Sulfation patterns of exogenous chondroitin sulfate affect chondrogenic differentiation of ATDC5 cells

Chondroitin sulfate (CS) has been used in cartilage tissue engineering techniques as a positive modulator of scaffolds. CS is a linear polysaccharide consisting of variously sulfated repeating disaccharides. The sulfation patterns of CS are closely related to their biological functions, but only monosulfated CS has been applied to scaffolds. In this study, we investigated the effects of various sulfation patterns of CS on chondrogenic differentiation using ATDC5 chondroprogenitor cells.

Disaccharide composition analysis of CS produced by ATDC5 cells at various differentiation steps was performed using high-performance liquid chromatography. ATDC5 cells were cultured with exogenously added, variously sulfated CS. Cell proliferation was analyzed by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) assay. Extracellular matrix production was evaluated by Alcian blue staining. Alkaline phosphatase (ALP) activity was evaluated using an ALP assay kit. Expression of chondrogenic markers was evaluated by real-time reverse transcription polymerase chain reaction (RT-PCR) or an enzyme-linked immunosorbent assay (ELISA) using a Type II Collagen Detection kit.

The major components of CS produced by ATDC5 cells were 4-O-monosulfated disaccharides throughout chondrogenic differentiation. Low proportions of 4,6-O-disulfated disaccharides were also detected. Compared to the control group, which did not contain GAGs, the WST-8 assay indicated fewer viable cells when treated with CS-E, which are rich in 4,6-O-disulfated disaccharides. CS-E significantly enhanced Alcian blue staining in a dose-dependent manner and decreased ALP activity after 21 days of culture. Real-time RT-PCR showed that CS-E significantly enhanced all chondrogenic markers, col2a1, aggrecan, and sox9, either at day 4 or day 14 of culture. The results of ELISA analysis confirmed that CS-E significantly enhanced the production of type II collagen.

ATDC5 cells produced four different monosulfated or disulfated disaccharides in their extracellular matrices. The sulfation patterns of exogenously added CS affected chondrogenic differentiation of ATDC5 cells. In particular, CS-E rich in disulfated disaccharides significantly promoted chondrogenic differentiation of ATDC5 cells. Thus, CS containing this disulfated structure may be a useful scaffold component for enhancing chondrogenesis in cartilage tissue engineering.