Purification of free chondroitin sulphate chains and their effects on chondrogenesis of BMSCs

Feng, Jingyi 2025. Purification of free chondroitin sulphate chains and their effects on chondrogenesis of BMSCs. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Abstract Background Cartilage defects can occur in various anatomical locations. Since articular cartilage has limited repair capacity, tissue engineering aims to restore it by mimicking its mature cell and matrix structure. Chondroitin sulphate (CS) has shown promise due to its role in replicating native cartilage extracellular matrix (ECM) and supporting chondrocyte function or stem cell chondrogenesis. The aims of this project were to develop and optimise strategy for CS purification from bovine articular cartilage, and to investigate the effects of CS on chondrocyte phenotype and bovine bone marrow stem cell (bBMSCs) chondrogenesis. Methods Bovine metacarpophalangeal joint cartilage was employed to extract proteoglycans by 4M guanidine-HCl and density gradient centrifugation. Alkaline treatment using sodium borohydride (NaBH4), released CS from aggrecan core protein. Further purification by anion-exchange chromatography and size-exclusion chromatography was conducted to separate CS from other molecules. Purified CS was analysed by glyco-analyses and proton nuclear magnetic resonance (1H-NMR) to determine the sulphation pattern. The effects of different concentrations of CS on chondrocyte phenotype was determined in 2D and 3D agarose cultures systems. This was extended to study the effect of CS chains on the chondrogenesis of bBMSC in monolayer, 2% agarose constructs and the TranswellTM system. RT-PCR was employed to examine chondrogenic gene marker expression, including col2a1, sox-9 and acan. Immunohistochemical staining was utilised to evaluate ECM composition. Results CS was isolated from bovine cartilage with purity of 96.47±1.90% (chondroitinase ABC digestion method) and 87% (carbazole reaction) where CS-4 was the predominant constituent, comprising 78.3% of purified CS. Purified CS facilitated chondrocyte sox-9 gene expression significantly (10-fold, p<0.001) on 0.1μg/cm2 CS-coated substrate. Chondrogenesis of bBMSCs was improved with the addition of 100μg/ml CS in chondrogenic medium by enhancing ECM deposition and the expression of sox-9 on both monolayer (2.2-fold, p<0.05) and the TranswellTM system (3.7-fold, p<0.001). Conclusion This thesis optimised a methodology to purify high-quality CS (predominantly CS-4) from bovine cartilage and demonstrated its role in enhancing ECM deposition and sox-9 expression in chondrocytes and BMSCs. The findings highlight CS as a promising biochemical cue for cartilage tissue engineering and regenerative medicine.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	20 January 2026
Last Modified:	20 Jan 2026 14:59
URI:	https://orca.cardiff.ac.uk/id/eprint/184063

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