Young-Gwang Ko

Electrospinning of microbial polyester for cell culture

Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture.

Stabilization, Carbonization, and Characterization of PAN Precursor Webs Processed by Electrospinning Technique

【In the present study, electrospun PAN precursor webs and the stabilized and carbonized nanofiber webs processed under different heat-treatment conditions were characterized by means of weight loss measurement, elemental analysis, scanning electron microscopy (SEM), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), thermogravimentric analysis (TGA), and X-ray diffraction (XRD) analysis. The result indicated that stabilization and carbonization processes with different temperatures and heating rates significantly influenced the chemical and morphological characteristics as well as the thermal properties of the stabilized and then subsequently carbonized nanofiber webs from PAN precursor webs. It was noted that the filament diameter and the carbon content of a carbonized nanofiber web as well as its weight change may be effectively monitored by controlling both stabilization and carbonization processes.】

Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges.

Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

Injectable pullulan hydrogel for the prevention of postoperative tissue adhesion.

Methods for reducing and preventing postoperative abdominal adhesions have been researched for decades; however, despite these efforts, the formation of postoperative peritoneal adhesions is continuously reported. Adhesions cause serious complications such as postoperative pain, intestinal obstruction, and infertility. Tissue adhesion barriers have been developed as films, membranes, knits, sprays, and hydrogels. Hydrogels have several advantages when used as adhesion barriers, including flexibility, low tissue adhesiveness, biodegradability, and non-toxic degraded products. Furthermore, compared with preformed hydrogels, injectable hydrogels can fill and cover spaces of any shape and do not require a surgical procedure for implantation. In this study, pullulan was modified through reaction with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to introduce carboxyl and phenyl groups as crosslinking sites. The grafting of tyramine on pullulan allows crosslinking branches on pullulan backbone. We successfully fabricated pullulan hydrogel with an enzymatic reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The chemical structure of modified pullulan was analyzed with ATR-FTIR and (1)H NMR spectroscopies. Rheological properties were tested by measuring storage modulus with varying H2O2, HRP, polymer solution concentrations and tyramine substitution rates. Cell viability and animal tests were performed. The modified pullulan hydrogel is an invaluable advance in anti-adhesion agents.

Growth behavior of endothelial cells according to electrospun poly(D,L-lactic-co-glycolic acid) fiber diameter as a tissue engineering scaffold

Investigating the effect of electrospun fiber diameter on endothelial cell proliferation provides an important guidance for the design of a fabric scaffold. In this study, we prepared biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) fibrous nonwoven mats with different fiber diameters ranged from 200 nm to 5 µm using the electrospinning technique. To control the fiber diameters of PLGA mats, 4 mixture solvents [hexafluoro-2-propanol, 2,2,2,-trifluoroethanol:dimethylformamide (9:1), 2,2,2,-trifluoroethanol:hexafluoro-2-propanol (9:1), chloroform] were used. Average diameters were 200 nm, 600 nm, 1.5 µm, and 5.0 µm, respectively. Stereoscopic structure and spatial characterization of fibrous PLGA mats were analyzed using atomic force microscopy and a porosimeter. The mechanical properties of PLGA mats were analyzed using a universal testing machine. The spreading behavior and infiltration of endothelial cells on PLGA mats were visualized by field emission scanning electron microscopy and hematoxylin and eosin staining. Cell proliferation on different PLGA fibers with different diameters was quantified using the MTT assay. Cells on 200 nm diameter PLGA mats showed rapid attachment and spreading. However, the cells did not penetrate the PLGA mat. Cells cultured on 600 nm and 1.5 µm diameter fibers could infiltrate the pores and cell proliferation was dramatically increased after 14 days. Secreted prostacyclin from endothelial cells on each mat was measured to examine the ability to inhibit platelet activation. This basic study on cell proliferation and fiber diameter with physical characterization provides a foundation for studies examining nonwoven fibrous PLGA mats as a tissue engineering scaffold.

Fabrication and characterization of thermoresponsive polystyrene nanofibrous mats for cultured cell recovery.

Rapid cell growth and rapid recovery of intact cultured cells are an invaluable technique to maintain the biological functions and viability of cells. To achieve this goal, thermoresponsive polystyrene (PS) nanofibrous mat was fabricated by electrospinning of PS solution, followed by the graft polymerization of thermoresponsive poly(N-isopropylacrylamide)(PIPAAm) on PS nanofibrous mats. Image analysis of the PS nanofiber revealed a unimodal distribution pattern with 400 nm average fiber diameter. Graft polymerization of PIPAAm on PS nanofibrous mats was confirmed by spectroscopic methods such as ATR-FTIR, ESCA, and AFM. Human fibroblasts were cultured on four different surfaces, PIPAAm-grafted and ungrafted PS dishes and PIPAAm-grafted and ungrafted PS nanofibrous mats, respectively. Cells on PIPAAm-grafted PS nanofibrous mats were well attached, spread, and proliferated significantly much more than those on other surfaces. Cultured cells were easily detached from the PIPAAm-grafted surfaces by decreasing culture temperature to 20°C, while negligible cells were detached from ungrafted surfaces. Moreover, cells on PIPAAm-grafted PS nanofibrous mats were detached more rapidly than those on PIPAAm-grafted PS dishes. These results suggest that thermoresponsive nanofibrous mats are attractive cell culture substrates which enable rapid cell growth and recovery from the culture surface for application to tissue engineering and regenerative medicine.

