Abstract
Generally, scaffold is used as cell carrier for tissue engineering. Co-conjugating CSC/DS to chitosan is a potential scaffold material for cartilage tissue engineering. The aim of this study is to co-conjugate multiple glycosaminoglycans (GAGs) to 2D chitosan membranes and 3D porous chitosan scaffolds, hoping to more intimately mimic the natural extracellular matrix (ECM) for culturing chondrocyte in vitro and cartilage tissue engineering and further investigating the roles of GAGs in regulating ECM production and related gene expression. The chondroitin-4-sulfate (CSA), chondroitin-6-sulfate (CSC), dermatan sulfate (DS), and heparin were co-immobilized to chitosan membranes for maintaining the differentiation of monolayer chondrocytes culture and improving the ECM production in vitro. With the aid of 4-factor, 2-level 24-1 fractional factorial design, we formulated 8 GAG/chitosan compositions (groups N1-N8) which were found to profoundly influence chondrocyte behavior. Within the level range between -1 and +1, low levels of CSA (code -1; 2.6 mg), was desired for collagen production but undesired for cell proliferation. High level CSC (code +1; 1.3 mg) was favorable for GAG production but not for cell proliferation. Conversely high level DS (code +1; 0.13 mg) and heparin (code +1; 0.33 mg) were desired for cell proliferation but undesired for GAG and collagen production. Additionally, the interactions between GAG species affected collagen and GAG production too. Among the 8 GAGs/chitosan membranes, the ones with low CSA and heparin levels (N1 and N4) led to proper chondrocyte phenotype, as judged by chondrocyte-like morphology, modest cell expansion, higher GAG and collagen production and proper cartilage marker gene expression. This result indicates the potential of N1 and N4 GAGs formulation for culturing chondrocyte in vitro. Since simultaneous quantification of 4 different GAGs in the 3D scaffolds is technically difficult, the number of GAG species was reduced to 2 (CSC and DS) to simplify the experimental design. To optimize the CSC/DS formulation and investigate the roles of CSC and DS in cartilage formation, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations. Conjugating CSC or DS did not affect the physical properties of scaffolds, cell adhesion and proliferation, but impacted collagen and GAGs production. Within the experimental range, the GAGs and collagen production were found to positively correlate with amount of CSC on scaffold, but were negatively correlated with that of DS. According to the analysis by RSM, co-conjugating CSC 2.8 mg/scaffold and DS 10 μg/scaffold to chitosan scaffold is optimal formulation for cartilage tissue engineering. Further result illustrates that the minor DS on the CSC/DS/chitosan scaffolds was needed for improving the collagen and GAGs production. Moreover, the optimal formulation of CSC/DS/chitosan scaffolds up-regulated the gene expression of BMP2、TGF-β1、Ihh、PTHrP、Sox9、aggrecan、collagen II、TIMP3 and MMP13, but down-regulated the decorin and BMP4 expression. These results indicate the co-conjugating CSC/DS to chitosan scaffold modulated ECM production and related gene expression in vitro by regulating the BMP、TGF-β and Hedgehog signaling transduction pathway.