Kwangwoo Nam

The use of high-hydrostatic pressure treatment to decellularize blood vessels.

A decellularization method using high-hydrostatic pressure (HHP) technology (>600MPa) is described. The HHP disrupts the cells inside the tissue. The cell debris can be eliminated with a simple washing process, producing clean, decellularized tissue. In this study, porcine aortic blood vessel was decellularized by HHP. The mechanical properties and in vivo performance of the decellularized tissue were evaluated. Mechanical properties of the decellularized tissue were not altered by the HHP treatment. Reduced inflammation of the decellularized tissue was confirmed by xenogenic transplant experimentation. An allogenic transplantation study showed that decellularized blood vessel endured the arterial blood pressure, and there was no clot formation on the luminal surface. In addition, cellular infiltration into the vessel wall was observed 4 weeks after implantation, suggesting that HHP treatments could be applied widely as a high-quality decellularization method.

The effect of decellularized bone/bone marrow produced by high-hydrostatic pressurization on the osteogenic differentiation of mesenchymal stem cells.

Decellularized bone/bone marrow was prepared to provide a microenvironment mimicking that of the bone marrow for three-dimensional culture in vitro. Bone/bone marrows were hydrostatically pressed at 980 MPa at 30 °C for 10 min to dismantle the cells. Then, they were washed with EGM-2 and further treated in an 80% EtOH to remove the cell debris and lipid, respectively. After being rinsed and shaken with PBS again, treated bone/bone marrows were stained with hematoxylin and eosin (H-E) to assess the efficacy of decellularization. Cells were determined to have been completely removed through H-E staining of their sections and DNA quantification. Rat mesenchymal stem cells (rMSCs) were seeded on the decellularized bone/bone marrows and cultured for 21 days. The adhesion of rMSCs on or into decellularized bone/bone marrows was confirmed and proliferated over time in culture. The osteogenic differentiation effect of decellularized bone/bone marrows on rMSCs in the presence or absence of dexamethasone was investigated. Decellularized bone/bone marrows without dexamethasone significantly increased alkaline phosphatase (ALP) activity, indicating promoted osteogenic differentiation of rMSCs. In an animal study, when decellularized bone/bone marrows were implanted into the rat subcutaneous, no immune reaction occurred and clusters of the hematopoietic cells could be observed, suggesting the decellularized bone/bone marrows can provide a microenvironment in vivo.

Preparation of a collagen/polymer hybrid gel designed for tissue membranes. Part I: controlling the polymer-collagen cross-linking process using an ethanol/water co-solvent.

The drawback with collagen/2-methacryloyloxyethyl phosphorylcholine (MPC) polymer hybrid gels (collagen/phospholipid polymer hybrid gels) prepared in alkaline morpholinoethane sulfonic acid (MES) aqueous solution is that the cross-linking rate between the polymer and the collagen is low. To solve this problem, ethanol has been adopted as the reaction solvent, to prevent 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride (EDC) hydrolysis. Alterations in the ethanol mole concentration changed the cross-linking rate between the MPC polymer and the collagen gel. Prevention of EDC hydrolysis is clearly observed; protonation of carboxyl groups implies that the ratio of ethanol to water should be controlled. The polymer shows signs of penetration into the collagen gel layer, thus forming a totally homogeneous phase gel. This affects the mechanical strength of the collagen gel, making the gel much stiffer and brittle with an increase in the swelling ratio, as compared with that prepared in MES buffer. However, it is possible to obtain a collagen/phospholipid polymer hybrid gel with a high polymer portion and the cross-linking rate can be successfully controlled.

The significance of hydrated surface molecular mobility in the control of the morphology of adhering fibroblasts

The effects of the hydrated molecular mobility and the surface free energy of polymer surfaces on fibronectin adsorption and fibroblast adhesion were investigated. ABA-type block copolymers composed of polyrotaxane (PRX) with different number of threaded α-cyclodextrin (α-CD), random copolymers with similar chemical composition to the PRX block copolymers, and conventional polymers were prepared to determine a wide range of hydrated molecular mobility (Mf) values estimated by quartz crystal microbalance-dissipation (QCM-D) measurements. Fibronectin adsorption was highly dependent on surface free energy, and high surface fibronectin density resulted in a large projected cell area on the polymer surfaces. However, the morphology of adhering fibroblasts was not explained by the surface free energy, but it was found to be strongly dependent on the Mf values of the polymer surfaces in aqueous media. These results emphasize the importance of Mf in the discussion of the elongated morphology of adhering fibroblasts on various polymer surfaces.

Effects of vibration on differentiation of cultured PC12 cells.

