Hui-Jeong Gwon

Synergistic Effect of Dual‐Functionalized Fibrous Scaffold with BCP and RGD Containing Peptide for Improved Osteogenic Differentiation

Over the last decade, bone tissue engineering scaffolds have been advanced owing to the bioceramic incorporation and biomimetic modification. In this report, a dual-functional fibrous scaffold with a bioceramic and biomolecule is developed, and a combined effect of a dual-modification is investigated. Biphasic calcium phosphate (BCP) is incorporated in electrospun poly (L-lactide) scaffolds, and Arg-Gly-Asp (RGD) peptide is then conjugated through the graft polymerization of acrylic acid by γ-ray irradiation. The scaffolds exhibit the intrinsic properties of BCP as well as RGD peptide, and only RGD peptide improves an adhesion and proliferation of the human mesenchymal stem cell. However, alkaline phosphatase activity and calcium formation are synergistically improved by the BCP and RGD peptide indicating that a favorable microenvironment is constructed for bone formation. Therefore, this combination strategy with bioceramic and biomolecule can be a useful tool for the bone tissue engineering.

Hyaluronic acid/chondroitin sulfate-based hydrogel prepared by gamma irradiation technique

Gamma-ray irradiation of novel hydrogels was used to develop a biocompatible hydrogel system for skin tissue engineering. These novel hydrogels are composed of natural polymers including hyaluronic acid (HA) and chondroitin sulfate (CS), and the synthetic polymer, poly(vinyl alcohol) (PVA). The γ-ray irradiation method has advantages, such as relatively simple manipulation without need of any extra reagents for polymerization and cross-linking. We synthesized HA and CS derivatives with polymerizable residues. The HA/CS/PVA hydrogels with various compositions were prepared by using γ-ray irradiation technique and their physicochemical properties were investigated to evaluate the feasibility of their use as artificial skin substitutes. HA/CS/PVA hydrogels showed an 85-88% degree of gelation under 15 kGy radiation. All HA/CS/PVA hydrogels exhibited more than 90% water content and reached an equilibrium swelling state within 24h. Hydrogels with higher concentrations of hyaluronidase solution and HA/CS content had proportionally higher enzymatic degradation rates. The drug release behaviors from HA/CS/PVA hydrogels were influenced by the composition of the hydrogel and drug properties. Exposure of human keratinocyte (HaCaT) culture to the extracts of HA/CS/PVA hydrogels did not significantly affect the cell viability. All HaCaT cell cultures exposed to the extracts of HA/CS/PVA hydrogels exhibited greater than 92% cell viability. The HaCaT growth in HA/CS/PVA hydrogels gradually increased as a function of culture time. After 7 days, the HaCaT cells in all HA/CA/PVA hydrogels exhibited more than 80% viability compared to the control group HaCaT culture on a culture plate.

Topical treatment of the buccal mucosa and wounded skin in rats with a triamcinolone acetonide-loaded hydrogel prepared using an electron beam.

In this study, a triamcinolone acetonide-loaded hydrogel was prepared by electron beam irradiation and evaluated for use as a buccal mucoadhesive drug delivery system. A poloxamer was modified to have vinyl end groups for preparation of the hydrogel via an irradiation cross-linking reaction. Carbopol was introduced to improve the mucoadhesive properties of the hydrogel. The in vitro release of triamcinolone acetonide from the hydrogel was examined at 37 °C. To investigate the topical therapeutic effect of triamcinolone acetonide on wounded rat skin and buccal mucosa, the appearance and histological changes were evaluated for 15 days after treatment with saline, triamcinolone acetonide solution, triamcinolone acetonide hydrogel, and blank hydrogel, respectively. Triamcinolone acetonide was released constantly from the gel formulation at 37 °C and reach 100% at about 48 h. After 15 days, in the skin of the group treated with the triamcinolone acetonide-loaded hydrogel, the wound was almost completely free of crust and a number of skin appendages, including hair follicles, had formed at the margins of the tissue. Moreover, the inflammatory response in the buccal mucosa was milder than that in the other groups, and the wound surface was completely covered with regenerating, hyperkeratotic, thickened epithelial cells. Our results indicate that the triamcinolone-acetonide hydrogel showed sustained drug release behavior, while causing no significant histopathological changes in buccal and skin tissues. Therefore, this hydrogel system may be a powerful means of drug delivery for buccal administration with controlled release and no tissue irritation.

