Kyu Back Lee

Nanostructure-Dependent Water-Droplet Adhesiveness Change in Superhydrophobic Anodic Aluminum Oxide Surfaces: From Highly Adhesive to Self-Cleanable

Water-droplet adhesiveness was freely controlled on a single platform of superhydrophobic anodized aluminum oxide (AAO) within the range from highly adhesive to self-cleanable. Changing the structure from nanopore to nanopillar arrays at the surface caused a dramatic increase in the receding angle and a decrease in the hysteresis of water contact angles. The presence of dead-end nanopores but not through nanoholes was recognized as one of the main causes of the adhesiveness of superhydrophobic surfaces. The adhesiveness-controllable superhydrophobic AAO can be an excellent platform on which to elucidate the physical nature of the wetting phenomenon related to the nanostructure and has promising potential in technological applications.

Characterization of low-molecular-weight hyaluronic acid-based hydrogel and differential stem cell responses in the hydrogel microenvironments

Hyaluronic acid is a natural glycosaminoglycan involved in biological processes. Low-molecular-weight hyaluronic acid (10 and 50 kDa)-based hydrogel was synthesized using derivatized hyaluronic acid. Hyaluronic acid was acrylated by two steps: (1) introduction of an amine group using adipic acid dihydrazide, and (2) acrylation by N-acryloxysuccinimide. Injectable hyaluronic acid-based hydrogel was prepared by using acrylated hyaluronic acid and poly(ethylene glycol) tetra-thiols via Michael-type addition reaction. Mechanical properties of the hydrogel were evaluated by varying the molecular weight of acrylated hyaluronic acid (10 and 50 kDa) and the weight percent of hydrogel. Hydrogel based on 50-kDa hyaluronic acid showed the shortest gelation time and the highest complex modulus. Next, human mesenchymal stem cells were cultured in cell-adhesive RGD peptide-immobilized hydrogels together with bone morphogenic protein-2 (BMP-2). Cells cultured in the RGD/BMP-2-incorporated hydrogels showed proliferation rates higher than that of control or RGD-immobilized hydrogels. Real-time RT-PCR showed that the expression of osteoblast marker genes such as CBFalpha1 and alkaline phosphatase was increased in hyaluronic acid-based hydrogel, and the expression level was dependent on the molecular weight of hyaluronic acid, RGD peptide, and BMP-2. This study indicates that low-molecular-weight hyaluronic acid-based hydrogel can be applied to tissue regeneration as differentiation guidance materials of stem cells.

Electrically-driven hydrogel actuators in microfluidic channels: fabrication, characterization, and biological application

The utility of electro-responsive smart materials has been limited by bubble generation (hydrolysis) during application of electrical fields and by biocompatibility issues. Here we describe the design of a device that overcomes these limitations by combining material properties, new design concepts, and microtechnology. 4-hydroxybutyl acrylate (4-HBA) was used as a backbone hydrogel material, and its actuating behavior, bending force, and elasticity were extensively characterized as a function of size and acrylic acid concentration. To prevent bubble generation, the system was designed such that the hydrogel actuator could be operated at low driving voltages (<1.2 V). A microfluidic channel with an integrated electroactive hydrogel actuator was developed for sorting particles. This device could be operated in cell culture media, and the sorting capabilities were initially assessed by sorting droplets in an oil droplet emulsion. Biocompatibility was subsequently tested by sorting mouse embryoid bodies (mEBs) according to size. The sorted and collected mEBs maintained pluripotency, and selected mEBs successfully differentiated into three germ layers: endoderm, mesoderm, and ectoderm. The electroactive hydrogel device, integrated into a microfluidic system, successfully demonstrated the practical application of smart materials for use in cell biology.

In vitro response of primary human bone marrow stromal cells to recombinant human bone morphogenic protein-2 in the early and late stages of osteoblast differentiation.

