Wan-Kyu Ko

Electrospun chitosan nanofibers with controlled levels of silver nanoparticles. Preparation, characterization and antibacterial activity

The ideal wound dressing would have properties that allow for absorption of exudates, and inhibition of microorganism for wound protection. In this study, we utilized an electrospinning (ELSP) technique to design a novel wound dressing. Chitosan (CTS) nanofibers containing various ratios of silver nanoparticles (AgNPs) were obtained. AgNPs were generated directly in the CTS solution by using a chemical reduction method. The formation and presence of AgNPs in the CTS/AgNPs composite was confirmed by x-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV) and thermogravimetric analysis (TGA). The electrospun CTS/AgNPs nanofibers were characterized morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These nanofibers were subsequently tested to evaluate their antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa) and gram-positive Methicillin-resistant Staphylococcus aureus (MRSA). Results of this antibacterial testing suggest that CTS/AgNPs nanofibers may be effective in topical antibacterial treatment in wound care.

Enhanced bone regeneration with a gold nanoparticle–hydrogel complex

Gold nanoparticles (GNPs) are widely used in diagnostics, drug delivery, biomedical imaging, and photo-thermal therapy due to their surface plasmon resonance, fluorescence, and easy-surface functionalization. According to recent studies, GNPs display a positive effect on the osteogenic differentiation of mesenchymal stem cells (MSCs) and MC3T3-E1 osteoblast-like cells. The aim of this study was to develop a new approach for bone tissue regeneration based on the utilization of a biodegradable hydrogel loaded with GNPs. We have used photo-curable gelatin hydrogels (Gel) in order to provide a proof of principle of GNPs in regeneration strategies for bone tissue repair. We have investigated the effects of these Gel-GNP composite hydrogels both in vitro and in vivo. The in vitro results showed that the hydrogels loaded with GNPs promote proliferation, differentiation, and alkaline phosphate (ALP) activities of human adipose-derived stem cells (ADSCs) as they differentiate towards osteoblast cells in a dose-dependent manner. Moreover, the in vivo results showed that these hydrogels loaded with high concentrations of GNPs had a significant influence on new bone formation. Through these in vitro and vivo tests, we found that the Gel-GNP can be a useful material for bone tissue engineering.

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.

The effect of gold nanoparticle size on osteogenic differentiation of adipose-derived stem cells.

There have been many medical applications based on gold nanoparticles (GNPs) over the past several centuries. Recently, researchers have focused on bone tissue engineering applications utilizing GNPs. The effect of various sizes of gold nanoparticles on the differentiation of human adipose-derived stem cells (ADSCs) into osteoblasts was investigated. The concentration of gold nanoparticles was fixed at 1 μM and varying sizes of 15, 30, 50, 75 and 100 nm (spherical GNPs) were used. The lack of cytotoxicity was confirmed by establishing viability of ADSCs using cell counting kit-8 (CCK-8) and live/dead assays. The results showed that each size of GNPs had no significant toxicity on ADSCs during 1 week of incubation. Osteogenic differentiation of ADSCs was confirmed by alkaline phosphatase (ALP) staining, ALP activity, calcium deposition, and real time PCR experiments. It was found, through dark field assays and microscope cell images, that 30 nm and 50 nm GNPs were preferentially up taken into the ADSCs. As expected, all sizes of gold nanoparticles promoted the differentiation of ADSCs toward osteoblasts more than control. Among all sizes, 30 and 50 nm GNPs appeared to have the highest differentiation rates. The data consistently demonstrated that 30 and 50 nm GNPs are the most effective in promoting osteogenic differentiation of ADSCs.

Inhibition of Osteoclast Differentiation by Gold Nanoparticles Functionalized with Cyclodextrin Curcumin Complexes

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under in vitro conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both in vitro and in vivo. We prepared β-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR-CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR-CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR-CGNPs. In addition, the CUR-CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR-CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the in vivo results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR-CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR-CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis.

