Kuen Yong Lee

Degradation of Partially Oxidized Alginate and Its Potential Application for Tissue Engineering

Alginate has been widely used in a variety of biomedical applications including drug delivery and cell transplantation. However, alginate itself has a very slow degradation rate, and its gels degrade in an uncontrollable manner, releasing high molecular weight strands that may have difficulty being cleared from the body. We hypothesized that the periodate oxidation of alginate, which cleaves the carbon‐carbon bond of the cis‐diol group in the uronate residue and alters the chain conformation, would result in promoting the hydrolysis of alginate in aqueous solutions. Alginate, oxidized to a low extent (∼5%), degraded with a rate depending on the pH and temperature of the solution. This polymer was still capable of being ionically cross‐linked with calcium ions to form gels, which degraded within 9 days in PBS solution. Finally, the use of these degradable alginate‐derived hydrogels greatly improved cartilage‐like tissue formation in vivo, as compared to alginate hydrogels.

Blood compatibility and biodegradability of partially N-acylated chitosan derivatives

Chitosan was selectively N-acylated with various carboxylic anhydrides, e.g., acetic, propionic, n-butyric, n-valeric and n-hexanoic anhydrides, in the presence of methanol. The degree of N-acylation of about 20-50% was obtainable without occurrence of gelation by using carboxylic anhydrides of 0.3-1.2 mol per glucosamine residue. In vitro blood compatibility tests of N-acyl chitosans were performed by rheological measurement, blood clotting test and scanning electron microscopic observation for human blood and plasma protein. The rheological measurement of coagulation of plasma protein, considering the shear flow effect of blood, gave precise and quantitative results compared with other methods. N-Acyl chitosans showed more blood compatible properties than N-acetyl chitosan and, in particular, N-hexanoyl chitosan was the most compatible. Enzymatic degradation was also investigated by adding a lysozyme solution to the N-acyl chitosan solution and film, incubating at 37 degrees C. N-Acyl chitosans had as high a susceptibility to lysozyme as N-acetyl chitosans. It was considered that the amount of derivatized groups and the physical form of N-acyl chitosans contributed to biodegradability. The molecular weight (Mw) of the material liberated from the N-acyl chitosan film by the action of lysozyme was 2 x 10(4) - 10 x 10(4).

Preparation of chitosan self-aggregates as a gene delivery system

Hydrophobically modified chitosan containing 5.1 deoxycholic acid groups per 100 anhydroglucose units was synthesized by an EDC-mediated coupling reaction. Formation and characteristics of self-aggregates of hydrophobically modified chitosan were studied by fluorescence spectroscopy and dynamic light scattering method. The critical aggregation concentration (cac) of the self-aggregate was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. The cac value in PBS solution (pH 7.2) was 1.7x10(-2) mg/ml. Mean diameter of self-aggregates in PBS solution (pH 7.2) was 162 +/- 18 nm with an unimodal size distribution. Charge complex formation between self-aggregates and plasmid DNA was confirmed by electrophoresis on an agarose gel. Migration of DNA on an agarose gel was completely retarded above a charge ratio ( +/-) of 4/1 at pH 7.2. The free DNA dissociated from the complexes was observed by electrophoresis above pH 8.0 at a fixed charge ratio of 4/1. An efficient of COS-1 cells was achieved by self-aggregates/DNA complexes.

Degradable and injectable poly(aldehyde guluronate) hydrogels for bone tissue engineering

Degradable and injectable hydrogels may be ideal for bone-tissue engineering, especially in the craniofacial region because of the ease of access for injection. Alginate hydrogels potentially could be used as injectable cell delivery vehicles, but they exhibit a limited range of mechanical properties and uncontrollable disintegration time. Therefore we synthesized new hydrogels, composed of poly(aldehyde guluronate) (PAG) and adipic acid dihydrazide, that have a wide range of mechanical stiffness and controllable degradation rate. MC3T3-E1 cells adhered and multiplied on PAG hydrogels in vitro. When primary rat calvarial osteoblasts were mixed with PAG hydrogels and subcutaneously injected into the backs of mice, mineralized bone tissues were formed 9 weeks following implantation. These hydrogels may find wide utility as an injectable delivery system for bone precursor cells as well as for other applications in tissue engineering.

Comparison of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in SCID mice

Therapeutic angiogenesis is a promising approach to treat patients with cardiovascular disease, and will likely be critical to engineering large tissues. Many growth factors have been found to play significant roles in angiogenesis, and vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the most extensively investigated angiogenic factors to date. However, the appropriate dose to obtain a desired response and the effectiveness of each factor, relative to the other, in promoting angiogenesis at a specific site in the body remains unclear. We have used alginate hydrogels as localized delivery vehicles for VEGF and bFGF, and compared the ability of these factors to promote new blood vessel formation in the subcutaneous tissue of severe combined immunodeficient (SCID) mice. We have found that the thickness of a granulation tissue layer formed around the gel and the number of blood vessels in the layer increased with the dose of VEGF in the gel, but the density of new blood vessels remained relatively constant. Sustained and localized delivery of bFGF from the gels, while similarly leading to an increase in the density of blood vessels in the granulation tissue, did not lead to as high of a blood vessel density as VEGF. The results of this study support previous studies demonstrating the utility of both VEGF and bFGF in promoting angiogenesis, and suggest VEGF is more appropriate for creating a dense bed of new blood vessels in this model.

