本研究利用在細胞表面塗佈合適的細胞外間質，來增強細胞間作用力，達成緊密之三維組織結構成形，進行體外模型之模擬。本實驗主要針對皮膚纖維細胞及軟骨細胞進行相關研究。對於皮膚纖維細胞，透過纖連蛋白(FN)與細胞上的整合素連接，並接著與明膠(G)相連，藉由層層塗佈(Layer-By-Layer)的方式，在細胞表面形成纖連蛋白-明膠奈米塗層，大大增強細胞間的結構，在培養日達到三天時，為其最佳狀態，另外，我們發現在塗佈過程前將細胞以較溫和的溶液(Accutase)作用與培養容器分離，可減緩細胞膜上蛋白被破壞，進而加強塗佈的效率，達到更佳的構築結果。在初代培養細胞中，藉由以上技術構築出的一般皮膚細胞與蟹足腫細胞相較於二維培養有明顯的差異，蟹足腫細胞顯示有較強之作用力與細胞外間質分泌能力，可模擬出實際在人體中的情形。軟骨細胞層片製作則利用第二型膠原蛋白(COL II)進行細胞表面塗佈，然而膠原蛋白會造成嚴重的細胞收縮，因此另外加入纖連蛋白來進行改善，在塗佈細胞培養7天後，可看出細胞株與原代細胞皆能完好堆疊，而未塗佈細胞則呈現鬆散結構，到第14天培養更發現有軟骨組織的形成，顯示細胞外間質塗佈有誘導軟骨生成的潛力，然而細胞層片收縮的情形依然存在，因此我們將更多的細胞外間質組合作為材料進行表面塗佈，其中，第二型膠原蛋白與糖聚蛋白(GAG)之組合，顯著改善組織收縮情形，表示該組合具有作為合適細胞層片建立之潛力。因此，我們的組織建構技術對於蟹足腫模型構築及軟骨組織誘導皆具有進行後續疾病研究之能力。

To construct in vitro three-dimensional tissue models to imitate the living tissues in our human bodies, several different extracellular matrices were selected to coat on the cell surface in this study to increase the interaction between the cells. Related studies on dermal fibroblasts and chondrocytes were conducted in this work. For dermal fibroblasts, fibronectin (FN) was used to connect with integrins on the cells, and then connected to gelatin (G) by layer-by-layer (LBL) coating, forming a FN-G nanofilm on the cell surface, which greatly improved the structure and was optimized when the culture day reached three days. Separating from the culture substrate with a mild solution (Accutase) can ease the breakdown of proteins on the cell membrane, improving coating efficiency and construction outcomes. For primary cells, the normal fibroblast created by the preceding procedures are notably different from the keloid fibroblast when it compared to two-dimensional culture, and the keloids have more force and capability for extracellular matrix secretion. Cell surface coated with type II collagen (COL II) was employed for chondrocyte layer formation. Collagen, on the other hand, would induce significant cell shrinking, thus fibronectin was added to moderate the severe shrinking phenomena. After 7-day culture, the coated cells revealed firmly stacked constructions, whereas the uncoated cells possessed a loose structure, and the creation of cartilage tissue was observed on the 14-day culture, showing that the coating of the extracellular matrix have the potential to induce the cartilage formation. However, the cell layer continued to shrink during the culture, a mixture of additional extracellular matrices were selected for cell coating. Among these, the combination of COL II and glycosaminoglycan (GAG) improved the constructed tissues greatly showing that the mixture has the potential to become a suitable cell layer. As a result, our tissue building technique may conduct follow-up illness research for both the keloid model and the induction of cartilage tissue.

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