Multifunctional hydrogel coatings on the surface of neural cuff electrode for improving electrode-nerve tissue interfaces

UNLABELLED Recently, implantable neural electrodes have been developed for recording and stimulation of the nervous system. However, when the electrode is implanted onto the nerve trunk, the rigid polyimide has a risk of damaging the nerve and can also cause inflammation due to a mechanical mismatch between the stiff polyimide and the soft biological tissue. These processes can interrupt the transmission of nerve signaling. In this paper, we have developed a nerve electrode coated with PEG hydrogel that contains poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with anti-inflammatory cyclosporin A (CsA). Micro-wells were introduced onto the electrode in order to increase their surface area. This allows for loading a high-dose of the drug. Additionally, chemically treating the surface with aminopropylmethacrylamide can improve the adhesive interface between the electrode and the hydrogel. The surface of the micro-well cuff electrode (MCE) coated with polyethylene glycol (PEG) hydrogel and drug loaded PLGA microspheres (MS) were characterized by SEM and optical microscopy. Additionally, the conductive polymers, poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT/PSS), were formed on the hydrogel layer for improving the nerve signal quality, and then characterized for their electrochemical properties. The loading efficiencies and release profiles were investigated by High Performance Liquid Chromatography (HPLC). The drug loaded electrode resulted in a sustained release of CsA. Moreover, the surface coated electrode with PEG hydrogel and CsA loaded MP showed a significantly decreased fibrous tissue deposition and increased axonal density in animal tests. We expect that the developed nerve electrode will minimize the tissue damage during regeneration of the nervous system. STATEMENT OF SIGNIFICANCE The nerve electrodes are used for interfacing with the central nervous system (CNS) or with the peripheral nervous system (PNS). The interface electrodes should facilitate a closed interconnection with the nerve tissue and provide for selective stimulation and recording from multiple, independent, neurons of the neural system. In this case, an extraneural electrodes such as cuff and perineural electrodes are widely investigated because they can completely cover the nerve trunk and provide for a wide interface area. In this study, we have designed and prepared a functionalized nerve cuff electrode coated with PEG hydrogel containing Poly lactic-co-glycol acid (PLGA) microspheres (MS) loaded with cyclosporine A (CsA). To our knowledge, our findings suggest that surface coating a soft-hydrogel along with an anti-inflammatory drug loaded MS can be a useful strategy for improving the long-term biocompatibility of electrodes.

Titanium dental implants surface-immobilized with gold nanoparticles as osteoinductive agents for rapid osseointegration

Gold nanoparticles (GNPs) are quite attractive materials for use as osteogenic agents due to their potential effects on the stimulation of osteoblast differentiation. In this study, an osseo-integrated titanium (Ti) implant surface coated with GNPs was used for promotion of bone regeneration. We prepared a silanized Ti surface by chemical treatment of (3-Mercaptopropyl) trimethoxysilane (MPTMS) and immobilized the GNP layer (Ti-GNP) on their surfaces via Au-S bonding. The GNP layer is uniformly immobilized on the surface and the layer covers the titanium oxide surface well, as confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The Ti-GNP was used to investigate the effectiveness of this system both in vitro and in vivo. The in vitro results showed that the Ti-GNP significantly enhances the osteogenic differentiation with increased mRNA expression of osteogenic differentiation specific genes in human adipose-derived stem cells (ADSCs). Furthermore, the in vivo results showed that Ti-GNP had a significant influence on the osseous interface formation. Through these in vitro and vivo tests, we found that Ti-GNP can be useful as osseo-integration inducing dental implants for formation of an osseous interface and maintenance of nascent bone formation.

Coenzyme q10 regulates osteoclast and osteoblast differentiation.

