Yu-Shik Hwang

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11

Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 μm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 μm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.

The use of murine embryonic stem cells, alginate encapsulation, and rotary microgravity bioreactor in bone tissue engineering

The application of embryonic stem cells (ESCs) in bone tissue engineering and regenerative medicine requires the development of suitable bioprocesses that facilitate the integrated, reproducible, automatable production of clinically-relevant, scaleable, and integrated bioprocesses that generate sufficient cell numbers resulting in the formation of three-dimensional (3D) mineralised, bone tissue-like constructs. Previously, we have reported the enhanced differentiation of undifferentiated mESCs toward the osteogenic lineage in the absence of embryoid body formation. Herein, we present an efficient and integrated 3D bioprocess based on the encapsulation of undifferentiated mESCs within alginate hydrogels and culture in a rotary cell culture microgravity bioreactor. Specifically, for the first 3 days, encapsulated mESCs were cultured in 50% (v/v) HepG2 conditioned medium to generate a cell population with enhanced mesodermal differentiation capability followed by osteogenic differentiation using osteogenic media containing ascorbic acid, beta-glycerophosphate and dexamethasone. 3D mineralised constructs were generated that displayed the morphological, phenotypical, and molecular attributes of the osteogenic lineage, as well mechanical strength and mineralised calcium/phosphate deposition. Consequently, this bioprocess provides an efficient, automatable, scalable and functional culture system for application to bone tissue engineering in the context of macroscopic bone formation.

Stem cells in bone tissue engineering.

Bone tissue engineering has been one of the most promising areas of research, providing a potential clinical application to cure bone defects. Recently, various stem cells including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose tissue-derived stem cells (ADSCs), muscle-derived stem cells (MDSCs) and dental pulp stem cells (DPSCs) have received extensive attention in the field of bone tissue engineering due to their distinct biological capability to differentiate into osteogenic lineages. The application of these stem cells to bone tissue engineering requires inducing in vitro differentiation of these cells into bone forming cells, osteoblasts. For this purpose, efficient in vitro differentiation towards osteogenic lineage requires the development of well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source for application to bone tissue engineering therapies. This review provides a critical examination of the various experimental strategies that could be used to direct the differentiation of ESC, BM-MSC, UCB-MSC, ADSC, MDSC and DPSC towards osteogenic lineages and their potential applications in tissue engineering, particularly in the regeneration of bone.

Microfluidic Techniques for Development of 3D Vascularized Tissue

Development of a vascularized tissue is one of the key challenges for the successful clinical application of tissue engineered constructs. Despite the significant efforts over the last few decades, establishing a gold standard to develop three dimensional (3D) vascularized tissues has still remained far from reality. Recent advances in the application of microfluidic platforms to the field of tissue engineering have greatly accelerated the progress toward the development of viable vascularized tissue constructs. Numerous techniques have emerged to induce the formation of vascular structure within tissues which can be broadly classified into two distinct categories, namely (1) prevascularization-based techniques and (2) vasculogenesis and angiogenesis-based techniques. This review presents an overview of the recent advancements in the vascularization techniques using both approaches for generating 3D vascular structure on microfluidic platforms.

Development of collagenase-resistant collagen and its interaction with adult human dermal fibroblasts

Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary source in biomedical application. Collagen has been widely used in the crosslinked form to extend the durability of collagen. The chemical treatment influences the structural integrity of collagen molecule resulting in the loss of triple helical characteristic. The structural characteristic of collagen is importantly related to its biological function for the interaction with cell. In this study, structural stability of collagen was enhanced thought EGCG treatment, resulting in high resistance against degradation by bacterial collagenase and MMP-1, which is confirmed by collagen zymography. The triple helical structure of EGCG-treated collagen could be maintained at 37 degrees C in comparison with collagen, which confirmed by CD spectra analysis, and EGCG-treated collagen showed high free-radical scavenging activity. Also, with fibroblasts culture on EGCG-treated collagen, the structural stability of EGCG-treated collagen provided a favorable support for cell function in cell adhesion and actin filament expression. These observations underscore the need for native, triple helical collagen conformation as a prerequisite for integrin-mediated cell adhesion and functions. According to this experiment, EGCG-treated collagen assumes to provide a practical benefit to resist the degradation by collagenase retaining its structural characteristic, and can be a suitable biomaterial for biomedical application.

Antioxidants, like coenzyme Q10, selenite, and curcumin, inhibited osteoclast differentiation by suppressing reactive oxygen species generation.

