Modulation of chondrocyte phenotype via baculovirus-mediated growth factor expression.

Baculovirus has emerged as a new gene delivery vector thanks to a number of advantages. This study demonstrated that baculovirus conferred efficient gene delivery and mediated expression of growth factors (TGF-beta1, IGF-1 and BMP-2) to therapeutic levels in rabbit chondrocytes. Interestingly, the cellular response to growth factor stimulation was dependent on the cell passage. The highly de-differentiated passage 5 (P5) chondrocytes failed to respond to the stimulation by either growth factor. The de-differentiated P3 cells also failed to maintain the chondrocyte phenotype, but baculovirus-mediated BMP-2 expression remarkably reversed the de-differentiation and enhanced the aggrecan and collagen II production in 2D and 3D cultures, as evidenced by cell morphology, histological staining and gene expression analyses. Baculovirus-mediated TGF-beta1 expression modestly enhanced the cartilage-specific matrix production, although to a lesser extent. Intriguingly, IGF-1, a well-known chondroinductive protein, failed to stimulate the P3 cells likely due to the loss of IGF-1 receptor expression. In summary, this study proved for the first time the potentials of baculovirus in modulating the differentiation status of chondrocytes in the context of cartilage tissue engineering, but also highlighted the importance of selecting appropriate cell passage and growth factor for genetic manipulation.

Preparation of nanoparticles composed of chitosan/poly-gamma-glutamic acid and evaluation of their permeability through Caco-2 cells.

In this study, a novel nanoparticle system for paracellular transport was prepared using a simple and mild ionic-gelation method upon addition of a poly-gamma-glutamic acid (gamma-PGA) solution into a low-molecular-weight chitosan (low-MW CS) solution. The particle size and the zeta potential value of the prepared nanoparticles can be controlled by their constituted compositions. The results obtained by the TEM and AFM examinations showed that the morphology of the prepared nanoparticles was spherical in shape. Evaluation of the prepared nanoparticles in enhancing intestinal paracellular transport was investigated in vitro in Caco-2 cell monolayers. It was found that the nanoparticles with CS dominated on the surfaces could effectively reduce the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers. After removal of the incubated nanoparticles, a gradual increase in TEER was noticed. The confocal laser scanning microscopy observations confirmed that the nanoparticles with CS dominated on the surface were able to open the tight junctions between Caco-2 cells and allowed transport of the nanoparticles via the paracellular pathways.

Physically crosslinked alginate/N,O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs.

In the study, a complex composed of alginate blended with a water-soluble chitosan (N,O-carboxymethyl chitosan, NOCC) was prepared to form microencapsulated beads by dropping aqueous alginate-NOCC into a Ca(2+) solution. These microencapsulated beads were evaluated as a pH-sensitive system for delivery of a model protein drug (bovine serum albumin, BSA). The main advantage of this system is that all procedures used were performed in aqueous medium at neutral environment, which may preserve the bioactivity of protein drugs. The swelling characteristics of these hydrogel beads at distinct compositions as a function of pH values were investigated. It was found that the test beads with an alginate-to-NOCC weight ratio of 1:1 had a better swelling characteristic among all studied groups. With increasing the total concentration of alginate-NOCC, the effective crosslinking density of test beads increased significantly and a greater amount of drug was entrapped in the polymer chains (up to 77%). The swelling ratios of all test groups were approximately the same ( approximately 3.0) at pH 1.2. At pH 7.4, with increasing the total concentration of alginate-NOCC, the swelling ratios of test beads increased significantly (20.0-40.0), due to a larger swelling force created by the electrostatic repulsion between the ionized acid groups (-COO(-)). It was shown that BSA was uniformly distributed in all test beads. At pH 1.2, retention of BSA in hydrogels may be improved by rinsing test beads with acetone (the amount of BSA released was below 15%). At pH 7.4, the amounts of BSA released increased significantly ( approximately 80%) as compared to those released at pH 1.2. With increasing the total concentration of alginate-NOCC, the release of encapsulated proteins was slower. Thus, the calcium-alginate-NOCC beads with distinct total concentrations developed in the study may be used as a potential system for oral delivery of protein drugs to different regions of the intestinal tract.

Novel method using a temperature-sensitive polymer (methylcellulose) to thermally gel aqueous alginate as a pH-sensitive hydrogel.

A novel method using a temperature-sensitive polymer (methylcellulose) to thermally gel aqueous alginate blended with distinct salts (CaCl2, Na2HPO4, or NaCl), as a pH-sensitive hydrogel was developed for protein drug delivery. It was noted that the salts blended in hydrogels may affect the structures of an entangled network of methylcellulose and alginate and have an effect on their swelling characteristics. The methylcellulose/alginate hydrogel blended with 0.7 M NaCl (with a gelation temperature of 32 degrees C) demonstrated excellent pH sensitivity and was selected for the study of release profiles of a model protein drug (bovine serum albumin, BSA). In the preparation of drug-loaded hydrogels, BSA was well-mixed to the dissolved aqueous methylcellulose/alginate blended with salts at 4 degrees C and then gelled by elevating the temperature to 37 degrees C. This drug-loading procedure in aqueous environment at low temperature may minimize degradation of the protein drug while achieving a high loading efficiency (95-98%). The amount of BSA released from test hydrogels was a function of the amount of alginate used in the hydrogels. The amount of BSA released at pH 1.2 from the test hydrogel with 2.5% alginate was relatively low (20%), while that released at pH 7.4 increased significantly (86%). In conclusion, the methylcellulose/alginate hydrogel blended with NaCl could be a suitable carrier for site-specific protein drug delivery in the intestine.