Radiation-induced biomimetic modification of dual-layered nano/microfibrous scaffolds for vascular tissue engineering

One of the interesting strategies for developing the artificial blood vessels is to generate multi-layered scaffolds for mimicking the structure of native blood vessels such as the intima, media, and adventitia. In this study, we prepared dual-layered poly(L-lactide-co-ɛ-caprolactone) (PLCL) scaffolds with micro- and nanofibers as a basic construct of the vessel using electrospinning methods, which was functionalized using a gelatin through acrylic acid (AAc) grafting by γ-ray irradiation. Based on the microfibrous platform (fiber diameter 5 μm), the thickness of the nanofibrous layer (fiber diameter 700 nm) was controlled from 1.1 ± 0.8 to 32.2 ± 1.7 μm, and the mechanical property of the scaffolds was almost maintained despite the increase in thickness of the nanofibrous layer. The successful AAc graft by γ-ray irradiation could allow the gelatin immobilization on the scaffolds. The proliferation of smooth muscle cells (SMC) on the scaffolds toward a microfibrous layer was approximately 1.3-times greater than in the other groups, and the infiltration was significantly increased, presenting a wide cell distribution in the cross-section. In addition, human umbilical vein endothelial cell (HUVEC) adhesion toward nanofibrous layer was well-managed over the entire surface, and the accelerated proliferation was observed on the gelatin-functionalized scaffolds presenting the well-organized gap-junctions. Therefore, our biomimetic dual-layered scaffolds may be the alternative tools for replacing the damaged blood vessels.

Hydrogel incorporated with chestnut honey accelerates wound healing and promotes early HO-1 protein expression in diabetic (db/db) mice

Proper wound management is an increasingly important clinical challenge and is a large and growing unmet need. Pressure ulcers, hard-to-heal wounds, and problematic surgical incisions are emerging with increasing frequency. At present, the wound-healing industry is experiencing a paradigm shift toward innovative treatments that exploit nanotechnology, biomaterials, and biologics. Here we determined the effectiveness of a radiation-processed hydrogel patch for the delivery of chestnut honey (CH) for the promotion of cutaneous wound healing in diabetic mice. CH is a natural compound that has antioxidant and bactericidal effects. Two full-thickness wounds were made on the dorsal side of diabetic (db/db) mice and the wounds were covered with hydrogel-incorporated CH. Time course observations revealed that mice treated with CH hydrogel showed accelerated wound closure and formation of granulated tissue, enhanced Ki-67 expression and early upregulated HO-1 proteins in the wound region compared with water hydrogel or non-treated mice. Taken together, these findings indicate that CH hydrogel can promote wound healing in diabetics with early HO-1 protein expression.

Preparation of Cellulose-based Nanofibers Using Electrospinning

Eelctrospinning is a straightforward method to prepare fibers with diameters as small as several tens of nanometers (Doshi & Reneker, 1995). In eclectrospinning, a high electrostatic voltage is imposed on a drop of polymer solution held by its surface tension at the end of a capillary. The surface of the liquid is distorted into a conical shape known as the Taylor cone. Once the voltage exceeds a critical value, the electrostatic force overcomes the solution surface tension and a stable liquid jet is ejected from the cone tip. Solvent evaporates as the jet travels through the air, leaving behind ultrafine polymeric fibers collected on an electrically grounded target (Fong et al., 1999, 2002; Shin et al., 2001). Electrospun mats have a larger specific surface area and small pore size compared to commercial non-woven fabrics. They are of interest in a wide variety of applications including semi-permeable membranes, tissue engineering scaffolds and drug delivery systems (Tsai et al., 2002; Gibson et al., 2001; Kenawy et al., 2002; Luu et al., 2003). Recently, electrospun nanofibers (NFs) based on cellulose and its derivatives have been studied as potential candidates for applications within the field of pharmaceuticals. For instance, several reports deal with the investigation of electrospun fiber mats as delivery vehicles, showing dosage forms with useful and controllable dissolution properties. This interest in cellulose-based NFs is primarily driven by its environmental value as a biomaterial. The cellulose is an abundant and renewable resource found in most parts of the world, which makes it a cheap raw material for various applications (Zeng et al., 2003; Jiang et al., 2004; Verreck et al., 2003; Liu & Hsieh, 2002). However, little research has been done on the use of cellulose and cellulose derivatives as a raw material within electrospinning. The complications involved in electrospinning of cellulose are mainly due to the many difficulties ascribed to the material, one being its reluctance to interact with conventional solvents. Therefore, the choice of solvent systems is very important. Ethyl-cellulose (EC) is a kind of cellulose ether, and it shows a non-biodegradable and biocompatible polymer. EC is one of the extensively studied encapsulating materials for the controlled release of pharmaceuticals (Prasertmanakit et al., 2009). The film made from EC has quite good permeability, it has been widely used industrial air filter (Park et al., 2007). Hydroxypropyl methylcellulose (HPMC) is frequently used as the basis for sustained release hydrophilic matrix tablets (Ford, 1999). HPMC backbone is composed of glucose

