近年來許多的臨床手術已經被發展出來用以治療骨軟骨損傷，但其成效相當有限，易產生不預期的纖維軟骨增生；雖然骨軟骨自體移植或同種異體移植物被視為黃金標準，但礙於不易取得或捐贈數量少，仍舊不是長久之計。隨著科技日新月異，許多科學家開始導入組織工程學的概念來改善現有傳統治療的缺點，在眾多材料方面，可注射式高分子提供了多項優點，如可在原位成型並與受體組織緊密接合、富有高度含水性以及良好的型態順應性等，除此之外，由於近年微創手術的興起，可注射式水凝膠之特性恰好適用於關節鏡治療，降低手術風險與縮短住院時程。
　　本篇文章中，我們成功地合成出無毒且具溫感性的異丙基丙烯醯胺與幾丁聚醣共聚物水凝膠，並以巰基修飾形成雙流鍵交聯。在過去，異丙基丙烯醯胺接枝幾丁聚醣共聚物已被證實具有良好的生物相容性，並且能夠於注射入體內後快速進行相轉變(由液態變為固態)，極具有成為細胞載體與植入物之潛力。然而，力學強度的不足遏止了其在生物醫學上的應用，為了克服該限制，我們使用醫療上常用的抗氧化劑N-乙酰基半胱氨酸，以碳二亞胺化學接枝至幾丁聚醣支鏈，形成化學性交聯以增強其機械性質。
　　透過Ellman試劑，我們發現巰基的接枝率會受到碳二亞胺濃度之影響，在氧化反應後，我們得到修飾過後的水凝膠能達到九倍以上的強度提升，其壓縮模量約為11.4千帕斯卡，並且再加上流變學分析，結果發現儲能模量與交聯程度有著正向關係，也再一次驗證強度的提升。此外，由掃描式電子顯微鏡觀察到微觀下每組水凝膠都具有良好的孔洞互連性，同時也導致其他物理性質，如溶脹能、相變溫度與蛋白質貼附能力的改變。利用存活螢光染色及細胞活性分析，我們發現該水膠對於三種不同的細胞類型(間質幹細胞、纖維母細胞、成骨細胞)皆不影響其生物機能並表現極佳的存活率；在動物皮下植入結果上，發現於注射後四周後實驗鼠並沒有出現急性的體外排斥現象，生長狀態亦無異常。最後，從微電腦斷層掃描技術與術後外觀評分，顯示混合間質幹細胞的水凝膠，不論是否經過修飾，在骨軟骨缺損模型上皆達到不錯的填補率與近似於原先軟骨的平滑表面。
　　總結以上，我們認為以異丙基丙烯醯胺接枝幾丁聚醣的溫感型水凝膠相當適合用作為軟骨組織工程的生醫材料，並且強化機械性質後更能夠促使該智慧型高分子用於其他不同組織需求。

　　Currently numerous clinical treatments for osteochondral injury have been developed but the unexpected outcome still existed such as fibrocartilage formation and insufficient donor autografts or allografts. As an advance of technological knowledge and biological science, many researchers have engaged in an innovative strategy, tissue engineering, considered as a potential to address the shortage of conventional treatments. Among various biomaterials, an injectable polymer has caught many attentions depending on the advantages including in situ gelation to attach host tissue, high hydration and shape adaptability. Additionally, minimal invasive surgery is preferable than traditional operation with less risks and shorter hospital stay. Combination with injectable hydrogels has been a promising strategy for clinical need statements.
　　In the present study, we fabricated a non-toxic thermo-sensitive NIPAAm-g-chitosan (NC) hydrogels with thiol modification for disulfide crosslinking strategy, abbreviated as “TNC”. Previously, NC copolymer has been proven to have excellent biocompatibility and rapid phase transition after injection, suitable to serve as cell carriers or implanted scaffolds. However, weak mechanical properties significantly restricted its application in the biomedical field. In order to overcome this limitation, we introduced thiol side chains into chitosan by covalently conjugating N-acetyl-cysteine (NAC) with carbodiimide chemistry to strengthen mechanical properties.
　　Ellman’s assay showed degree of thiol substation increased as carbodiimide concentration leading to higher cross-linking. After oxidation of thiols for creating disulfide bonds, modified NC hydrogels did improve the compressive modulus over 9 folds (11.4kPa). Additionally, oscillatory frequency sweep showed the positive correlation of storage modulus and cross-linking density. Interconnected microstructure appeared in NC based hydrogels shown in SEM image resulting in tunable physical properties such as swelling capacity, phase transition temperature and protein absorption. In vitro Live/Dead and MTS assay represented a great proliferation and viability for mesenchymal stem cells (MSC), fibroblast and osteoblast. In vivo subcutaneous implantation further confirmed biocompatibility and biodegradability of synthetic hydrogels with minor foreign body reaction at 4 week. Subsequently osteochondral defect in rat model with Micro-CT analysis and gross scoring system illustrated a better coverage and smoother cartilage surface in MSC encapsulated NC and TNC groups compared to sham group. In sum, we suggested that the thiol-modified thermo-sensitive polysaccharide hydrogels are promising to be a cell-laden biomaterial for cartilage tissue regeneration and the strengthened stiffness with disulfide crosslinking broadened the application of NIPAAm-based smart polymer towards diverse ranges of tissue.

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