探討交聯型玻尿酸水膠對於人類脂肪幹細胞軟骨分化的影響

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

張倩玫(Chien-Mei Chang)

指導教授 ： 何美泠

高雄醫學大學/醫學院/醫學系生理學科碩士班/碩士(2015年)

摘要

關節軟骨本身無血管及神經組織，自我修復的能力差。因此，以細胞為基礎的關節軟骨組織工程已經成為治療軟骨再生的策略之一。 人類脂肪幹細胞已知為具有價值的細胞來源，在於其多種分化潛能，以及從脂肪組織分離下來的幹細胞含量比例高。而引導人類脂肪幹細胞走向軟骨化的條件就成了重要因素。 玻尿酸為關節軟骨主要胞外基質成分之一，我們先前研究指出人類脂肪幹細胞在富含玻尿酸微環境的條件下，能被引導走向軟骨化。過去的報告也指出胞外基質所形成的硬度會可以誘導幹細胞軟骨化，以玻尿酸為基礎所合成的基材，在哪一範圍的硬度最適合人類脂肪幹細胞走向軟骨化尚未清楚。因此本研究假設玻尿酸經由不同修飾程度交聯的水膠會誘導人類脂肪幹細胞與胞外基質的合成，並引導脂肪幹細胞走向軟骨化，研究目的主要評估經由不同修飾程度交聯而成玻尿酸水膠是否會影響人類脂肪幹細胞軟骨化的程度。 材料使用 1% (w/v)的玻尿酸利用化合物methacrylic anhydride以不同比例修飾，經紫外光交聯成不同比例的水膠，並利用物性測試儀測量不同修飾程度水膠；另外，測量每一不同修飾程度水膠的吸水膨潤比。接著進一步評估不同修飾程度的水膠對人類脂肪幹細胞的細胞相容性以及軟骨化基因表現的影響。由應力測試結果得知，水膠的楊氏係數隨著修飾程度的增加而增加，顯示修飾度越高，水膠的硬度也增加；反觀吸水膨潤比中，修飾程度的增加，其吸水膨潤比的比例下降。hADSCs包覆於水膠中培養5-7天後測試其生物相容性，在MTS assay結果當中，修飾程度較低的組別在第5天的時候細胞存活率有明顯的下降；利用Live/Dead staining，結果顯示細胞在培養7天後仍然維持存活的現象。在基因表現部份我們評估軟骨化基因collagen type II及 aggrecan，結果發現在第5天，隨著水膠硬度的增加， collagen type II及 aggrecan有顯著上升的表現量。本實驗結果顯示，由不同修飾程度交聯而成的水膠的確會影響水膠本身的物理特性;而在細胞相容性評估中也顯示人類脂肪幹細胞在合成的水膠中可維持其細胞存活率; 基因表現顯示，越高修飾度的玻尿酸水膠，其軟骨化基因有較高表現量，而未來需進一步藉由組織切片染色以評估幹細胞在水膠內長期培養下，是否會受到硬度的影響而影響軟骨胞外基質的合成。

關鍵字

軟骨 ； 組織工程 ； 玻尿酸 ； 脂肪幹細胞 ； 水膠 ； 基材硬度

並列摘要

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® 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.

並列關鍵字

cartilage ； tissue engineering ； hyaluronic acid ； adipose derived stem cell ； hydrogel ； matrix stiffness

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