Transition-Metal-Free and Chemoselective NaO^<i>t</i>Bu–O₂‑Mediated Oxidative Cleavage Reactions of <i>vic</i>-1,2-Diols to Carboxylic Acids and Mechanistic Insight into the Reaction Pathways

A method for efficient oxidative cleavage of <i>vic</i>-1,2-diols using a NaO^<i>t</i>Bu–O₂ system resulted in the formation of carboxylic acids in high yields. The present protocol is an eco-friendly alternative to a conventional transition-metal-based method. This new strategy allows large-scale production with nonchromatographic purification while also suppressing competitive reaction pathway such as benzilic acid rearrangement

Investigation of wound healing process guided by nano-scale topographic patterns integrated within a microfluidic system

<div><p>When living tissues are injured, they undergo a sequential process of homeostasis, inflammation, proliferation and maturation, which is called wound healing. The working mechanism of wound healing has not been wholly understood due to its complex environments with various mechanical and chemical factors. In this study, we propose a novel <i>in vitro</i> wound healing model using a microfluidic system that can manipulate the topography of the wound bed. The topography of the extracellular matrix (ECM) in the wound bed is one of the most important mechanical properties for rapid and effective wound healing. We focused our work on the topographical factor which is one of crucial mechanical cues in wound healing process by using various nano-patterns on the cell attachment surface. First, we analyzed the cell morphology and dynamic cellular behaviors of NIH-3T3 fibroblasts on the nano-patterned surface. Their morphology and dynamic behaviors were investigated for relevance with regard to the recovery function. Second, we developed a highly reproducible and inexpensive research platform for wound formation and the wound healing process by combining the nano-patterned surface and a microfluidic channel. The effect of topography on wound recovery performance was analyzed. This <i>in vitro</i> wound healing research platform will provide well-controlled topographic cue of wound bed and contribute to the study on the fundamental mechanism of wound healing.</p></div

Cell culture on various nano-pattern densities.

<p><b>(A)</b> SEM images of the nano-patterns (1:1, 1:2 and 1:5). Dimensions of the nanostructure were measured as width 560 ± 20 nm and depth 325 ± 3 nm. The gap between the structures varies for each case. A gap equal to the width of the nanostructure was considered the 1:1 ratio nano-pattern. Further, we constructed 1:2 and 1:5 ratio nano-patterns in which the dimension of the gap was 2 or 5 times broader than the width of the ridge. <b>(B)</b> Microscopic images of NIH-3T3 fibroblasts cultured on the nano-pattern (cell density = 300–400 cells/mm², scale bar = 100 μm). <b>(C)</b> Fluorescence images of stained cells on nano-patterned surface using nuclei (DAPI) and F-actin (Phalloidin).</p

Orientation and elongation of cells on the nano-patterns.

<p>NIH-3T3 fibroblasts on nano-patterns were investigated based on their elongation and orientation following two indices to validate the contact guidance effect from various densities of nano-patterned surfaces. <b>(A)</b> Definition of the indices for cell body orientation and elongation. Orientation angle, <i>θ</i>, is the angle between the nano-pattern and longitudinal length of the cell body. Elongation index, <i>E</i>, is the aspect ratio of the long axis to the short axis of the cell body. <b>(B)</b> Orientation angle and elongation index of cells on the various nano-patterns: 1:1, 1:2 and 1:5 nano-pattern ratios and control (no-pattern). Cellular shape was captured using a microscope at 12 and 24 hours after cell seeding. Average values of <b>(C)</b> orientation angle, <i>θ</i>, and <b>(D)</b> elongation index, <i>E</i> (n = 30, *p < 0.05 and **p < 0.01).</p

Cell migration on nano-patterned surfaces.

<p><b>(A)</b> Micrographs of NIH-3T3 fibroblast migration on the nano-patterned surfaces. Colored lines indicate the distance and orientation of cell migration. After cell seeding, cell movements were captured every 2 hours for 6 hours. <b>(B)</b> Distribution of cell migration orientation and distance (n = 39). Orientation angle was the averaged value, and distance was summed over 3 measurement points over a total of 6 hours. <b>(C)</b> Average values of the migrated distance and orientation of cells cultured on nano-patterns and the control surface (n = 39, **p < 0.01).</p

Wound generation and healing processes in the microfluidic devices.

<p><b>(A)</b> Schematic of wound formation and healing processes in a nano-pattern integrated microfluidic device. First, the wound is formed due to the layered flow of trypsin/EDTA. The enzyme induces cell detachment from the nano-patterned surface within a selective region. Trypsin/EDTA is replaced with culture medium after wound formation is finished. Leading cells on the wound edge recover into the cell-free area (wound healing process). <b>(B)</b> Micrographs of the wound formation and healing processes. Sequentially, from the top: the cell culture, wound formation and wound healing stages.</p

Nano-pattern integrated microfluidic system used to reproduce the wound healing process.

<p><b>(A)</b> CAD design of the microfluidic device, which includes 2 inlets, 1 outlet and a cell culture region. <b>(B)</b> Process used to fabricate the microfluidic channel and nano-pattern. The device and nano-patterns were irreversibly combined through plasma bonding methods. <b>(C)</b> 3D image of the combined microfluidic device with the nano-pattern. Schematic of the <i>in vitro</i> wound formation process in the microfluidic channel using the layered flow of trypsin/EDTA. Due to trypsinization, cells detached from the microfluidic channel, allowing the selective formation of a cell-free area.</p

Depiction of hepatic bile duct and CCA.

<p>(A) Upon infection of the common bile duct, <i>C. sinensis</i> produces ESPs, which stimulate CCA. (B) CCA and ESPs stimuli can be simulated using a microfluidic platform, culturing HuCCT1 cells (in the bile duct channel) on a COL1 hydrogel incorporated in the ECM channel. The cultured HuCCT1 cells form aggregates on COL1 in the bile duct channel and invade into the COL1 in response to ESPs stimuli.</p

3D invasion of HuCCT1 cells into COL1 ECM over 6 days.

<p>(A) Phase-contrast image of protruding cells on day 3. (B) Summary data showing the total numbers of COL1-invading cells over 6 days. *<i>P</i><0.05 and *** p<0.001 compared with the control. Significance was analyzed by Student's <i>t</i>-test. Error bars, ± SEM. (C) Immunofluorescence image of HuCCT1 cell penetration (red arrowheads) into COL1 under control (top) and Dx1 (bottom) conditions. Scale bars = 100 µm.</p

3D culture of HuCCT1 cells in a microfluidic device.

<p>(A) The PDMS device was bonded with a glass coverslip, and microchannels were coated with PDL. The gel port was filled with pre-polymerized COL1 solution and then the device was incubated at 37°C for 30 minutes. A cell suspension (1×10⁶ cells/ml) was injected into the bile duct channel. The cells were stacked onto the polymerized COL1 hydrogel by gravity by standing the device vertically for 2 hours. The medium was replaced with serum-free RPMI-1640 containing different concentrations of ESPs. (B) (i) Dimensions of the COL1 hydrogel and bowl-like structure that precisely fixes the number of cells on the COL1 hydrogel. (ii) Phase-contrast image of cells stacked on the collagen gel. Scale bar = 200 µm. (iii) SEM image of the fibrous structure of COL1.</p