurement to evaluate the maturation of stem cell osteogenic differentiation at the single cell level and could encourage further AFM applications in tissue engineering related to mechanobiology.

Application of the deep learning for the prediction of rainfall in Southern Taiwan.

Precipitation is useful information for assessing vital water resources, agriculture, ecosystems and hydrology. Data-driven model predictions using deep learning algorithms are promising for these purposes. Echo state network (ESN) and Deep Echo state network (DeepESN), referred to as Reservoir Computing (RC), are effective and speedy algorithms to process a large amount of data. In this study, we used the ESN and the DeepESN algorithms to analyze the meteorological hourly data from 2002 to 2014 at the Tainan Observatory in the southern Taiwan. The results show that the correlation coefficient by using the DeepESN was better than that by using the ESN and commercial neuronal network algorithms (Back-propagation network (BPN) and support vector regression (SVR), MATLAB, The MathWorks co.), and the accuracy of predicted rainfall by using the DeepESN can be significantly improved compared with those by using ESN, the BPN and the SVR. In sum, the DeepESN is a trustworthy and good method to predict rainfall; it could be applied to global climate forecasts which need high-volume data processing.

Mechanical stretch induces hair regeneration through the alternative activation of macrophages.

Tissues and cells in organism are continuously exposed to complex mechanical cues from the environment. Mechanical stimulations affect cell proliferation, differentiation, and migration, as well as determining tissue homeostasis and repair. By using a specially designed skin-stretching device, we discover that hair stem cells proliferate in response to stretch and hair regeneration occurs only when applying proper strain for an appropriate duration. A counterbalance between WNT and BMP-2 and the subsequent two-step mechanism are identified through molecular and genetic analyses. Macrophages are first recruited by chemokines produced by stretch and polarized to M2 phenotype. Growth factors such as HGF and IGF-1, released by M2 macrophages, then activate stem cells and facilitate hair regeneration. A hierarchical control system is revealed, from mechanical and chemical signals to cell behaviors and tissue responses, elucidating avenues of regenerative medicine and disease control by demonstrating the potential to manipulate cellular processes through simple mechanical stimulation.

Alteration of mesenchymal stem cells polarity by laminar shear stimulation promoting ß-catenin nuclear localization.

Mesenchymal stem cell (MSC) is mechanosensitive and the respond to mechanical force is pattern specific. We previously reported that oscillatory shear stress at 0.5 ±â€¯4 dyne/cm2 guided MSCs polarity vertical to net flow direction before apolaric stage at 30 min resulting in phosphorylation of ß-catenin and inhibition of Wnt signaling. This time, we explored laminar shear stress (LS) at 0.5 dyne/cm2 polarized MSCs by guiding F-actin orientation parallel to the flow direction before apolarity at 30 min accompanied with activation of Wnt signaling. Time-dependent microarray analysis supported cell-cell junctional complex of MSCs was the major mechanosensor on MSCs to respond 0.5 dyne/cm2 LS. Three-dimensional immunofluorescence image confirmed LS promoting ß-catenin nuclear localization during 15 min to 1 h with a peak at 30 min. Functional analysis of proteomic study on MSC with 30 min LS stimulation indicated that upregulation of ß-catenin downstream proteins related to cardiovascular development, endothelial cell protection and angiogenesis. Conditioned medium from MSCs with 30 min LS stimulation improved the viability of human endothelial cells from oxidative damage. In conclusion, 0.5 dyne/cm2 LS on MSCs for 30 min guides MSCs lack of polarity and promotes ß-catenin nuclear translocation favoring Wnt activation and paracrine cardiovascular support.

Optimization of Pulley-Type Ring Resonator with Waveguide Offset.

In this work, we dealt with the optimization of the pulley-type ring resonator using the offset of the straight input and output waveguide at the junction with the curved waveguide. We adopted the finite-difference time-domain method to simulate the structure. It was found that the coupling loss could be significantly reduced and the critical coupling could be precisely tuned. This results in the possibility of the Q-factor being higher than that of the structure without waveguide offset. In this study, the Q-factor of the ring resonator is increased from 9180 to 11,302. The corresponding enhancement is 23.1%.

