光交聯聚乙二醇共聚酯類水膠做為間葉幹細胞誘導成骨組織支架之研究

The study of photocrosslinked PEG-based polyester hydrogel as scaffold for mesenchymal stem cell-induced osteogenesis

張展昆(Chan-Kun Chang)

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/碩士(2010年)

https://doi.org/10.6843/NTHU.2010.00171

摘要

本實驗在聚乙二醇(polyethylene glycol, PEG)兩端共聚合上己內酯(ε-caprolactone)，並於其末端進行烯基化(acrylation)，使其分子量2500的高分子鏈上具有碳-碳雙鍵成為PCL-PEG-PCL-DA (PEC-DA)。此共聚物溶於水後經UV照射可形成光交聯水膠。成品和烯基化的聚乙二醇-聚乳酸(PLA-PEG-PLA-DA, PEL-DA)、烯基化的聚乙二醇(PEG-DA)兩種光交聯水膠比較。將應用這些水膠包埋臍帶血間葉幹細胞，同時誘導其分化成骨母細胞，以期達到骨生成之目的。使用三種材料10%(w/v)、20%(w/v)濃度的水膠做比較，結果顯示水膠的膨潤率隨水膠濃度增加而降低、交聯度和壓縮應力隨水膠濃度增加而增加。透過熱性質分析證實其交聯程度隨水膠濃度增加而增加。PEG-DA與PEC-DA水膠在磷酸緩衝液中的降解情形類似，28天後最多降解30%，pH最低降到6.5；PEL-DA水膠28天後最多降解40%，pH最低降到3.7。使用300 ppm的光起始劑在細胞包埋過程中能使細胞受最少影響。三種材料各濃度包埋人類間葉幹細胞的包埋率在90%以上、一天後的細胞存活率在90%以上。28天的誘導培養期間，首次觀察到細胞在PEL-DA 20%水膠中能伸展，具最佳細胞形態；細胞在PEC-DA水膠中也可伸展但較不明顯；在PEG-DA水膠中則無。包埋細胞後PEL-DA水膠降解最快，無法維持28天。各材料20%水膠內的細胞到28天後細胞存活率較10%水膠低。膠原蛋白一型、鹼性磷酸酶基因表現和細胞內鹼性磷酸酶活性的測定都可確認在各水膠中的間葉幹細胞誘導成骨母細胞。在PEC-DA 10%水膠內的細胞誘導成骨母細胞的表現最好。

關鍵字

光交聯水膠 ； 聚乙二醇共聚酯類 ； 己內酯 ； 臍帶血間葉幹細胞 ； 骨生成

並列摘要

In this research, polyethylene glycol (PEG) was copolymerized with ε-caprolactone and acrylated to obtain C-C double bond at both ends. The product PEC-PEG-PEC-DA(PEC-DA) was able to form hydrogel upon UV irradiation. This hydrogel was compared with those formed by PEG and PEG-PLA(PEL) as scaffolds to encapsulate umbilical mesenchymal stem cells and to induce toward osteogenesis. In the study, 10%(w/v) and 20%(w/v) hydrogels of three materials were prepared. Swelling ratio decreased as hydrogel concentration increased. Meanwhile, thermodiagrams showed that crosslinking density increased as hydrogel concentration increased. Monitoring scaffold degradation in phosphate buffer showed that PEG-DA and PEC-DA have similar weight loss about 30% on day 28 and the pH decreased to 6.5. PEL-DA hydrogel maximum weight loss was about 40% and the pH decreased to 3.7. The photoinitiator used at 300 ppm showed little impact on encapsulated cell and encapsulation rate of cell was above 90%. The relative viabilities of cell after 1 day encapsulation in hydrogels of three scaffold materials were above 90%. During 28 days induction culture, we observed cell spread within PEL-DA 20% hydrogel. Cells also spread within PEC-DA hydrogels to a less degree, but not in PEG-DA hydrogels. After cell encapsulation, PEL-DA hydrogels degrade fastest and could not last for 28 days. Cell within 20% hydrogels has less viability then 10% hydrogels of three materials. Assays for collagen type I gene expression, alkaline phosphatase (ALP) gene expression and ALP activity confirm osteogenesis. PEC-DA 10% hydrogel is the best scaffold material for cell encapsulation in this study.

並列關鍵字

photocrosslinked hydrogel ； PEG-based polyester ； epsilon-caprolactone ； umbilical mesenchymal stem cell ； osteogenesis

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