人類間葉幹細胞於組織工程支架上軟骨分化能力之評估

Abstract

本研究使用第二型膠原蛋白-透明質酸複合物 (type II collagen-hyaluronan, CII-HA)及豬小腸黏膜下層 (small intestinal submucosa, SIS) 作為誘導幹細胞軟骨化的基材，比較來自人類四種不同組織的間葉幹細胞軟骨分化的潛力，分別為骨髓間葉幹細胞 (bone marrow derived mesenchymal stem cells, BMCS)，脂肪幹細胞 (adipose derived adult stem cells, ADAS)，胎盤間葉幹細胞 (placenta-derived mesenchymal stem cells, PDMC) 及牙齦纖維母細胞 (gingival fibroblasts, GF)。在二維系統進行軟骨誘導分化14天後的結果顯示，胎盤間葉幹細胞最具有軟骨化潛力，其次為牙齦纖維母細胞，而後將此兩種細胞植入以冷凍乾燥法 (freeze-drying) 製成的第二型膠原蛋白-透明質酸複合支架及八層豬小腸黏膜下層進行三維的誘導軟骨分化培養，培養28天後結果顯示此兩種幹細胞植入第二型膠原蛋白-透明質酸複合支架具有較好的誘導軟骨化效果。最後在免疫缺乏小鼠皮下進行體內培養證實胎盤間葉幹細胞植入第二型膠原蛋白-透明質酸複合支架為最佳的組織工程軟骨。

The chondrogenesis differentiation potential of mesenchymal stem cells (MSC) isolated from four different human tissues were compared on two biomaterials, type II collagen-hyaluronan composite (CII-HA) films and small intestinal submucosa (SIS) sheets. The four human MSCs were bone marrow-derived mesenchymal stem cells (BMSC), adipose-derived adult stem cells (ADAS), gingival fibroblasts (GF) and placenta-derived mesenchymal stem cells (PDMC). The CII-HA composite films or three-dimensional (3D) scaffold were fabricated in this study. Upon TGF-β3 induction, PDMC demonstrated the best chondrogenesis differentiation potential on both materials, followed by GF. PDMC and GF were further seeded in CII-HA composite scaffolds and 8-layer SIS scaffolds for evaluation of neocartilage formation in vitro. After 28 days, CII-HA composite scaffolds seeded with either MSCs were surfaced with a cartilaginous-like layer. Histology also showed better neocartilage formation when MSCs were grown in CII-HA composite scaffolds. NOD SCID mice subcutaneous implantation further confirmed that the combination of PDMC and CII-HA composite scaffolds promoted the formation of tissue-engineered cartilage.