A number of factors must be added to human bone marrow stromal cells (hBMSCs) in vitro to induce osteogenesis, including ascorbic acid (AA), β‐glycerophosphate (GP), and dexamethasone (Dex). Bone morphogenic protein (BMP)‐2 is an osteoinductive factor that can commit stromal cells to differentiate into osteoblasts. However, it is still not clear whether the addition of BMP‐2 alone in vitro can induce hBMSCs to complete osteoblast differentiation, resulting in matrix mineralization. This study compares the effects of BMP‐2 and Dex, alone and combined, on the early and late stages of hBMSC differentiation. We found that BMP‐2 causes a significant induction of alkaline phosphatase (ALP) activity in hBMSCs, with a transcriptional upregulation of known BMP‐2‐responsive genes, and the stable expression of cbfa1 in the nucleus and the regions surrounding the nucleus in the early phase of osteoblast differentiation. However, continuous treatment with BMP‐2 alone at doses ranging from 100 to 300 ng/mL results in a less efficient enhancement of in vitro matrix mineralization, despite a significant induction of ALP activity at a concentration of 100 ng/mL. Our results reflect how the effects of BMP‐2 on hBMSCs can vary depending on the stage of osteoblast differentiation, and highlight the need to understand the role of BMP‐2 in primary hBMSCs derived from diverse sources in order to increase the efficiency of using BMP‐2 in osteoinductive therapies.

Nanotopographical control for maintaining undifferentiated human embryonic stem cell colonies in feeder free conditions

Recently emerging evidence has indicated surface nanotopography as an important physical parameter in the stem cell niche for regulating cell fate and behaviors for various types of stem cells. In this study, a substrate featuring arrays of increasing nanopillar diameter was devised to investigate the effects of varying surface nanotopography on the maintenance of undifferentiated human embryonic stem cells (hESC) colonies in the absence of feeder cells. Single hESCs cultured across gradient nanopattern (G-Np) substrate were generally organized into compact colonies, and expressed higher levels of undifferentiated markers compared to those cultured on the unstructured control substrate. In particular, hESC demonstrates a propensity to organize into more compact colonies expressing higher levels of undifferentiated markers towards a smaller nanopillar diameter range (D = 120-170 nm). Cell-nanotopography interactions modulated the formation of focal adhesions and cytoskeleton reorganization to restrict colony spreading, which reinforced E-cadherin mediated cell-cell adhesions in hESC colonies. Maintaining compact hESC colony integrity revealed to be indispensable for hESC undifferentiated state as the loss of cell-cell adhesions within spreading hESC on the control substrate exhibited morphological and gene expression signatures of epithelial-to-mesenchymal transition-like processes. Findings in this study demonstrated a feasible approach to screen the optimal nanotopographical cues for maintaining undifferentiated hESC colonies in feeder free conditions, which provides a platform for further investigations into developing hESC feeder free culture systems for the purpose of regenerative medicine.

Effects of cross-linking molecular weights in a hyaluronic acid–poly(ethylene oxide) hydrogel network on its properties

We examined the effects of cross-linking molecular weights on the properties of a hyaluronic acid (HA)-poly(ethylene oxide) (PEO) hydrogel. Swelling behaviors, mechanical strength and rheological behaviors of the HA-PEO hydrogel were evaluated by employing different cross-linking molecular weights (100 kDa and 1.63 mDa) of the HAs in the hydrogel networks. The low molecular weight of HA was obtained in advance by treating high molecular weight HA with a hydrogen chloride solution. Methacrylation of HA was obtained by grafting aminopropylmethacrylate to its caroboxylic acid functional groups. While reduction of the HA molecular weights was confirmed by gel permeation chromatography, the degree of methacrylate grafting to the HA was measured by (1)H-nuclear magnetic resonance. Synthesis of the HA-PEO hydrogel was successfully achieved via the Michael-type addition reaction between the methacrylate arm groups in the HA and the six thiol groups in PEO. The hydrogel formation was not dependent upon the HA molecular weights and its gelation behaviors were markedly different. Compared to the properties of the high molecular weight HA-based PEO one, the low molecular weight HA-based hydrogel induced quicker hydrogelation, as observed from the behaviors of the elastic and viscous modulus. Furthermore, the low molecular weight HA-based hydrogel demonstrated stronger mechanical properties as measured with a texture analyzer, lower water absorption as measured with a microbalance and smaller pore sizes on its surface and cross section as observed with scanning electron microscopy. The information about the effects of the cross-linking molecular weights of the gel network on the properties of the HA-based PEO hydrogel may lead to better design of hydrogels, especially in tissue engineering applications.