Different types of physiological‐mechanical stress, such as shear stress in vascular endothelial cells or hydrostatic pressure in chondrocytes are well known as regulators of cell function. In this study, the effects of vibration, a type of non‐physiological mechanical stimulation, on differentiation of rat pheochromocytoma (PC12) cells are reported. A nano‐vibration system was designed to produce nanometer‐scale vibration. The frequency and amplitude of the nano‐vibrations were monitored by a capacitance displacement sensor connected to an oscilloscope. When PC12 cells exposed to nerve growth factor were subjected to vibration at 10 kHz, differentiation and elongation of their neurites were promoted earlier in the culture. Vibration promoted differentiation of PC12 cells. This approach could therefore also be promising for determining of the effects of the physical environment on cell differentiation. Biotechnol. Bioeng. 2011; 108:592–599.

Fabrication of a heterostructural fibrillated collagen matrix for the regeneration of soft tissue function

Most collagen matrices used in tissue engineering and regenerative medicine are gels or sponges. Our goal is to prepare an artificial extracellular matrix (ECM) that possesses similar structure to the native ECM of soft tissue in order to replicate its physical and biological properties. Here, we report the preparation of collagen matrices with fibrillated structures based on the fibrillogenesis/gelling method. We focused on regenerating the function of native ECM of soft tissues by creating a multifunctional collagen. A collagen matrix is a sponge/gel composite structure which allows the cell to infiltrate only on one side of the matrix without shrinkage. This results in healing and regeneration of the tissue surrounding the matrix, similar to the native ECM in which cell infiltration occurs from one side during implantation. Furthermore, high mechanical strength can prevent deformation or shrinkage, and decelerate the degradation during the early phase of implantation, which eventually cause device failure.

Preparation of poly(vinyl alcohol)/DNA hydrogels via hydrogen bonds formed on ultra-high pressurization and controlled release of DNA from the hydrogels for gene delivery

Poly(vinyl alcohol) (PVA) hydrogels interacting with DNA mediated by hydrogen bonds (PVA/DNA hydrogel) were developed using ultra-high pressure (UHP) technology. The goal was to create a new method of gene delivery by controlled release of DNA. Mixed solutions of DNA and PVA at various concentrations were pressurized at 10 000 atmospheres at 37°C for 10 min. PVA/DNA hydrogels with good formability were produced at PVA concentrations of more than 5% w/v. The presence of DNA in the obtained hydrogels was confirmed by spectroscopic analysis and nucleic acid dye staining. DNA release from the hydrogels was investigated using PVA/DNA hydrogel samples of 5% and 10% w/v formed by UHP treatment or by conventional freeze–thaw methods. The DNA release curves from both types of samples showed a rapid phase in the initial 15 h followed by a sustained release phase. However, there was a difference in the amount of DNA released. Less DNA was released by the pressurized hydrogels than by the freeze–thaw hydrogels. Also, the cumulative amount of DNA released decreased as the PVA content in the hydrogels increased. These results indicate that DNA release from the hydrogels can be modulated by changing the preparation method and the PVA content. Furthermore, it was demonstrated that DNA release could be controlled by varying the amount and duration of pressurizing used to form the hydrogels. Intact fractions of plasmid DNA released from the hydrogels were separated by agarose gel electrophoretic analysis. These results suggest that, using controlled release, DNA from PVA/DNA hydrogels formed by UHP treatment can be transfected into cells.

Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix

The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 μm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

High-hydrostatic pressure technique is an effective method for the preparation of PVA–heparin hybrid gel

To develop an antithrombotic material for preparation of small-diameter vascular graft, we describe a novel method to prepare a poly(vinyl alcohol) (PVA)-heparin hydrogels prepared by high-hydrostatic pressure (HHP, 980 MPa), which is designed for sustained release of heparin. Antithrombogenic test revealed that HHP method would not affect the antithrombin III (ATIII) activity of the released heparin. The distribution of heparin in the polymer matrix was homogeneous than freeze-thawing gel, due to the fast gelling affect of PVA which takes approximately 10 min for gel formation. The formation of intra- and intermolecular hydrogen bonds between PVA chains has trapped the heparin inside, suppressing the phase separation between PVA and heparin. Furthermore, evenly distribution of heparin induced the formation of heparin and PVA molecular complex, which brought the sustained release of heparin from the PVA despite the high swelling ratio. Our results show that it is possible to prepare a PVA-heparin hybrid gel which can be applied as an effective material for an antithrombotic system without using any chemical agent.

Preparation of a Nanoscaled Poly(vinyl alcohol)/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery

Our previous research showed that poly(vinyl alcohol) (PVA) nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps) prepared by high hydrostatic pressurization (980 MPa), which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity.