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。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。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。此外,我们發現在甲殼素表面上培養之脂肪幹細胞可增進其幹細胞特性及分化能力。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,兼具有良好的機械強度及生物活性,可在體外培養環境中成功誘導脂肪幹細胞之軟骨分化。本計畫目的為以動物實驗探討甲殼素與軟骨組織細胞外基質混合生物材料結合脂肪幹細胞應用於組織工程之可行性。第一年之實驗擬以2種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4種,接著將人類脂肪幹細胞種入複合鷹架材料,並採用免疫缺陷小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。第二年再由其中選出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,自第二年之實驗中再選出1種鷹架材料,進一步驗證該鷹架材料確實適合與脂肪幹細胞結合應用於軟骨組織工程。本研究預期以動物實驗驗證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, including 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 current paradigm of tissue engineering is culturing cells in 3D porous scaffolds in vitro to develop mature tissue for implantation.Chitosan is a widely used biomaterial. It has good mechanical property and has been applied in 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 extracellular matrix (ECM) can induce adipose stem cells toward chondrogenic differentiation without exogenous growth factors. Moreover, we observed enhanced stemness and differentiation capacities when culturing ASCs on chitosan films. Therefore, we fabricated a cross-linked composite biomaterial made of chitosan and cartilage ECM with good mechanical properties and bioactivity, and it could successfully induce chondrogenic differentiation of adipose stem cells in vitro.The aim of this project is to validate the aforementioned chitosan-cartilage ECM composite scaffolds, when combined with adipose stem cells, can provide cartilage regeneration in animal models. In the first year, we will fabricate 4 constructs with different chitosan/cartilage ECM compositions and different cross-linking degrees. After examining their physical properties, human adipose stem cells will be seeded into some of the scaffolds. In a subcutaneous implantation model of SCID mice, we will test the cartilage repair potential of these scaffolds with or without ASC seeding. In the second year, we will further choose 2 scaffolds based on the experimental results of the first year and test them in a knee articular cartilage defect model of rabbits. In the third year, we will further verify a 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 verify that chitosan-cartilage ECM composite scaffolds provide environmental stimuli necessary for chondrogenic differentiation of adipose stem cells in vivo. 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
animal model