Efficient generation of hepatic cells from mesenchymal stromal cells by an innovative bio-microfluidic cell culture device.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent and have great potential in cell therapy. Previously we reported the differentiation potential of human MSCs into hepatocytes in vitro and that these cells can rescue fulminant hepatic failure. However, the conventional static culture method neither maintains growth factors at an optimal level constantly nor removes cellular waste efficiently. In addition, not only is the duration of differentiating hepatocyte lineage cells from MSCs required to improve, but also the need for a large number of hepatocytes for cell therapy has not to date been addressed fully. The purpose of this study is to design and develop an innovative microfluidic device to overcome these shortcomings. METHODS: We designed and fabricated a microfluidic device and a culture system for hepatic differentiation of MSCs using our protocol reported previously. The microfluidic device contains a large culture chamber with a stable uniform flow to allow homogeneous distribution and expansion as well as efficient induction of hepatic differentiation for MSCs. RESULTS: The device enables real-time observation under light microscopy and exhibits a better differentiation efficiency for MSCs compared with conventional static culture. MSCs grown in the microfluidic device showed a higher level of hepatocyte marker gene expression under hepatic induction. Functional analysis of hepatic differentiation demonstrated significantly higher urea production in the microfluidic device after 21 days of hepatic differentiation. CONCLUSIONS: The microfluidic device allows the generation of a large number of MSCs and induces hepatic differentiation of MSCs efficiently. The device can be adapted for scale-up production of hepatic cells from MSCs for cellular therapy.

Fluid-induced, shear stress-regulated extracellular matrix and matrix metalloproteinase genes expression on human annulus fibrosus cells.

BACKGROUND: Mechanical loading plays an important role in the regulation of extracellular matrix (ECM) homeostasis as well as pathogenesis of intervertebral disc (IVD) degeneration. The human annulus fibrosus (hAF) in the IVD is subjected to contact shear stress during body motion. However, the effects of shear stress on hAF cells remain unclear. This aim of the study was to investigate the expression of the ECM (COLI, COLIII and aggrecan) and matrix metalloproteinase (MMP-1, MMP-3 and ADAMTS-4) genes in hAF cells following fluid-induced shear stress in a custom-fabricated bio-microfluidic device. METHODS: hAF cells were harvested from degenerated disc tissues in routine spine surgery, staged by magnetic resonance imaging, expanded in monolayers and then seeded onto the bio-microfluidic device. The experimental groups were subjected to 1 and 10 dyne/cm(2) shear stress for 4 h, and no shear stress was applied to the control group. We used real time polymerase chain reaction for gene expression. RESULTS: Shear stress of 1 dyne/cm(2) exerted an anabolic effect on COLI and COLIII genes and catabolic effects on the aggrecan gene, while 10 dyne/cm(2) had an anabolic effect on the COLI gene and a catabolic effect on COLIII and aggrecan genes. The COLI gene was upregulated in a stress-dependent manner. Expression of MMP-1 was significantly higher in the 10 dyne/cm(2) group compared to the control group (P < 0.05), but was similar in the control and 1 dyne/cm(2) groups. Expression of MMP-3 and ADAMTS-4 were similar in all three groups. CONCLUSION: Taken together, hAF cells responded to shear stress. The findings help us understand and clarify the effects of shear stress on IVD degeneration as well as the development of a new therapeutic strategy for IVD degeneration.

Low power consumption and high-contrast light scattering based on polymer-dispersed liquid crystals doped with silver-coated polystyrene microspheres.

Polymer-dispersed liquid crystals (PDLCs) have attracted considerable attention for optical device applications in recent years. However, the high operating voltage of PDLCs limits their applications. This study reports a simple approach used for the first time to decrease the operating voltage of PDLCs by means of doping 3 µm-diameter silver-coated polystyrene microspheres (Ag-coated PSMSs) into PDLCs. Ag-coated PSMSs construct an induced electric field between each other when an external electric field is applied. This induced electric field can enhance the effective electric field so the operating voltage can be actively reduced from 77 V to 40 V. Such PDLCs also possess a high contrast ratio of >50 and a high on-state transmittance of ~73%. Therefore, PDLCs doped with Ag-coated PSMSs maintain a high contrast ratio and improve their electro-optical properties.

Mechanosensitive TRPM7 mediates shear stress and modulates osteogenic differentiation of mesenchymal stromal cells through Osterix pathway.

