Evaluation of Chondrogenesis in Collagen/Chitosan/Glycosaminoglycan Scaffolds for Cartilage Tissue Engineering

Yoo, Jae Ho; Lee, Myung Chul; Lee, Jong Eun; Jeon, Kyung Chull; Kim, Yong Min; Jung, Mi Young; Ahn, Hyun Jeong; Seong, Sang Cheol; Choi, Sun Jong; Lee, Ji Ho

Abstract

PURPOSE
The scaffold is essential for cartilage tissue engineering. Collagen, chitosan, or glycosaminoglycan( GAG) has separately been proposed as in vitro scaffolds. However, the influence of collagen:chitosanchondroitin sulfate(Col:Chi-CS) composites on cell behavior has not yet been thoroughly examined. Therefore, the aim of this study is to develop a novel Col:Chi-CS blended scaffold that binds covalently with CS for cartilage tissue engineering.
MATERIALS AND METHODS
The behavior of rabbit chondrocytes seeded in vitro into collagen/chitosan/GAG scaffolds with different chitosan contents (collagen:chitosan ratios of 20:1, 5:1, and 1.25:1) was investigated. The porous scaffolds containing collagen and chitosan were fabricated by using a freeze drying technique and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide(EDC) in the presence of CS. The physicochemical/ mechanical properties of scaffolds were determined by analyzing scanning electron microscopy, compression modulus, immobilized GAG content, and water-binding capacity. Rabbit chondrocytes seeded onto these scaffolds were cultured for 1, 3, 7, and 14 days. The cell proliferation rate was evaluated with 3H-thymidine uptake and total GAG content assay was done via DMB assay using ELISA method. For the histological assessment of extracellular matrix, staining with safranin-O/fast green and immunohistochemistry were used.
RESULTS
Scanning electron microscope(SEM) views of the scaffolds showed that all three had interconnected pores of mean diameter 164, 353, and 567 micrometer at collagen:chitosan ratios of 20:1, 5:1, and 1.25:1. GAG was covalently bound onto these scaffolds at 6.4%(w/w) in all three cases, i.e., regardless of chitosan content. However, increased chitosan content resulted in enhanced mechanical properties and increased pore size. Biochemical analysis of these scaffolds showed that proliferation rate and GAG synthesis increased with time, and this became most significant in the collagen:chitosan(20:1)-CS scaffold on day 14. The histology of the cell-seeded constructs showed a significantly higher percentage of cells with spherical morphology, which is specific to mature chondrocyte, especially in the collagen:chitosan(20:1)-CS scaffold at each time point. This finding was consistent with the observation that the pericellular matrix was stained positive for proteoglycans and type II collagen on day 14.
CONCLUSION
The novel collagen:chitosan(20:1)-CS scaffold seems to be a useful carrier material for cartilage tissue engineering.