Yongdoo Park

Microfluidic Chip-Based Fabrication of PLGA Microfiber Scaffolds for Tissue Engineering

In this paper, we have developed a method to produce poly(lactic- co-glycolic acid) (PLGA) microfibers within a microfluidic chip for the generation of 3D tissue engineering scaffolds. The synthesis of PLGA fibers was achieved by using a polydimethylsiloxane (PDMS)-based microfluidic spinning device in which linear streams of PLGA dissolved in dimethyl sulfoxide (DMSO) were precipitated in a glycerol-containing water solution. By changing the flow rate of PLGA solution from 1 to 50 microL/min with a sheath flow rate of 250 or 1000 microL/min, fibers were formed with diameters that ranged from 20 to 230 microm. The PLGA fibers were comprised of a dense outer surface and a highly porous interior. To evaluate the applicability of PLGA microfibers generated in this process as a cell culture scaffold, L929 fibroblasts were seeded on the PLGA fibers either as-fabricated or coated with fibronectin. L929 fibroblasts showed no significant difference in proliferation on both PLGA microfibers after 5 days of culture. As a test for application as nerve guide, neural progenitor cells were cultured and the neural axons elongated along the PLGA microfibers. Thus our experiments suggest that microfluidic chip-based PLGA microfiber fabrication may be useful for 3D cell culture tissue engineering applications.

Synthesis of Cell‐Laden Alginate Hollow Fibers Using Microfluidic Chips and Microvascularized Tissue‐Engineering Applications

Biomimetic microstructures can be used for tissue assembly, tissue and organ regeneration, and as drug carriers. To date, diverse microscale structures that use microengineering technologies (e.g., microor nanoparticles, microor nanofibers, and 2D patterns) have been used as drug delivery systems for the controlled release of therapeutic drugs, as scaffolds for support of cells in defective tissues, and as basic materials for the study of cell–cell interactions. In living organisms, microscale and porous hollow fibers, such as blood vessels, are important for the delivery of small amounts of liquid to specific places. Such structures also have industrial applications including the filtering and channeling of small volumes of liquid. In spite of their diverse potential applications, the study of hollow fibers has been limited to specific areas, such as hemodialysis, because fabrication is very difficult. In addition, it is difficult to load cells and proteins into hollow fibers without loss of biological activity. Previously, we demonstrated the fabrication of microscale fibers or hollow fibers based on an ‘‘on-the-fly’’ method that uses microfluidic chips. However, the material that we originally used was not biodegradable or biocompatible. Subsequently, we reported the production of alginate solid fibers and cell encapsulation in these fibers. In addition, Sugiura et al. have reported the fabrication of an alginate

Nerve regeneration following spinal cord injury using matrix metalloproteinase-sensitive, hyaluronic acid-based biomimetic hydrogel scaffold containing brain-derived neurotrophic factor.

Spinal cord injury leads to the permanent loss of motor and sensory function in the body. To enhance spinal cord regeneration, we used a hyaluronic acid-based hydrogel as a three-dimensional biomimetic scaffold for peptides and growth factors. Three components were used to provide guidance cues: a matrix metalloproteinase peptide crosslinker, an IKVAV (Ile- Lys-Val-Ala-Val) peptide derived from laminin, and brain-derived neurotrophic factor (BDNF). Human mesenchymal stem cells (hMSCs) were cultured in hydrogels in vitro for 10 days to induce neuronal differentiation of hMSCs. Based on gene-expression data, the matrix metalloproteinase-sensitive peptide, IKVAV peptide, and BDNF were critical in the differentiation of hMSCs. Remodeling activity was found to be a key factor in guiding neural differentiation of stem cells. To test this approach in vivo, we used the spinal cord injured rat model and five different hydrogel compositions. Samples were injected into the intrathecal space, and animals were monitored for 6 weeks. Compared to all other groups, animals injected with BDNF-containing hydrogels showed the greatest improvement on locomotive tests (Basso-Beattie-Bresnahan score) during the initial stage after injury. These results suggest that hyaluronic acid-based hydrogels containing IKVAV and BDNF create microenvironments that foster differentiation of stem cells along the neural cell lineage, and they could be used to facilitate nerve regeneration after spinal cord injury.

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.

Regeneration of ischemic heart using hyaluronic acid-based injectable hydrogel.

An injectable hydrogel was applied to regenerate a myocardial infarction and functional recovery of the heart. A myocardial infarction was induced in rat by circumflex artery ligation. A hyaluronic acid-based hydrogel was injected into the epicardium of the infarcted area. Then, cardiac functions and regeneration of the myocardium in sham-operated (SHAM), myocardial infarction (MI), and gel-injected group (GEL) (n = 6) were evaluated 4 weeks after the injection. Measurements of the thickness of the wall showed that the thickness in the GEL group increased by up to 200% compared with that in the MI group (p < 0.001). The infarcted area of the left ventricular in the GEL group decreased by 53% compared with the MI group (p < 0.001). The number of arterioles and capillaries in the border zone of the GEL group increased by 152% and 148%, whereas the apoptotic index decreased by 42% (p < 0.05). Measurement of the heart functions, such as ejection fraction, arterial elastance (Ea), dP/dt max, and dP/dt min, indicated that the injection of a hydrogel significantly facilitated the functional recovery compared with the MI group. Because of its simplicity, easy applicability, and a great regenerating potential, this injectable hydrogel promises as a treatment for myocardial infarction.