Microenvironments that modulate fate commitments of mesenchymal stromal cells (MSCs) are composed of chemical and physical cues, but the latter ones are much less investigated. Here we demonstrate that intermittent fluid shear stress (IFSS), a potent and physiologically relevant mechanical stimulus, regulates osteogenic differentiation of MSCs through Transient receptor potential melastatin 7 (TRPM7)-Osterix axis. Immunostaining showed the localization of TRPM7 near or at cell membrane upon IFSS, and calcium imaging analysis demonstrated the transient increase of cytosolic free calcium. Expressions of osteogenic marker genes including Osterix, but not Runx2, were upregulated after three-hour IFSS. Phosphorylation of p38 and Smad1/5 was promoted by IFSS as well. TRPM7 gene knockdown abolished the promotion of bone-related gene expressions and phosphorylation. We illustrate that TRPM7 is mechanosensitive to shear force of 1.2 Pa, which is much lower than 98 Pa pressure loading reported recently, and mediates distinct mechanotransduction pathways. Additionally, our results suggest the differential roles of TRPM7 in endochondral and intramembranous ossification. Together, this study elucidates the mechanotransduction in MSCs fate commitments and displays an efficient mechano-modulation for MSCs osteogenic differentiation. Such findings should be taken into consideration when designing relevant scaffolds and microfluidic devices for osteogenic induction in the future.

Programmable Laser-Assisted Surface Microfabrication on a Poly(Vinyl Alcohol)-Coated Glass Chip with Self-Changing Cell Adhesivity for Heterotypic Cell Patterning.

Organs are composed of heterotypic cells with patterned architecture that enables intercellular interaction to perform specific functions. In tissue engineering, the ability to pattern heterotypic cells into desired arrangement will allow us to model complex tissues in vitro and to create tissue equivalents for regeneration. This study was aimed at developing a method for fast heterotypic cell patterning with controllable topological manipulation on a glass chip. We found that poly(vinyl alcohol)-coated glass showed a biphasic change in adhesivity to cells in vitro: low adhesivity in the first 24 h and higher adhesivity at later hours due to increased serum protein adsorption. Combining programmable CO2 laser ablation to remove poly(vinyl alcohol) and glass, we were able to create arrays of adhesive microwells of adjustable patterns. We tested whether controllable patterns of epithelial-mesenchymal interaction could be created. When skin dermal papilla cells and fibroblasts were seeded respectively 24 h apart, we were able to pattern these two cells into aggregates of dermal papilla cells in arrays of microwells in a background of fibroblasts sheet. Seeded later, keratinocytes attached to these mesenchymal cells. Keratinocytes contacting dermal papilla cells started to differentiate toward a hair follicle fate, demonstrating patternable epithelial-mesenchymal interaction. This method allows fast adjustable heterotypic cell patterning and surface topology control and can be applied to the investigation of heterotypic cellular interaction and creation of tissue equivalent in vitro.

Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering.

Liver transplantation is the ultimate treatment for severe hepatic failure to date. However, the limited supply of donor organs has severely hampered this treatment. So far, great potentials of using mesenchymal stem cells (MSCs) to replenish the hepatic cell population have been shown; nevertheless, there still is a lack of an optimal three-dimensional scaffold for generation of well-transplantable hepatic tissues. In this study, we utilized a cryo-chemical decellularization method which combines physical and chemical approach to generate acellular liver scaffolds (ALS) from the whole liver. The produced ALS provides a biomimetic three-dimensional environment to support hepatic differentiation of MSCs, evidenced by expression of hepatic-associated genes and marker protein, glycogen storage, albumin secretion, and urea production. It is also found that hepatic differentiation of MSCs within the ALS is much more efficient than two-dimensional culture in vitro. Importantly, the hepatic-like tissues (HLT) generated by repopulating ALS with MSCs are able to act as functional grafts and rescue lethal hepatic failure after transplantation in vivo. In summary, the cryo-chemical method used in this study is suitable for decellularization of liver and create acellular scaffolds that can support hepatic differentiation of MSCs and be used to fabricate functional tissue-engineered liver constructs.

Gene expression of human lung cancer cell line CL1-5 in response to a direct current electric field.

BACKGROUND: Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. Highly metastatic human lung cancer cells, CL1-5, exhibit directional migration and orientation under dcEFs. To understand the transcriptional response of CL1-5 cells to a dcEF, microarray analysis was performed in this study. METHODOLOGY/PRINCIPAL FINDINGS: A large electric-field chip (LEFC) was designed, fabricated, and used in this study. CL1-5 cells were treated with the EF strength of 0 mV/mm (the control group) and 300 mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. It was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. Some up-regulated genes such as ACVR1B and CTTN, and some down-regulated genes such as PTEN, are known to be positively and negatively correlated to cell migration, respectively. The protein-protein interactions of adherens junction-associated EF-regulated genes suggested that platelet-derived growth factor (PDGF) receptors and ephrin receptors may participate in sensing extracellular electrical stimuli. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction. CONCLUSIONS/SIGNIFICANCE: In this study, some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response.

