明膠/玻尿酸/軟骨素三共聚物於間質性幹細胞之軟骨組織工程應用

Abstract

相較於硬骨於體內之自然修復能力,自體修復之軟骨無法回復受損前的組 織功能。小範圍的軟骨缺損若無法即早修復,其將導致嚴重之關節炎疾病。然目 前臨床上之治療方式,皆無法根本性的修補軟骨缺損。 時至今日,軟骨組織工程之研究,仍未有適用於生物反應器之最佳組合。 明膠/玻尿酸/軟骨素三共聚物已於過去的研究展示其能維持軟骨細胞之型態。本 研究中,將使用間質性幹細胞做為軟骨組織工程之細胞來源,播種間質性幹細胞 於三共聚物支架,並結合誘導幹細胞軟骨分化之小分子藥物 Kartogenin ,作為 分化訊號。 於生物反應器培養 21 日後,本組合成功產生俱軟骨特性之構物。其軟骨相 關基因包含: Aggrecan 、︑ SOX9 以及 Col2a1 皆有顯著性之上昇,此外聚集之軟 骨性團塊可於掃描式電子顯微鏡下檢視,而其組織切片亦能發現類似軟骨陷窩之 結構。於免疫染色中,亦可檢視到軟骨蛋白聚醣 (aggrecan) 以及第二型膠原蛋白 (type II collagen) 表現。 相較於傳統上使用蛋白質誘發分化,使用小分子藥物於培養基消耗量大之 生物反應器為一較俱臨床可行性之選項。本研究以明膠/玻尿酸/軟骨素、 KGN 以及間質性幹細胞之組合提供了小分子藥物調控幹細胞之一範例。

Contrary to the capability of bone repair, the cartilage regeneration in body couldn’t recover the cartilage lesions to proper function, and the failure of small defect repairing could lead to severe osteoarthritis disease. However, current clinical treatment options are not satisfying to fully recover the lesions. Till now, there is no optimized combination of scaffold and signal for cartilage tissue engineering in bioreactor system. Previously we have demonstrated tri- copolymer composed of gelatin, hyaluronic acid and chondroitin-6-sulfate can retain the phenotype of chondrocyte. In this study we demonstrated the combination of mesenchymal stem cells (MSCs) with tri-copolymer and Kartogenin (KGN) , a small molecule drug which promotes MSCs toward chondrogenetic differentiation. The combination successfully induced the chondrogenesis. After 21 days culture in homemade perfusion bioreactor system, the construct shows chondrogenetic characteristic in both genetic and morphology aspect. Aggrecan, Sox-9 and Col2a1 are all up-regulated, and the condensed cell mass are found in the porous scaffold structure. The lacunae-like structure were also found by Hematoxylin/Eosin staining. Since culturing in the bioreactor with cytokine or growth factor is impractical due to its need for great amount of medium, culturing with small molecule has become much more feasible. We expect that this research provides an example for small molecule regulated practice and thus provide a new combination for cartilage tissue engineering.