Polyelectrolyte Complexes of Sodium Alginate with Chitosan or Its Derivatives for Microcapsules

Chitosan, a cationic polysaccharide, was heterogeneously deacetylated with a 47% sodium hydroxide solution and followed by a homogeneous reacetylation with acetic anhydrides to control the N-acetyl content of the chitosan having a similar molecular weight. The chitosans having different degrees of N-acetylation were complexed with sodium alginate, an anionic polysaccharide, and the formation behavior of polyelectrolyte complexes (PECs) was examined by the viscometry in various pH ranges. The maximum mixing ratio (Rmax) increased with a decrease in the degree of N-acetylation of the chitosan at the same pH, and with a decrease in pH at the same degree of N-acetylation. Similarly, N-acylated chitosans were also prepared. The N-acyl chitosans scarcely affected the formation behavior of PECs with sodium alginates. For the application of the PECs produced, the microencapsulation of a drug was performed and the release property of drug was tested. The microcapsules were prepared in one step by the extrusion of a solution of guaifenesin and sodium alginate into a solution containing calcium chloride and chitosan through interpolymeric ionic interactions. The drug release during the drug-loaded microcapsules storage in saline was found to depend on the pH where the microcapsules were formed and the kind of N-acyl groups introduced to the chitosan.

Structural changes and their effect on mechanical properties of silk fibroin/chitosan blends

Silk fibroin/chitosan blend films were prepared by the solvent casting method. Miscibility between silk fibroin and chitosan was examined by dynamic mechanical thermal analysis. Structural changes of silk fibroin by the addition of chitosan were investigated by IR spectroscopy. The conformational transition of silk fibroin from random coil form to β-sheet structure induced by blending with chitosan resulted in the increase of crystallinity and density of the blend films. The blend film containing 30 wt % chitosan exhibited a maximum increase in crystallinity and density. It was found that the tensile strength and initial tensile modulus of blend films were greatly enhanced with increasing the chitosan content and showed a maximum value at the composition of 30 wt % chitosan.

Effect of surface properties on the antithrombogenicity of silk fibroin/S-carboxymethyl kerateine blend films.

Polymeric blends of silk fibroin (SF) and S-carboxymethyl kerateine (SCMK) were prepared by the solvent casting method to study the effect of surface properties on the antithrombogenicity. The films of SF/SCMK showed better antithrombogenic properties than SF or SCMK alone. Among them, the film containing 50 wt% SCMK showed the best antithrombogenicity. When the SF/SCMK films were treated with methanol, the antithrombogenicity of the films was scarcely affected except the SF-rich ones. The enhanced antithrombogenic properties were explained in terms of polarity of the surface. The blend films showed an enhancement of polar contribution to surface free energy (gamma(P)S and polar stabilization energy (I(SW)). SF-rich films showed high gamma(P)S and I(SW) values when treated with methanol. This change of surface properties was considered to be due to the fact that the conformational transition from random coil structure to beta-structure of proteins may have affected the surface properties, especially the polar properties.

DSC studies on bound water in silk fibroin/S-carboxymethyl kerateine blend films

The physical state of water, absorbed in silk fibroin (SF)/S-carboxymethyl kerateine (SCMK) blend films, was studied by differential scanning calorimetry (DSC) to investigate the change of internal structures of the blend films. The amount of non-freezing bound water in the blend film was decreased with the addition of SCMK because of the change in the secondary structure of SF from random coil form to β-structure form. This transition could be also monitored by thermal analysis using DSC. The maximum change of the secondary structure was observed at a blend ratio of 50/50 (wt/wt).

Cell-interactive polymers for tissue engineering

Tissue engineering is one exciting approach to treat patients who need a new organ or tissue. A critical element in this approach is the polymer scaffold, as it provides a space for new tissue formation and mimics many roles of natural extracellular matrices. In this review, we describe several design parameters of polymer matrices that can significantly affect cellular behavior, as well as various polymers which are frequently used to date or potentially useful in many tissue engineering applications. Interactions between cells and polymer scaffolds, including specific receptor-ligand interactions, physical and degradation feature of the scaffolds, and delivery of soluble factors, should be considered in the design and tailoring of appropriate polymer matrices to be used in tissue engineering applications, as these interactions control the function and structure of engineered tissues.