Nanofiber mats composed of a chitosan-poly(d,l-lactic-co-glycolic acid)-poly(ethylene oxide) blend as a postoperative anti-adhesion agent.

Postoperative tissue adhesion causes serious complications and suffering in 90% of patients after peritoneum surgery, while commercial anti-adhesion agents cannot completely prevent postoperative peritoneal adhesions. This study demonstrates electrospining of a blended solution of chitosan, poly(d,l-lactic-co-glycolic acid) (PLGA), and poly(ethylene oxide) (PEO) to fabricate a chitosan-based nanofibrous mat as a postoperative anti-adhesion agent. Rheological studies combined with scanning electron microscopy reveal that the spinnability of the chitosan-PLGA solution could be controlled by adjusting the blend ratio and concentration with average fiber diameter from 634 to 913 nm. Biodegradation of the nanofiber specimens showed accelerated hydrolysis by chitosan. Proliferation of fibroblasts and antimicrobial activity of nanofibers containing chitosan was analyzed. Abdominal defects with cecum adhesion in rats demonstrated that the blend nanofiber mats were effective in preventing tissue adhesion as a barrier (4 weeks after abdominal surgery) by coverage of exfoliated peritoneum and insufficient wound sites at the beginning of the wound healing process. Chitosan-PLGA-PEO blend nanofiber mats will provide a promising key as a postoperative anti-adhesion agent.

Hydrophobization of silk fibroin nanofibrous membranes by fluorocarbon plasma treatment to modulate cell adhesion and proliferation behavior

AbstractSaturated fluorocarbon (CF4) immobilized silk fibroin (SF) nanofibrous membranes were prepared and characterized for biomedical applications. Biocompatible barrier membranes that provide both hydrophobic and hydrophilic surface properties on each side are critical to prohibit soft tissue invasion into localized bone defect. As a barrier membrane, SF nanofibrous mat was fabricated by electrospinning method, and then subsequently modified with water vapor treatment for insolubilization in water and CF4 gas plasma treatment for surface hydrophobization. Morphology of SF nanofibrous mats were observed by scanning electron microscopy. Conformational change of insolubilized SF nanofibers was confirmed by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and 13C nuclear magnetic resonance (NMR) spectroscopy. Immobilized fluorine atoms on CF4 plasma treated SF nanofibrous membranes were detected using electron spectroscopy for chemical analysis (ESCA). Water contact angle of the SF nanofiber membrane surface was analyzed by varying plasma input power and time. Insolubilized SF nanofibrous membrane maintained nonwoven mat structure without deformation after water immersion. SF nanofibrous membranes showed significant increment of water contact angle from 99.7° to 141.2° by CF4 gas plasma treatment. Fibroblasts on plasma untreated SF nanofibrous membranes were well attached and spread than a control tissue culture polystyrene dish. Fibroblasts on the CF4 gas plasma treated SF nanofibrous membrane showed significantly lower proliferation behavior than plasma untreated SF nanofibrous membranes. Fluorocarbon immobilized SF nanofibrous barrier membrane will be useful for biomedical applications such as a guided bone regeneration.

Guiding bone regeneration using hydrophobized silk fibroin nanofiber membranes

Biocompatible barrier membranes with both hydrophobic and hydrophilic surface properties provide critical backup for guided regeneration at localized bone defects without soft tissue invasion. As a surface modified functional barrier, saturated fluorocarbon (CF4)-immobilized nanofibrous silk fibroin (SF) membranes were fabricated by electrospinning for a fibrous non-woven mat, water vapor treatment for insolubilization, and followed by CF4 gas plasma treatment for top surface hydrophobization. Plasma-treated SF nanofiber membranes maintained a non-woven mat structure without shrinkage and deformation in a five-month biodegradation test. From in vivo rabbit cranium perforation model, nanofibrous SF membranes prevented soft tissue invasion and facilitated volumetric bone regeneration compared with the control groups. New bone ingrowth in bone defects at 4 and 8 weeks after surgery was visualized by trichrome staining. Medical application of fluorocarbon-immobilized nanofibrous SF barrier membranes could be one of the practical approaches for guided bone regeneration.

Preparation and Characterization of Polyurethane Foam Dressings Containing Natural Antimicrobial Agents for Wound Healing

The foam dressing material continuously absorbs the exudate of the wound area and maintains moisture environment, so that there is a weak point of the bacterial growth. Therefore, antimicrobial property is one of the important factors in the development of moisture environment foam dressing. The purpose of this study is to evaluate the effectiveness of wound dressing after manufacturing antimicrobial foam dressing materials containing asiatic acid, madecassic acid, and aciaticoside of the Centella asiatica. In order to fabricate superior absorbent layer compared to conventional polyurethane foam dressings, the optimum mechanical properties, pore morphology, absorbency and absorption rate were established on blending ratio of the foam mixture and the antimicrobial component. The foam containing the active ingredients of the Centella asiatica showed high antimicrobial activity and excellent wound recovery rate in animal model experiments. The polyurethane foam dressing material containing active ingredients of the Centella asiatica is expected to be used as an antimicrobial wound treatment.