Characterization of hydroxyapatite-coated bacterial cellulose scaffold for bone tissue engineering

The goal of this study was to develop a novel hydroxyapatite (HA) coated bacterial cellulose (BC) scaffold for bone tissue regeneration. HA-coated BC was prepared by immersing in 30 mL of 5× simulated body fluid at 37°C for 12 h. The resulting HA-coated BC scaffolds were characterized by scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATRFTIR) spectroscopy, and thermal gravimetric analysis (TGA). HA spherical globules were newly formed on the surface of the BC, and a fibrous network of BC scaffolds still maintained their dimensions for cell adhesion and proliferation. ATR-FTIR spectroscopy analysis showed bands assigned to specific signals for phosphate and carbonate ions from HA. HA-coated BC scaffolds of thermal gravimetric analysis presented residue of around 25%. The ability for bone regeneration of HA-coated BC scaffolds was evaluated using a rat calvarial defect model for 4 and 8 weeks. After implantation, both BC and HAcoated BC scaffolds showed new bone formation derived from existing bone, and found new bone even inside the scaffold. Furthermore, a new bone area was signigicantly increased in the HA-coated BC scaffolds compared with those from BC scaffolds, and bone-like materials were frequently found in HA-coated BC scaffolds. Therefore, the HA-coated BC scaffolds can be used as an effective tool for bone tissue regeneration.

Promotion of human mesenchymal stem cell differentiation on bioresorbable polycaprolactone/biphasic calcium phosphate composite scaffolds for bone tissue engineering

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ∼ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.

OPTIMAL PRODUCTION OF L-threo-2,3-DIHYDROXYPHENYLSERINE (L-threo-DOPS) ON A LARGE SCALE BY DIASTEREOSELECTIVITY-ENHANCED VARIANT OF L-THREONINE ALDOLASE EXPRESSED IN Escherichia coli

This study examined the efficient production and optimal separation procedures for pure L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) from a mixture of diastereomers synthesized by whole-cell aldol condensation reaction, harboring diastereoselectivity-enhanced L-threonine aldolase in Escherichia coli JM109. The addition of the reducing agent sodium sulfite was found to stimulate the production of L-threo-DOPS without affecting the diastereoselectivity ratio, especially at the 50 mM concentration. The optimal pH for diastereoselective synthesis was 6.5. The addition of Triton X-100 also strongly affected the synthesis yield, showing the highest conversion yield at a 0.75% concentration; however, the diastereoselectivity of the L-threonine aldolase was not affected. Lowering the temperature to 10°C did not significantly affect the diastereoselectiviy without affecting the synthesis rate. At the optimized conditions, a mixture of L-threo-DOPS and L-erythro-DOPS was synthesized by diastereoselectivity-enhanced L-threonine aldolase from E. coli in a continuous process for 100 hr, yielding an average of 4.0 mg/mL of L-threo-DOPS and 60% diastereoselectivity (de), and was subjected to two steps of ion exchange chromatography. The optimum separation conditions for the resin and solvent were evaluated in which it was found that a two-step process with the ion-exchange resin Dowex 50 W × 8 and activated carbon by washing with 0.5 N acetic acid was sufficient to separate the L-threo-DOPS. By using two-step ion-exchange chromatography, synthesized high-purity L-threo-DOPS of up to 100% was purified with a yield of 71%. The remaining substrates, glycine and 3,4-dihydroxybenzaldehyde, were recovered successfully with a yield of 71.2%. Our results indicate this potential procedure as an economical purification process for the synthesis and purification of important L-threo-DOPS at the pharmaceutical level.

Biodegradable polycaprolactone/cuttlebone scaffold composite using salt leaching process

We prepared biodegradable polycaprolactone/cuttlebone scaffold composite by salt leaching process. In the first step, a co-continuous blend of biodegradable materials, polycaprolactone (PCL) and cuttlebone (CB), and an amount of sodium chloride salt particles were mixed using a stirrer. Next, the extraction of mineral salts using de-ionized distilled water was performed using a biodegradable PCL/CB scaffold with fully interconnected pores. Finally, the durable morphology of the scaffolds was fabricated by freeze-drying process at −53 °C for 24 hrs in a vacuum. In addition, the quadrilateral pres ranged from about 250 to 300 μm in diameter. Scanning electron microscopy (SEM) and mercury intrusion porosimeter techniques were carried out to characterize the pore morphology. By increasing the CB and sodium chloride salt particle content, the number of interconnected pores, material properties, and pore morphology were dramatically changed. The average compressive strengths (load at 50% strain) of the different porous PCL/CB scaffolds were found to decrease from 133 to about 79 (load at 50% strain, gf) with an increase in porosity. The values of the porosity increased as the sodium chloride salt volume fraction increased