聚吡咯-氫氧基磷灰石-膠原蛋白複合材料 3-D 導電支架之製備及其在骨組織工程之應用

摘要複合型材料細胞支架可結合多種材料之特性，以提供組織工程中功能性的細胞生長環境。本研究先將聚吡咯 (Polypyrrole, PPy) 經由氧化還原法聚合到氫氧基磷灰石 (Hydroxyapatite, HAp) 表面上形成導電粉末材料 PPy-HAp。並以掃描式電子顯微鏡 (SEM) 觀察微結構，X 光粉末繞射儀 (XRD) 分析晶格結構，傅立葉轉換紅外線光譜儀 (FTIR) 及熱重分析儀 (TGA) 確認各材料在複合材料中的存在，並利用四點探針儀確認其具有導電度。接著再以大鼠第一型膠原蛋白與 PPy-HAp 進行共混，冷凍乾燥成型後，再以 EDC/NHS 交聯出三維 PHC 細胞支架。以四點探針儀量確認其具有導電度，SEM 可觀察支架具有多孔微結構，液體置換法計算出支架孔洞率為 95~97 %，TNBS 測量到交聯率為 15~23 %，膠原蛋白酶測試到 28 天降解率為 13~41 %，以 TGA 確認各材料在複合材料中的存在，進一步以體外和活體內測試得知 PHC 細胞支架具有良好的生物相容性。細胞增生實驗結果得知 0.5 PHC 細胞支架可以使人類脂肪間葉幹細胞穩定增生，並且可以藉由其導電能力，提升 18~30 % 細胞增生的能力，硬骨分化實驗結果得知 0.5 PHC 可以藉由電刺激來提升 ALP 活性、鈣生成量、ALP、RUNX2、BMP2 基因表現量讓 hASC 達到硬骨分化的效果。

關鍵字

複合支架；氫氧基磷灰石；聚吡咯；電刺激；硬骨分化；脂肪間葉幹細胞；膠原蛋白

並列摘要

Abstract The multifunctional scaffolds, which combining characteristics of a variety of materials are increasingly play an important role in tissue engineering. In this research, we prepared polyprrole-hydroxyapatite (PPy-HAp) composite by oxidative polymerization, the microstructure and lattice structure of the composite were analyzed by scanning electron microscope (SEM) and X-ray powder diffraction (XRD), repectively. Fourier transform infrared spectrometer (FTIR) analysis was use to confirm that both PPy and HAp existed in the composite material. The conductance of the composite material was measure by Four-point probe analyzer, and use thermogravimetry analyzer (TGA) to confirme the components of PPy-HAp. Then extracted the type I collagen from wistar rat-tail tendons and mixed with PPy-HAp powder, after that, the mixture were cast by freezer and freeze drying to form the PHC scaffolds. After EDC/NHS crosslinked, Four-point probe analyzer and SEM were analyzed the conductance and microstructure of scaffolds, repectively. The porosity was calculated by liquid replacement law, and TNBS reagent was estimated the extent of crosslinking, and collagenase solution for degradation rate test and TGA analysis components in the scaffolds. By series of analysis, the results shows that the PHC scaffold is a degradable, has conductive capacity and porous- rech structure. further more, by in vitro and in vivo of cell toxicity test was informed that the PHC scaffold has good biocompatibility, and alamarBlue® test also indicated that 0.5 PHC scaffold can provided hASC a good growth environment, and can upgrade cell movement and proliferate ability by electrical stimulation. In the end, the osteogenic differentiation experiment found that under the electrical stimulation, 0.5 PHC can provide more osteogenic differentiation level without additional osteogenic differentiation reagent, and after 34 days of experiment period, the porous structure of scaffold can be still maintained and let cells stability growth in the scaffold.

並列關鍵字

electrical stimulation ； composite scaffold ； polypyrrole ； collagen ； adipose derived mesenchymal stem cells ； osteogenic differentiation ； hydroxyapatite

參考文獻

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聚吡咯-氫氧基磷灰石-膠原蛋白複合材料 3-D 導電支架之製備及其在骨組織工程之應用

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