開發去細胞軟骨基質與表層包覆玻尿酸之膠原蛋白基質以作為人工軟骨的支架
中文摘要
本篇論文的目的，是希望能研發出新的人工軟骨以供治療軟骨疾病，所使用 的材料支架有兩種，分別是以豬軟骨基質及自豬皮萃取而得的膠原蛋白基質 為主體，再經之後不同的處理。在軟骨基質方面，使用了三種酵素，進行不 同時間、溫度的處理，希望找出最佳的去細胞方法，並以 H&E stain 、 nested PCR 、 fluorimetric DNA assay 評估去細胞程度；在膠原蛋白基質方面，使用 兩種添加玻尿酸的方式，希望添加對軟骨細胞的生長有利的玻尿酸，並分析 物理性質，最後再將軟骨細胞植入這兩種材料支架中培養，並觀察軟骨細胞 的生長表現。我們以 MTT assay 觀察細胞在不同時間的生長量，也以 H&E st ain 觀察細胞在基質中的分布，再以 RT-PCR 的方式分析細胞的 mRNA 表現，另一方面，則是將去細胞基質植入老鼠體內以評估其生體相容性。
結果顯示，以玻尿酸酶加胰蛋白酶或分解酶加胰蛋白酶，併用界面活性劑處 理 24 小時，能有效去除細胞；使用表層包覆玻尿酸的方式，可得到較堅實的 膠原蛋白基質結構，但孔洞直徑會隨玻尿酸量的增加而減少；而兩種材料支 架都具孔洞性適合細胞生長。目前已將軟骨細胞植入，但有關觀察細胞生長 的實驗，及去細胞基質的生體相容性實驗，仍在進行中，期望細胞在兩種材 質內都能有良好的生長表現。Development of Acellular Cartilage Matrix and HA Coated Collagen Matrix as the Scaffold for the Artificial Cartilage
英文摘要
The objective of this thesis was to develop new artificial cartilage for treating cartilage disease . We used two kinds of material scaffolds, which were based on porcine cartilage matrix and c ollagen matrix extracted from porcine skin, followed by various treatments. In the part of porc ine cartilage matrix, we used three enzymes and treated by various time and temperature for fi nding the best method to remove the porcine cells. Then we applied H&E stain, nested PCR, a nd fluorimetric DNA assay to estimate the degree of decellularization. In the other part of coll agen matrix extracted from porcine skin, we used two ways of adding hyaluronic acid for addi ng hyaluronic acid which was advantageous to chondrocyte growth. Then we analyzed the ph ysical properties. Finally, we seeded chondrocytes in the two kinds of material scaffolds, and o bserved the growth of chondrocytes. We applied MTT assay for the amount, H&E stain for the distribution, and RT-PCR for the mRNA expression. On the other hand, the acellular cartilage matrix was implanted into the lesion of Wistar rats to evaluate the biocompatibility.
The results showed that when treated with hyaluronidase and trypsin or with dispase and tryps in, and combined surfactant, for 24 hr could effectively remove the cells, and when coated wit h hyaluronic acid could have firm structure but the pore diameters decreased with the increase of hyaluronic acid content. Two scaffolds had porous structure for cell growth. Now we have s eeded chondrocytes in the two scaffolds, but the experiments about the observation of cell gro wth and the biocompatibility of acellular cartilage matrix are still in progress. We expect that c ell would grow well in the both two scaffolds.