Size-controllable quartz nanostructure for signal enhancement of DNA chip

A mask-free, cost-effective dry-etching method for the fabrication of height- and spacing-controlled, pillar-like nanostructures was established in order to detect DNA molecules. The height and spacing of the quartz nanostructure were regulated by successive O(2) and CF(4) reactive ion etching times. The height and spacing of the nanostructures were tuned between 118 and 269 nm and between 107 and 161 nm, respectively. Probe DNA was immobilized on the structure and hybridized with fluorescently-labeled target DNA. Increases in the height and spacing of the nanopillar structure positively correlated with the fluorescence intensity of bound DNA. Usage of the nanostructure increased the DNA detection limit by up to 100-fold.

In vivo canine studies of a sinkhole valve and vascular graft coated with biocompatible PU-PEO-SO3

PU-PEO-SO3 was applied as a coating material over a newly designed Sinkhole bileaflet PU heart valve and a porous PU vascular graft. Performance and biocompatibility were evaluated using an in vivo canine shunt system between the right ventricle and pulmonary artery. The survival periods in three implantations were 14, 24, and 39 days, during which no mechanical failure occurred in any Sinkhole valve or vascular graft. Scanning electron microscopy (SEM) studies demonstrated much less platelet adhesion and thrombus formation on PU-PEO-SO3 grafts than on PU vascular grafts. Cracks in the valve leaflet were occasionally observed on PU surfaces, but not on PU-PEO-SO3. After a 39 day implantation, calcium deposition on vascular grafts was decreased as compared with valve leaflets, and calcification on PU-PEO-SO3 was much lower than on PU. These results suggest that Sinkhole valves and vascular grafts are promising, and PU-PEO-SO3 as a coating material is more blood compatible, biostable, and calcification resistant in vivo than in untreated PU.

New approach to radial expansive force measurement of self expandable esophageal metal stents

The accurate measurement of radial expansive force is crucial for optimal design and implantation of self expandable esophageal metal stents. In the present study, a new method of measurement under experimental conditions simulating actual stent implantation has been developed. This method offers precise and reproducible measurements and can be applied to a wide variety of stent types. In particular, the method enables one to measure expansive pressure as well as the true radial expansive force up to the radial compression ratio of 72%, covering the range of compression often encountered in a partially obstructed lumen. The test results for various kinds of metal stents are presented and compared. Based on these results, three important points of observation critical in explaining and predicting the expansion characteristics of stents have been reported. Further understanding and characterization of these findings will be necessary for developing new stents with outstanding clinical efficacy.

Local Delivery System of Immune Modulating Drug for Unresectable Adenocarcinoma: In Vitro Experimental Study and In Vivo Animal Study

The purpose of the study was to evaluate the efficacy and safety of a developed drug delivery system containing OK-432 through in vitro and animal study. An OK-432-impregnated polycarbonate/polyurethane stent membrane was used to develop a drug delivery system (DDS) enabling the locoregional release of OK-432. Polyethyleneglycol was used as a detergent and porosity generator. The stability of OK-432 in solvent, releasing kinetics of drug, and cytotoxicity of the DDS were evaluated. OK-432-impregnated DDS was implanted in mice in which a human adenocarcinoma cell line was injected and grown in their back. Flow cytometry and enzyme-linked immunosorbent assay were used for quantifying the amount of drug. OK-432 exposed to phosphate-buffered saline and OK-432 exposed to N,N-dimethylacetamide showed similar results on dot graphs and histograms. However, OK-432 exposed to tetrahydrofurane showed different dot graphs and histograms, which means that the antigenicity of the drug was changed. The release rate of OK-432 was maintained at a constant level for 6 weeks. The local delivery of OK-432 was found to have an antitumor effect on a human adenocarcinoma cell line in an animal study, but no effect on this cell line in in vitro cell culture. Histologic examination showed minimal inflammatory reaction in surrounding tissue. Our study shows that local treatment using this OK-432 release system is safe and effective in reducing adenocarcinoma in a mouse model.