The effect of cross-linked hyaluronan hydrogel on chondrogenic differentiation of human adipose-derived stem cells

• Main Authors: Chien-Mei Chang, 張倩玫
• Other Authors: Mei-Ling Ho
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/92t2e2

碩士 === 高雄醫學大學 === 醫學系生理學科碩士班 === 103 === Articular cartilage is a flexible connective tissue that lacks of blood vessels, nerves and lymph system, resulting in limited ability for self-repair. Therefore, cartilage-tissue engineering has become one of the potential regenerative therapies. Human adipose derived stem cells (ADSCs) as the cell source were preferred due to the harvest rate and multipotent differential potential. Another important factor in cartilage engineering is the signaling which induces the chondrogenic differentiation. Hyaluronic acid (HA) is the major extracellular matrix (ECM) component of cartilage. Our previous study indicated that HA-microenvironment enhanced chondrogenesis of ADSCs. The ECM stiffness has been reported to influence stem cells fate, including proliferation and differentiation. Based on the previous finding, the aim of this study was to develop HA based hydrogel with different matrix stiffness and test the cytocompatibility of the hydrogel on ADSCs. HA 1% (w/v) was first modified with different amount of methacrylic anhydride, and then formed a cross-linked hydrogel (CLMH) by exposed to UV light. The Young’s modulus of CLMH was evaluated under unconfined compression with a texture analyzer. Cell survival was measured by LIVE/DEAD&;#174; Viability/Cytotoxicity Kit, and MTS assay. The mRNA levels of chondrogenic differentiation genes were measured by real-time RT-PCR. The Young’s modulus of CLMH showed that the matrix stiffness of CLMH increased with higher modification rate. The results of swelling ratio analysis showed that the CLMH exhibited different swollen behaviors with changes in the modification rate. The MTS assay were used to evaluate the viability of human ADSCs encapsulated inside the gel, the result showed that CLMH increased the cell viability on day 5 comparing to day 1. Live/Dead assay also showed that the ADSCs remain survived in all CLMH groups after 7 days culture. This study showed that increasing the modification rate of HA hydrogel results in raising the construct stiffness. Lots of ADSCs showed alive in hydrogels at day 7. Collagen type II and aggrecan were used as marker to evaluate the chondrogenic differentiation of hADSCs, and the results showed that upregulation of these two markers with higher stiffness of CLMH. Long-term culture to confirm the ECM synthesis in the construct is needed.