Coenzyme Q10 (CoQ10), a powerful antioxidant, is a key component in mitochondrial bioenergy transfer, generating energy in the form of ATP. Many studies suggest that antioxidants act as inhibitors of osteoclastogenesis and we also have previously demonstrated the inhibitory effect of CoQ10 on osteoclast differentiation. Despite the significance of this effect, the molecular mechanism when CoQ10 is present at high concentrations in bone remodeling still remains to be elucidated. In this study, we investigated the inhibitory effect of CoQ10 on osteoclastogenesis and its impact on osteoblastogenesis at concentrations ranging from 10 to 100 μM. We found that nontoxic CoQ10 markedly attenuated the formation of receptor activator of nuclear factor κB ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone-marrow-derived monocytes (BMMs) and RAW 264.7 cells. Osteoclastogenesis with CoQ10 was significantly suppressed the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor, which are genetic markers of osteoclast differentiation and scavenged intracellular reactive oxygen species, an osteoclast precursor, in a dose-dependent manner. Furthermore, CoQ10 strongly suppressed H2 O2 -induced IκBα, p38 signaling pathways for osteoclastogenesis. In bone formation study, CoQ10 acted to enhance the induction of osteoblastogenic biomarkers including alkaline phosphatase, type 1 collagen, bone sialoprotein, osteoblast-specific transcription factor Osterix, and Runt-related transcription factor 2 and, also promoted matrix mineralization by enhancing bone nodule formation in a dose-dependent manner. Together, CoQ10 acts as an inhibitor of RANKL-induced osteoclast differentiation and an enhancer of bone-forming osteoblast differentiation. These findings highlight the potential therapeutic applications of CoQ10 for the treatment of bone disease.

Inhibition of Osteoclast Differentiation and Bone Resorption by Bisphosphonate-conjugated Gold Nanoparticles

In recent years, gold nanoparticles (GNPs) have been reported to affect the regeneration of bone tissue. The goal of this study was to improve bone tissue regeneration by using targeted GNPs. We fabricated a functionalized GNPs conjugated with alendronate (ALD), of the bisphosphonate group. Subsequently, the ALD, GNPs, and ALD conjugated GNPs (GNPs-ALD) were analyzed by ultraviolet-visible absorbance (UV-vis) spectrophotometer, Attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR), and thermo gravimetric analysis (TGA). The prepared GNPs-ALD were used to investigate their inhibitory effects on the receptor activator of nuclear factor- κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). Additionally, the GNPs-ALD were applied to ovariectomy (OVX)-induced osteoporotic mice and the experiments were evaluated. ALD was found to be successfully conjugated to the GNPs surface, and it displayed significant adhesion onto the bone surface. The in-vitro study indicated that the GNPs, ALD and GNPs-ALD suppressed osteoclast formation in a dose-dependent manner. Furthermore, in the OVX mouse model, the mice treated GNPs-ALD had higher bone density as compared to other OVX mice groups. The results from these tests indicated that GNPs-ALD can be useful agents for preventing and treating osteoporosis.

Generation of functionalized polymer nanolayer on implant surface via initiated chemical vapor deposition (iCVD)

Initiated chemical vapor deposition (iCVD) was utilized to generate a 200nm thick, uniform, functionalized polymer nanolayer comprised of glycidyl methacrylate (GMA) on the surface of titanium implants as a means to improve cellular attachment. Dot-patterned GMA-coated specimens were prepared as well as fully coated specimens. In vitro cellular responses, including cell morphology, protein adsorption, cell proliferation assays, alkaline phosphate activity (ALP) assays, and calcium deposition assays were studied using adipose derived stem cells. The mechanical stability of the thin film was investigated by XPS and FE-SEM analysis of the GMA-coated implant after implantation to an extracted bone from a pig. The GMA-coated specimens displayed increased protein adsorption, higher alkaline phosphatase activities, and higher calcium deposition as compared to control sample with no cytotoxicity. Additionally, no defect was observed in the test of mechanical stability. Notably, dot-patterned GMA-coated samples displayed higher alkaline phosphatase activities than others. Functionalized polymer nanolayer deposition via iCVD is a flexible and robust technique capable of mass production of biocompatible layers. These properties make this technique very suitable for implant applications in a variety of ways.