Oh Hyeong Kwon

Rapid cell sheet detachment from Poly(N-isopropylacrylamide)-grafted porous cell culture membranes

Fabrication of functional tissue constructs using sandwiched layers of cultured cells could prove to be an attractive approach to tissue engineering. Rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheet and prevents functional damage. To accelerate the required culture substrate hydrophilic and hydrophobic structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform IR and electron spectroscopy for chemical analysis revealed that PIPAAm was successfully grafted to surfaces of porous membranes. Atomic force microscopy (AFM) results showed that PIPAAm-grafted membranes had smoother surfaces than ungrafted controls while retaining their porous structure. The mean roughness of PIPAAm-grafted and -ungrafted porous membrane surfaces determined by digital AFM autocalculation was 4.40 +/- 0.4 and 5.9 +/- 0.4 nm, respectively. Tissue culture polystyrene (TCPS) dishes grafted with PIPAAm were compared with PIPAAm-grafted porous membranes in cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-grafted TCPS surfaces compared to only 30 min to detach cell sheets from PIPAAm-grafted porous membranes. With porous membranes, the water accesses the PIPAAm-grafted surface from underneath and peripheral to the attached cell sheet, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cell sheet. With TCPS PIPAAm-grafted surfaces the water is supplied from only the periphery of a cell sheet, slowing detachment.

Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes.

Fabrication of functional tissue constructs from designed three-dimensional structures of cells using the layered method of cultured cell sheets could prove to be an attractive approach to tissue engineering. Rapid recovery of cell sheets is considered to be important as a basic technology for practical assembly of tissue-mimicking structures. To accelerate required culture substrate hydrophilic/hydrophobic functional changes according to the hydrated/dehydrated structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted with poly(ethylene glycol) (PEG) onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform infrared and electron spectroscopy for chemical analysis revealed that PIPAAm and PEG were successfully grafted to surfaces of porous membranes. PIPAAm-grafted porous membranes (PIPAAm-PM) were compared with porous membranes co-grafted with various amounts of PEG and PIPAAm (PIPAAm(PEG)-PM) for cell sheet detachment experiments. Approximately 35min incubation at 20 degrees C was required to completely detach cell sheets from PIPAAm-PM in a static condition, while only 19min to detach cell sheets from PIPAAm(PEG0.5%)-PM, which is co-grafted with PIPAAm and 0.5wt% of PEG. With porous membranes, water molecules were accessed by the PIPAAm molecules grafted on the surfaces from both underneath and peripheral to the attached cell sheet, resulting in more rapid hydration of grafted PIPAAm molecules and detachment of cell sheet than that for nonporous tissue culture polystyrene (TCPS) dish. With PIPAAm(PEG)-PMs, grafted PEG chains should accelerate the diffusion of water molecules to PIPAAm grafts, showing more rapid detachment of cell sheet compare to PIPAAm-PMs.

Electrospun PHBV/collagen composite nanofibrous scaffolds for tissue engineering

Electrospinning has recently emerged as a leading technique for the formation of nanofibrous structures made of synthetic and natural extracellular matrix components. In this study, nanofibrous scaffolds were obtained by electrospinning a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type-I collagen in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HIFP). The resulting fibers ranged from 300 to 600 nm in diameter. Their surfaces were characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis and atomic force microscopy. The PHBV and collagen components of the PHBV/collagen nanofibrous scaffold were biodegraded by PHB depolymerase and a type-I collagenase aqueous solution, respectively. The cell culture experiments indicated that the PHBV/collagen nanofibrous scaffold accelerated the adhesion and growth of NIH3T3 cells more effectively than the PHBV nanofibrous scaffold, thus making the former a good scaffold for tissue engineering.

Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces

Here we demonstrate a novel cell coculture method without any apparent limitation in cell-type combinations that exploits thermally responsive polymer-grafted patterns to alter cell-cell and cell-surface interactions. Thermally responsive acrylamide polymer is first covalently patterned onto culture surfaces by masked electron beam irradiation. One cell type is then cultured to confluency at 37 degrees C. Reducing cell culture temperature below 32 degrees C selectively swells temperature sensitive polymer-grafted domains, detaching adherent cells only from these grafted patterns. Another cell type is then seeded over the same surface at 37 degrees C. These subsequently seeded cells adhere only to the now-exposed polymer-grafted domains. Initially seeded cells remaining adherent on nonpatterned surfaces and cells added in the second seeding are then cocultured at 37 degrees C in well-ordered patterns.

Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces.

Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.

Nanofabrication of Microbial Polyester by Electrospinning Promotes Cell Attachment

The biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as nanofibrous mats by electrospinning. Image analysis of the electrospun nanofibers fabricated from a 2 wt% 2,2,2-trifluoroethanol solution revealed a unimodal distribution pattern of fiber diameters with an observed average diameter of ca. 185 nm. The fiber diameter of electrospun fabrics could be controlled by adjusting the electrospinning parameters, including the solvent composition, concentration, applied voltage, and tip-to-collector distance. Chondrocytes derived from rabbit ear were cultured on a PHBV cast film and an electrospun PHBV nano-fibrous mat. After incubation for 2 h, the percentages of attached chondrocytes on the surfaces of the flat PHBV film and the PHBV nanofibrous mat were 19.0 and 30.1%, respectively. On the surface of the electrospun PHBV fabric, more chondrocytes were attached and appeared to have a much greater spreaded morphology than did that of the flat PHBV cast film in the early culture stage. The electrospun PHBV nanofabric provides an attractive structure for the attachment and growth of chondrocytes as cell culture surfaces for tissue engineering.

Enzyme modification by polymers with solubilities that change in response to photoirradiation in organic media.

We have synthesized a hybrid subtilisin the solubility of which can be regulated by photoirradiation through coupling with a photoresponsive copolymer that carries spiropyran groups in its side chains. The copolymer was synthesized by polymerization of methacrylate, methacrylic acid, and spiropyran–carrying methacrylate. It was then covalently bonded to the amino groups of subtilisin Carlsberg via its carboxyl groups using a carbodiimide coupling agent. The hybrid subtilisin was perfectly soluble in toluene and efficiently catalyzed transesterification. After ultraviolet irradiation, the hybrid subtilisin precipitated and was easily and quantitatively recovered by centrifugation. Recovered hybrid subtilisin, resolubilized by visible light irradiation, retained its original transesterification activity even after several cycles of precipitation and solubilization.

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.

Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media

Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes.

Resorbable Scaffolds from Three Different Techniques: Electrospun Fabrics, Salt-Leaching Porous Films, and Smooth Flat Surfaces

Nanofibrous scaffolds of poly[(L-lactide)-co-(1,5-dioxepan-2-one)] generated by electrospinning have been compared with porous films obtained by solvent cast/salt leaching and homogeneous films. A comparison between the fibrous materials and the homogeneous solvent-cast films revealed that the surface of the nanofibers was more hydrophobic and that the nanofibers were degraded more rapidly in the presence of proteinase. It was obvious that the strain-to-break was reduced by the nanofiber formation, it decreased from 370% to 130% independent of fiber diameter. These values were however considerably higher than the strain-to-break of the solvent-cast/salt leaching scaffold. In addition, the nanofibrous material accelerated the adhesion and growth of the mesenchymal stem cell compared to the smooth material.