無血清組成物於關節軟骨組織工程效應之探討

Study of the Effect of Serum-free Componets on Articular Cartilage Tissue Engineering

黃瑞彬(Jui-Pin Huang)

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/碩士(2008年)

https://doi.org/10.6843/NTHU.2008.00191

摘要

本研究探討提供適當的無血清組成物，以利提升工程軟骨在臨床的應用性。於此，我們以多孔洞性三維膠原蛋白二型支架為基礎，專注在生長因子以及硫酸軟骨素這兩類無血清成份的使用。首先，利用即時定量聚合酶連鎖反應(qPCR)分析方法，於無血清基礎下將7種常用的生長因子，使用二水準部份因子實驗設計法以及陡升步伐實驗設計法，發展一組最加強軟骨細胞重要的特徵基因，collagen II，表現量的組合，乃是含五種生長因子濃度分別為EGF 8.31 ng/ml, TGF-β1 20.00 ng/ml, BMP-2 28.85 ng/ml, PDGF-bb 8.11 ng/ml, VEGF 12.69 ng/ml的無血清培養基；我們更從生化合成分析、組織切片染色觀察，發現均優越於使用含胎牛血清培養基培養後的細胞，證實有利於提升工程軟骨品質。此外運用於誘導軟骨走向後的人類臍帶血間葉幹細胞(hUMSC)，發現亦能維持正常軟骨細胞特徵。研究另一部份，我們純化低分子量硫酸軟骨素C 型(LMWCSC)並以50 ug/ml的濃度添加於無血清培養基中，透過qPCR分析結果發現實驗組除了能夠加速提升軟骨特徵基因，亦能抑制分解基因的表現，推測LMWCSC對於提升工程軟骨修復的時程具有潛力，有利於軟骨組織醫學上醫療的開發。因此，搭配生長因子組合或LMWCSC的無血清培養基對於軟骨組織工程的發展將具有應用性。

關鍵字

軟骨組織工程 ； 軟骨細胞 ； 人類臍帶血間葉幹細胞 ； 膠原蛋白二型支架 ； 生長因子 ； 低分子量硫酸軟骨素C型 ； 無血清培養基 ； 實驗設計法 ； 即時定量聚合酶連鎖反應

並列摘要

This study aimed on the development of serum-free conditions for clinical application of articular cartilage tissue engineering. Based on genipin-crosslinked collagen type II (COL II) scaffold and quantitative polymerase chain reaction (qPCR) analysis, 7 kinds of growth factors (GFs) and low-molecular weight chondroitin sulphate type C (LMWCSC) were chosen for the investigation. First, adapted from 2-level fractional factorial design and steepest ascent method, we selected a set of GFs and made a designed serum-free medium (dSFM) formulation which contained EGF 8.31 ng/ml, TGF-β1 20.00 ng/ml, BMP-2 28.85 ng/ml, PDGF-bb 8.11ng/ml, and VEGF 12.69 ng/ml. This dSFM could highly upregulate gene expression level of COL II, the unique marker of normal chondrocyte. From other parameter such as biochemical assay and histocytochemistry observation we also confirmed the better quality of engineered cartilage as compared to serum-containing group. In addition, we found this dSFM could maintain the induced human umbilical cord blood-derived mesenchymal stem cell (hUMSC) based on gene expression and biosynthetic rate. Next, we investigate the effect of LMWCSC on chondrocyte. We prepared it and found this small molecule could help chondrocyte to redifferentiate more rapidly. Meanwhile, several degradative genes were inhibited by LMWCSC. These data indicate that supplement with LMWCSC into medium are potent for promoting excreting ECM of normal cartilage as well as protecting from degradative damage, thus further development is promising. In summary, the choice of serum-free components into medium formulation could help cells develop normally and clinical application of cartilage tissue engineering.

並列關鍵字

cartilage tissue engineering ； chondrocyte ； collagen type II scaffold ； quantitative polymerase chain reaction ； experimental design ； growth factors ； low-molecular weight chondroitin sulphate type C ； serum-free medium

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