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  • 學位論文

軟骨基質對間葉幹細胞軟骨分化之影響

The Effects of Cartilage Matrix on Chondrogenic Differentiation of Mesenchymal Stem Cells

指導教授 : 方旭偉 張至宏
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摘要


細胞外基質參與調控間葉幹細胞之分化過程。我們假設:取自退化性關節膝蓋之軟骨碎片,具有促進骨髓間葉幹細胞軟骨生成之潛能。將獲取自置換全人工膝關節之非受損部位的軟骨碎片(30與60毫克)與間葉幹細胞混合,包裹於纖維蛋白膠中,形成的複合物植入於裸鼠皮下。組織切片結果顯示,軟骨碎片-纖維蛋白膠-間葉幹細胞複合物具有阿爾襄藍染色之類軟骨生成結構;然而,只有包裹間葉幹細胞之複合物,則濃縮成類似間葉幹細胞團塊培養之形貌。包裹60毫克軟骨碎片複合物的第二型膠原蛋白基因表現,於移植4星期後有顯著的增加;相反地,沒有含軟骨碎片的複合物則無法表現第二型膠原蛋白,顯示出這樣的間葉幹細胞無法朝向軟骨細胞分化。研究證實:取自退化性關節炎膝蓋之軟骨碎片,在不需要外加生長因子的條件下,可促進骨髓間葉幹細胞之軟骨生成作用。 此外,我們調查以軟骨碎片促進不同組織來源之間葉幹細胞軟骨生成的可行性,包括人類臍帶間質幹細胞以及乳牙間質幹細胞。間葉幹細胞與軟骨碎片包埋於膠原蛋白膠中,進行體外培養。含有臍帶間質幹細胞與軟骨碎片之複合物,於含生長因子的環境中培養6星期後,第二型膠原蛋白基因表現明顯增加,顯示含軟骨碎片與生長因子的環境,有助於臍帶間質幹細胞之軟骨分化。然而,軟骨碎片與生長因子激起的協同效應,使乳牙間葉幹細胞產生纖維軟骨組織。 另一方面,我們進一步將軟骨組織轉化成去細胞軟骨基質,並探討利用去細胞軟骨基質作為生物支架的可行性。將去細胞軟骨基質粉與人類滑液膜間葉幹細胞混合於膠原蛋白膠中,進行體外培養。組織切片結果顯示,去細胞軟骨基質粉與生長因子引發一個協同作用在產生組織工程軟骨,即時聚合酵素鏈鎖反應發現,去細胞軟骨基質粉在不含生長因子的條件下,具有促進第二型膠原蛋白基因表現的潛能,而在含生長因子的環境下,去細胞軟骨粉能降低滑液膜間葉幹細胞軟骨生成引起的肥大化現象。我們認為去細胞軟骨基質粉可以作為一生物功能性支架,應用於軟骨組織工程。 總結來說,存在於軟骨碎片中之生物性元素,例如生長因子與功能性蛋白,可能促進間葉幹細胞之軟骨生成作用。而去細胞軟骨基質也影響滑液膜間葉幹細胞之軟骨分化,並可以作為一個功能性支架。因此,我們認為應用軟骨基質於軟骨組織修復或再生,是一個非常值得努力與重視的課題。

並列摘要


Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). We hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of bone marrow-derived MSCs. Non-worn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage-like structure with positive Alcian blue staining in the cartilage fragment-fibrin-MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In summary, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction. Moreover, we investigated the facility in using cartilage fragments for chondrogenesis of MSCs from different tissues including human Wharton's jelly and exfoliated deciduous teeth. The MSCs and cartilage fragments were embedded into type I collagen gel for in vitro culture. The gene expression of type II collagen in the constructs containing Wharton’s jelly-derived MSCs and cartilage fragments with growth factors induction were significantly increased after 6 weeks of culture. This suggested that cartilage fragments with growth factors induction benefited the chondrogenic differentiation of Wharton’s jelly-derived MSCs. However, cartilage fragments and growth factors evoked a synergistic effect on human exfoliated deciduous teeth-derived MSCs to produce fibro-cartilage tissue formation. Besides, we further transformed the cartilage tissues into acellular cartilage matrix (ACM) and explored the feasibility of using ACM as a biological scaffold. The ACM powders and human synovium-derived mesenchymal stem cells (SMSCs) were mixed into collagen gel for in vitro culture. Histological results showed a synergistic effect from ACM powders and chondrogenic growth factors in the formation of engineered cartilage. The findings of real-time PCR suggested that ACM powders had the potential of promoting type II collagen gene expression in the growth factors-absent environment. Moreover, under the growth factors induction, the ACM powders could reduce the hypertrophy in chondrogenesis from SMSCs. We suggested that the ACM powders could be applied as biological and functional scaffold for cartilage tissue engineering. In conclusion, biological elements in cartilage fragments, such as growth factors and functional proteins, may promote the chondrogenic differentiation of MSCs. Moreover, ACM still influenced the chondrogenesis of SMSCs and could serve as a functional scaffold for further applications. Therefore, we considered it was worthwhile putting more efforts to work with cartilage matrix for cartilage repair or regeneration.

參考文獻


1. Aigner T, Stöve J., "Collagens--major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair," Adv Drug Deliv Rev., vol. 55(12), 2003, pp. 1569-93.
2. Rosowski M, Falb M, Tschirschmann M, Lauster R. "Initiation of mesenchymal condensation in alginate hollow spheres--a useful model for understanding cartilage repair?," Artif Organs., vol. 30(10), 2006, pp. 775-84.
3. Ahmed TA, Hincke MT. "Strategies for articular cartilage lesion repair and functional restoration," Tissue Eng Part B Rev., vol. 16(3), 2010, pp. 305-29.
5. Richmond JC. "Surgery for osteoarthritis of the knee," Rheum Dis Clin North Am., vol. 34(3), 2008, pp. 815-25.
6. Bastiaansen-Jenniskens YM, Koevoet W, de Bart AC, van der Linden JC, Zuurmond AM, Weinans H, Verhaar JA, van Osch GJ, Degroot J. "Contribution of collagen network features to functional properties of engineered cartilage," Osteoarthritis Cartilage, vol. 16(3), 2008, pp. 359-66.

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