Coenzyme Q10 (CoQ10), selenium, and curcumin are known to be powerful antioxidants. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts causes bone loss-related diseases. During osteoclast differentiation, the reactive oxygen species (ROS) acts as a secondary messenger on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared the relative inhibitory activities of CoQ10, sodium selenite, and curcumin on osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMMs) and RAW 264.7 cells. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. These also acted to significantly suppress the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor (OSCAR), which are genetic markers of osteoclast differentiation in a dose-dependent manner. These antioxidants also suppressed ROS-induced IκBα signaling pathways for osteoclastogenesis. Specially, curcumin displayed the highest inhibitory effect on osteoclast differentiation when concentrations were held constant. Together, CoQ10, selenite, and curcumin act as inhibitors of RANKL-induced NFATc1 which is a downstream event of NF-κB signal pathway through suppression of ROS generation, thereby suggesting their potential usefulness for the treatment of bone disease associated with excessive bone resorption.

In situ fabrication of alendronate-loaded calcium phosphate microspheres: Controlled release for inhibition of osteoclastogenesis

Bioabsorbable calcium phosphate (CaP) microspheres that can incorporate alendronate (ALD) through an in situ loading process and can control the ALD release rate have been described. ALD loading into CaP microspheres could be accomplished by emulsification (water-in-oil) and a subsequent CaP nucleation/growth process within the water droplets, which was initiated by a urea-mediated solution precipitation technique. ALD-loaded microspheres with a mean size range of 163-195 μm were obtained in a spherical shape. Inductively coupled plasma mass spectroscopy (ICP-MS), spectrophotometric analysis, and thermogravimetric analysis (TGA) showed that the amount of ALD loaded into the microspheres increased when the ALD feed content increased. Energy-dispersive X-ray spectroscopy (EDX) analysis revealed that the in situ loading process enabled the ALD loading throughout the microspheres. X-Ray diffraction (XRD) analysis demonstrated that crystalline hydroxyapatite (HAp) and amorphous CaP phases coexisted within the microspheres. In addition, the increased loading of ALD resulted in a larger proportion of the amorphous CaP phase within the microspheres. The ALD release rate could be controlled depending on the dissolution rate of microspheres, and ALD could be released over a period of 40 days. The evaluation of the biological activity showed that ALD-loaded CaP microspheres directly blocked osteoclast formation by releasing ALD to monocytic precursor cells and effectively inhibiting their differentiation into osteoclasts.

Type I atelocollagen grafting onto ozone‐treated polyurethane films: Cell attachment, proliferation, and collagen synthesis

An approach is presented for the graft copolymerization of type I atelocollagen onto the surface of polyurethane (PU) films treated with ozone. Through inducing oxidization to modify PU surface by ozone, peroxide groups are easily generated on the surface. Those peroxides are broken by redox-polymerization, and provide active species which initiate graft polymerization by reacting with amines in the collagen molecules. The ozone oxidation time and voltage could readily control the amount of peroxide production. The surface density of generated peroxides on PU surface was determined by iodide method. The maximum concentration of peroxide was about 10.20 x 10(-8)mol/cm(2) when ozone oxidation was performed at 60 V for 30 min. After the reaction of PU by ozone oxidation, type I atelocollagen was graft-copolymerized onto the PU film. All the physical measurements on the collagen-grafted surface indicated that the PU surface was effectively covered with type I atelocollagen. The interaction of the collagen-grafted PU surface with fibroblasts could be greatly enhanced by the surface graft polymerization with type I atelocollagen. Attachment and proliferation of fibroblasts on the grafted type I atelocollagen were significantly enhanced, and it is assumed that the atelocollagen matrix supported the initial attachment and growth of cells. In the early stage of proliferation, collagen synthesis in fibroblasts was not activated and remained at a relatively low level due to the grafted type I atelocollagen, increasing only with fibroblast differentiation.

Enhanced Neural Cell Adhesion and Neurite Outgrowth on Graphene-Based Biomimetic Substrates

Neural cell adhesion and neurite outgrowth were examined on graphene-based biomimetic substrates. The biocompatibility of carbon nanomaterials such as graphene and carbon nanotubes (CNTs), that is, single-walled and multiwalled CNTs, against pheochromocytoma-derived PC-12 neural cells was also evaluated by quantifying metabolic activity (with WST-8 assay), intracellular oxidative stress (with ROS assay), and membrane integrity (with LDH assay). Graphene films were grown by using chemical vapor deposition and were then coated onto glass coverslips by using the scooping method. Graphene sheets were patterned on SiO2/Si substrates by using photolithography and were then covered with serum for a neural cell culture. Both types of CNTs induced significant dose-dependent decreases in the viability of PC-12 cells, whereas graphene exerted adverse effects on the neural cells just at over 62.5 ppm. This result implies that graphene and CNTs, even though they were the same carbon-based nanomaterials, show differential influences on neural cells. Furthermore, graphene-coated or graphene-patterned substrates were shown to substantially enhance the adhesion and neurite outgrowth of PC-12 cells. These results suggest that graphene-based substrates as biomimetic cues have good biocompatibility as well as a unique surface property that can enhance the neural cells, which would open up enormous opportunities in neural regeneration and nanomedicine.

Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-κB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal-regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.