Chitosan-based Hyaluronic Acid Hybrid Polymer Fiber for a Novel Three Dimensionalscaffold in Cartilage Tissue Engineering

[Introduction] In cartilage tissue enginnering, a number of studies have shown the importance of selecting the appropriate biomaterials as scaffolds for chondrocyte attachment, for supporting its proliferation and for its synthesis of extra-cellular matrix (ECM). Although a variety of biomaterials, including naturally occurring and synthetic, have been introduced as potential scaffolds for cartilage repair, we believe that the ideal cell-carrier substance should be one which closely mimics the natural environment in the cartilage-specific ECM. To mimic the ECM, we focused on the application of hyaluronic acid to a scaffold material. In this study, we hypothesized that chitosan-based hyaluronic acid hybrid biomaterial, which we have originally developed, could provide excellent support for chondrocyte adhesion and the synthesis of ECM. The objectives of this study are to quantity the degree of rabbit chondrocyte attachment onto the chitosan-based hyaluronic acid hybrid polymer fibers and to evaluate the synthesis of ECM in the new material. [Materials and Methods] Polymer fiber preparation: Chitosan polymer fibers (Chitosan group) and chitosan-based hyaluronic acid hybrid polymer fibers (HA 0.04% group, Chitosan coated with hyaluronic acid 0.04%; HA 0.07% group, Chitosan coated hyaluronic acid 0.07%) were originally developed in our laboratory by the wetspinning method. The diameter of each fiber was 0.03 mm. Cell adhesion study: Chondrocytes were isolated from the articular surfaces of a Japanese white rabbit (2.0 kg) using a method described by Ochi, et al. The fibrous samples were cut into 10 mm pieces and packed in Teflon tubes (25mm length, 4.8mm inner diameter) and then 0.1ml of chondrocyte suspension containing 0.5 x 10cells was loaded on the column at room temperature. The cells were allowed to adhere in a humidified incubator (37? and 5% CO2) for 1 hour. Each column was gently rinsed with 1ml of 1M phosphate-buffered saline, and the number of unattached cells in the rinsed solution was quantified by microscopic observation using a hemocytometer. In vitro chondrocyte culture: The polymer fiber sheets were stacked in a perpendicular pattern with six layers (8 x 8 mm, 1mm thickness). These three dimensional (3-D) scaffolds were used for chondrocyte culture and further investigations . Chondrocyte suspension containing 0.6 x 10cells was embedded on the scaffolds. RNA isolation and semiquantitative RT-PCR analysis: RNA was extracted from samples incubated for 14 days with the TRIspin method described by Reno et al. Semiquantitative RT-PCR analysis was performed as previously reported by Hart et al. with rabbit specific primer sets for type I, II collagen, aggrecan, and a housekeeping gene, glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Data were expressed as a normalized ratio by comparing the integrated density values for the genes in question with these values for GAPDH so as to yield a semiquantitative assessment of gene expression. Morphological observations and immunohistochemistry (IHC): Cell morphology in the scaffold was observed by light microscopy and scanning electron microscopy (SEM) after 14 days in culture. Immunohistochemical stain was performed to detect expression of the type II collagen production. Statistical analysis: The means and standard errors for all results were calculated. Statistical comparisons were performed using one-way ANOVA and Fisher’s PLSD tests. Differences were considered significant for p< .05. For comparison, parallel samples of n=5 were used for each group of polymer fibers. [Results] Cell adhesion study: Adhesivities of the chondrocytes were expressed by percentage of chondrocytes trapped in the column. The value in the HA 0.04% and HA 0.07% groups (95.1 ± 1.3%, and 90.5 ± 3.2%, respectively) were significantly higher than that in the Chitosan group (vs 79.3 ± 2.2%, p < .05). Semiquantitative RT-PCR analysis: As shown in Table 1, culture samples at 14 days in all groups showed the mRNA expression of type I and II collagens. There were no statistically significant differences in the mRNA levels among the three groups. On the other hand, the HA 0.07% group resulted in a statistically significant increase in mRNA levels of aggrecan as compared to the other groups (p < .05). Morphological observations and IHC: Phase contrast micrographs showed the adhesion of the chondrocytes to the polymer fibers. Light micrographs demonstrated the cell proliferation and rich extra-cellular matrix synthesis on the 3-D scaffold (Fig. 2A). Scanning electron micrographs clearly revealed the cell proliferation with characteristic round morphology of the chondrocytes and the dense fiber of the type II collagen produced by the chondrocytes in the 3-D scaffold (Fig. 2B). Immunohistochemical staining with anti-type II collagen antibody demonstrated rich production of the type II collagen in the ECM from the chondrocytes. Table1. Mean normalized ratio (experimental integrated density / GAPDH integrated density) of mRNA for ECM molecules Group Type II collagen Aggrecan Type I collagen Chitosan 1.51 ± 0.07 No detection 0.45 ± 0.04 HA 0.04% 1.59 ± 0.07 1.07 ± 0.17 0.60 ± 0.11 HA 0.07% 1.38 ± 0.12 1.59 ± 0.09* 0.42 ± 0.11 *p < .05 vs Chitosan and HA 0.04% groups