Electrotaxis of lung cancer cells in a multiple-electric-field chip.

We report a microfluidic cell culture chip that was used for long-term electrotaxis study on a microscope. The cellular response under three different electric field strengths was studied in a single channel microfluidic chip. Electric field (EF) inside the microchamber was numerically simulated and compared to the measured value. Lung cancer cell lines with high and weak metastasis potential, CL1-5 and CL1-0, respectively, were used to demonstrate the function of the multi-field chip (MFC). The two cell lines exhibited greatly different response under the applied EF of E=74-375 mV/mm. CL1-5 cells migrated toward the anode while CL1-0 cells did not show obvious response. Under the applied EF, cell orientation was observed accompanying the cell migration. Judging from the different temporal responses of the orientation and the migration, it is proposed that the two EF-induced responses may involve different signaling pathways.

The enhancement of dermal papilla cell aggregation by extracellular matrix proteins through effects on cell-substratum adhesivity and cell motility.

Generally, cells tend to aggregate on a substratum with lower cell adhesivity. However, it also leads to compromised cell growth and higher cell loss after seeding. This study is aimed at tackling this dilemma by extracellular matrix (ECM) protein coating of a lower adhesive substratum poly(ethylene-co-vinyl alcohol) (EVAL) that has been shown to facilitate hair follicle dermal papilla (DP) spheroid formation. We found that coating with either fibronectin (Fn), collagen I, or collagen IV yields higher adhesivity and cell growth than that with laminin. However, cells can only aggregate on uncoated or Fn-coated EVAL. Quantitatively, Fn coating increases the number of spheroids by 67%. Analysis of cell migration reveals that collagen I, collagen IV and laminin coatings reduce cell motility, while Fn coating keeps cells highly motile. Inhibition of cell migration hinders spheroid formation. In addition, disruption of Fn function does not significantly compromise intercellular adhesion. Hence, Fn enhances cell aggregation by enhancing cell attachment, cell growth and cell motility. Our study demonstrates that intercellular organization as spheroids or flat monolayers is switchable by specific ECM protein coating and preserving cell motility is vital to cell aggregation. In addition to generation of spheroidal DP microtissues for hair follicle regeneration and large-scale production of aggregates of other cells, this strategy can help to regulate the tissue-substrate adhesivity and tissue spreadability on the surface of implantable materials.

Label-free quantification of asymmetric cancer-cell filopodium activities in a multi-gradient chip.

We use novel super-resolution bright-field optical microscopy to observe the filopodium activities of human lung cancer cells in a multi-gradient cell culture chip. Temporal variations of the filopodium numbers are measured without fluorescent labelling. By carefully designing the fluidic field inside the culture chip, we establish stable concentration gradients of the injected reagents. The reagents are injected via a separated central inlet, and the concentration gradients are different at different positions in the chip. The same chip can be used for both control and treated experiments. Using epidermal growth factor as the treatment, we verify that the protrusions of filopodia indicate the direction of concentration gradients experienced by a living cancer cell; while the treatment of bovine serum albumin shows no specific effect on the growth of filopodia. The combination of label-free, high-resolution optical microscopy and a micro cell culture chip establishes a convenient and versatile platform for dynamical cancer-cell analyses.

A transparent cell-culture microchamber with a variably controlled concentration gradient generator and flow field rectifier.

Real-time observation of cell growth provides essential information for studies such as cell migration and chemotaxis. A conventional cell incubation device is usually too clumsy for these applications. Here we report a transparent microfluidic device that has an integrated heater and a concentration gradient generator. A piece of indium tin oxide (ITO) coated glass was ablated by our newly developed visible laser-induced backside wet etching (LIBWE) so that transparent heater strips were prepared on the glass substrate. A polymethylmethacrylate (PMMA) microfluidic chamber with flow field rectifiers and a reagent effusion hole was fabricated by a CO(2) laser and then assembled with the ITO heater so that the chamber temperature can be controlled for cell culturing. A variable chemical gradient was generated inside the chamber by combining the lateral medium flow and the flow from the effusion hole. Successful culturing was performed inside the device. Continuous long-term (>10 days) observation on cell growth was achieved. In this work the flow field, medium replacement, and chemical gradient in the microchamber are elaborated.

Using a microfluidic device for 1 microl DNA microarray hybridization in 500 s.

This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 microl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO2 laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA-DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 mul target was used to hybridize with an array that can hold 5000 probes.