The enhancement of mature vessel formation and cardiac function in infarcted hearts using dual growth factor delivery with self-assembling peptides

For successful treatment of myocardial infarction (MI), it is important to prevent cardiac fibrosis and maintain cardiac function by protecting cardiomyocytes and inducing angiogenesis. To establish functional and stable vessels, various growth factors, ones stimulating both endothelial cells (EC) and vascular smooth muscle cells (VSMC), are required. Self-assembling peptides form fibers (<10 nm) and provide 3-dimensional microenvironments that can recruit EC and VSMC to promote vascularization and long-term delivery of growth factors. Here we demonstrate myocardial protection of infarcted heart using dual growth factor delivery with self-assembling peptides. After coronary artery ligation in rats, growth factors (PDGF-BB and FGF-2) with self-assembling peptides were injected. There were 6 rats in each group. Hearts were harvested at 4 and 8 weeks for functional and histological analysis. Infarct size and cardiomyocyte apoptosis in dual growth factors along with self-assembling peptides group were dramatically reduced compared to sham. The capillary and arterial density of this group recovered with angiogenic synergism and cardiac functions had almost recovered. In conclusion, dual growth factors along with self-assembling peptides lead to myocardial protection, stable vessel formation, and improvement in cardiac function.

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.

In vivo evaluation of MMP sensitive high-molecular weight HA-based hydrogels for bone tissue engineering.

Hyaluronic acid (170 kDa)-based hydrogel was synthesized using acrylated hyaluronic acid (HA) and matrix metalloproteinase (MMP) sensitive HA-based hydrogels were then prepared by conjugation with two different peptides: cell adhesion peptides containing integrin-binding domains (Arg-Gly-Asp: RGD) and a cross-linker with MMP degradable peptides to mimic the remodeling characteristics of natural extracellular matrices by cell-derived MMPs. Mechanical properties of these hydrogels were evaluated with different weight percentages (2.5 and 3.5 wt %) by measuring elastic modulus, viscous modulus, and swelling ratio. Human mesenchymal stem cells (hMSCs) were then cultured in MMP-sensitive or insensitive HA-based hydrogels and/or immobilized cell adhesive RGD peptides in vitro. Actin staining and image analysis proved that cells cultured in the MMP-sensitive hydrogel with RGD peptides showed extensive cell spreading and sprouting. Gene expression analysis showed that bone specific genes such as alkaline phosphatase, osteocalcin, and osteopontin increased in MMP-sensitive hydrogels as biomolecules such as BMPs and cells were added in the gels. For in vivo calvarial defect regeneration, five different samples (MMP insensitive hydrogel, MMP sensitive hydrogel, MMP sensitive hydrogel with BMP-2, MMP sensitive hydrogel with hMSC, and MMP sensitive hydrogel with BMP-2 and hMSC) were prepared. After 4 weeks of implantation, the Masson-Trichrome staining and micro computed tomography scan results demonstrated that the MMP sensitive hydrogels with BMP-2 and hMSCs have the highest mature bone formation. The MMP sensitive HA-based hydrogel could become useful scaffolds in bone tissue engineering with improvements on tissue remodeling rates and regeneration activity.

Regeneration of chronic myocardial infarction by injectable hydrogels containing stem cell homing factor SDF-1 and angiogenic peptide Ac-SDKP

Regeneration of chronic myocardial infarction (CMI) is one of the challenging issues due to its limited regeneration activity compared to acute or sub-acute stage. In this study, we examined whether combination of stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) injected with biomimetic hydrogels promote regeneration of cardiac function in a CMI model. We evaluated the regeneration of chronically infarcted myocardium using injectable biomimetic hydrogels containing two therapeutic factors; stromal-derived factor-1 (SDF-1) and Ac-SDKP for stem cell homing and angiogenesis, respectively. Injection of the two therapeutic factors into the infarct region of the left ventricle showed that the biomimetic hydrogels containing two therapeutic factor exhibited significantly improved left ventricle function, increased angiogenesis, decreased infarct size and greatest wall thickness within the infarct region at 4 weeks post-treatment. From these results, it is clear that hydrogels containing two therapeutic factors showed synergistic effects on regeneration in the chronic heart failure model. In conclusion, these results suggest that combination of stem cell homing factor with angiogenic peptides recruit stem cells to the microenvironments, increase the expression of angiogenic genes, enhance the matured vessel formation and improve the cardiac function in chronic MI.

Temperature-induced gel formation of core/shell nanoparticles for the regeneration of ischemic heart.

Vascular endothelial growth factor (VEGF)-loaded core/shell nanoparticles were prepared and their gelation behavior in response to temperature was characterized for the regeneration of ischemic heart. The core is composed of lecithin containing VEGF and the shell is composed of Pluronic F-127 (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer). When Capryol 90 (propylene glycol monocaprylate) was added to an aqueous solution of the core/shell nanoparticles, a temperature-induced gel composed of core/shell nanoparticles was observed to form at body temperature. This phenomenon was utilized for the stable localization of core/shell nanoparticles at the ischemic area. For an in vivo assessment, VEGF-loaded core/shell nanoparticles with and without inducement of the gel formation were applied to a subacute myocardial infarction model in rats and functional analysis of the heart was monitored by means of a PV catheter four weeks later. The results showed that the VEGF-loaded core/shell nanoparticles and their gel improved the heart functions, particularly with regard to the ejection fraction and cardiac output.