Abstract
摘要:軟骨再生治療有很大的相關醫療需求,頭頸部方面有墊高鼻子、氣管缺損、小耳症等需要修補軟骨的手術;四肢方面則包括年輕人運動傷害或外傷引起的關節軟骨缺損,以及老年人之退化性關節炎、關節骨缺血壞死等。鑒於目前組織重建之方法均未臻理想,以組織工程的方法進行手術被視為未來最可行之解決方法。組織工程的主要的技術模式在於取得足夠數量的細胞,種植於三維的多孔性鷹架材料,在體外透過適當方法培養成組織或半成熟的組織,然後植入體內以修補受損的組織器官。就可行性而言,軟骨組織本身無血管、神經之分佈,使其進行組織工程的困難度較其他大多數有血管、神經之分佈的組織為低。甲殼素為常用的生醫材料,具有良好的機械強度,可與軟骨細胞結合應用於軟骨組織工程,但軟骨細胞常面臨來源有限、取得不易等問題。幹細胞研究的開展為組織工程的細胞來源打開了技術瓶頸,脂肪組織中具有豐富的間質幹細胞,且容易取得與分離,十分適合應用於組織工程,然而甲殼素結合間質幹細胞應用於軟骨組織工程的結果並不成功。我们之前的研究指出,將軟骨組織以機械力量打碎並予以去細胞處理並重組製成一多孔狀鷹架材料,在體外實驗中不必外加生長因子即可引導脂肪幹細胞朝軟骨細胞分化。故我们假設將甲殼素與軟骨組織細胞外基質混合製成的生物材料將兼具有良好的機械強度及生物活性,可與脂肪幹細胞結合成功應用於軟骨組織工程。本計畫目的為研究如何結合甲殼素與軟骨組織細胞外基質製造適宜的生物材料,並以體外及體內實驗探討其結合脂肪幹細胞應用於組織工程之可行性。先期研究中我們已成功由天然來源之軟骨組織抽出細胞外基質,再結合甲殼素製造出適合組織工程採用的多孔狀鷹架材料。第一年之實驗擬以不同比例之甲殼素與軟骨基質混合,再以交聯劑 genipin製造交聯度不同之複合鷹架材料共24種,我們評估這些鷹架材料的三維結構、孔洞大小、機械強度等。接著將人類脂肪幹細胞種入複合鷹架材料,並於體外培養中驗證該生物材料與脂肪幹細胞之生物相容性及估軟骨分化程度。第二年之實驗擬自上述鷹架材料中選出4種,採用SCID鼠之皮下植入動物模式測試其應用於軟骨組織工程的潛力;再由其中選出2種鷹架材料,與脂肪幹細胞結合後採用大白兔之膝關節軟骨缺損動物模式進行實驗。第三年之實驗採用迷你豬之膝關節軟骨缺損動物模式,進一步驗證上述實驗結果最佳之鷹架材料確實適合應用於軟骨組織工程。本研究預期完成甲殼素與天然軟骨組織混合而適用於軟骨組織工程之鷹架材質,能提供脂肪幹細胞分化所需要之良好環境,再加上足夠的機械性質,使我們可以真正創造接近自然的軟骨組織供手術修補之用。
Abstract: Regenerative therapy of cartilage exhibits huge clinical demands, such as nasal augmentation, airway defect or microtia correction in the head and neck area; articular cartilage defects resulting from sports injury, osteoarthritis, and avascular necrosis. Current treatments for cartilage injury are not ideal, and tissue engineering approach is deemed as the most promising solution in the future. The paradigm of tissue engineering is culturing cells in 3D porous scaffolds in vitro to develop mature tissue for implantation. In terms of the feasibility, cartilage has been regarded as a more attainable target of tissue engineering because it is avascular and aneural.Chitosan is a widely used biomaterial. It has good mechanical property and has been applied to cartilage tissue engineering with chondrocytes. However, chondrocyte represents a limited cell source with low proliferative capability. Stem cell research provides a superior cell source for tissue engineering. Adipose tissue is enriched with mesenchymal stem cells, which are easily accessible by liposuction. We have demonstrated that a porous scaffold material derived solely from cartilage can induce adipose stem cells toward chondrogenic differentiation without exogenous growth factors. Therefore, we hypothesized that a composite biomaterial made of chitosan and cartilage matrix exhibits good mechanical properties and bioactivity, and it can be successfully applied in cartilage tissue engineering with adipose stem cells.The aim of this project is to develop chitosan-cartilage composite scaffolds, which will be combined with adipose stem cells for in vitro and in vivo assays of cartilage tissue engineering. In our preliminary study, we have successfully extracted extracellular matrix from native pig cartilage and mix it with chitosan to develop porous composite scaffolds. In the first year, we will fabricate 24 constructs with different compositions and different crosslinking degrees. After examining their physical properties, human adipose stem cells will be seeded into the scaffolds to test their biocompatibility as well as their capability of inducing chondrogenic differentiation. In the second year, we will adopt a subcutaneous implantation model of SCID mice to test the chondrogenic potential of 4 chosen scaffolds based on the first year study. We will further validate 2 scaffolds in knee articular cartilage defect model of rabbits. In the third year, we will further verify the scaffold with the best results in the previous studies in a large animal model – knee articular cartilage defect of miniature pigs.In summary, we aim to develop chitosan-cartilage composite scaffolds, which exhibit good mechanical properties and provide environmental stimuli necessary for chondrogenic differentiation of adipose stem cells. If the project succeeds, we can make a tissue equivalent that mimics the native cartilage tissue for surgical repair in the future.
Keyword(s)
軟骨再生
組織工程
甲殼素
脂肪幹細胞
細胞外基質
動物模式
cartilage regeneration
tissue engineering
chitosan
adipose stem cell
extracellular matrix