水膠(Hydrogel)有很高的含水量和相對低的細胞貼附率。有許多研究指出水膠可提供微觀環境來做軟骨分化，特別的是有些還會將水膠混摻透明質酸和膠原蛋白。在我們的研究中，我們試著去調控表面貼附力和細胞間的接觸作用來誘導骨髓間葉幹細胞(RM1)往軟骨分化。幾丁聚醣(Chitosan, CS)被選來適合細胞貼附的基底材料，另外我們使用表面改質，將兩端帶有Carboxylate的PEG(CP)接枝到CS表面上，以達到細胞貼附的改變。而在三維培養中，先製備好在不同孔徑大小的尼龍(Nylon mesh membrane)披覆上CS或是CP接枝上CS表面，來營造出一個三維的環境，利用此環境來控制細胞間的接觸作用，尼龍的孔徑大小我們選用30μm，100μm和180μm。在180μm的尼龍擁有較低的表面對體積的比率。在100μm和180μm中，我們觀察到細胞會逐漸聚集。在披覆CS的尼龍培養在Basal medium中，藉由GAGs染色和基因表現的分析指出有軟骨分化的趨勢。並且發現在180μm中對於間葉幹細胞的軟骨分化最好，也告知我們細胞間接觸作用的重要性。有趣的是，在披覆CS的尼龍培養在Chondrogenic medium中，並未大幅度地提升軟骨分化。在CP的尼龍中，細胞在第六個小時就開始出現聚集的現象，而且Aggrecan和Col2a1有更高的表現量，若再添加Chondrogenic medium來培養，發現又更能促進RM1分化成軟骨細胞，GAGs染色和基因表現量都是最好的。從這些結果指出當我們稍微的降低表面貼附率就可以增加細胞間的接觸作用，而且推論出促進軟骨分化的重要因素就是要提升細胞間的接觸作用。Hydrogels display high water retention and relatively low cell adhesivity. Several studies have demonstrated that hydrogels can provide an instructive microenvironment for the chondrogenesis of mesenchymal stem cells (MSC), especially when hyaluronan and collagen II were incorporated. In this study we try to elucidate that it is surface adhesivity, cell-cell contact, or bio-specific matrix-cell interaction dominates instructive differentiation.Chitosan was selected as the base substrate for cell attachment. Cell adhesion was altered through surface modification of chitosan by carboxylate ended polyethylene glycol (CP). Nylon meshes of different mesh sizes coated with chitosan or CP modified chitosan were prepared to provide 3-D environments for cell-cell contact control. The size of Nylon mesh selected are 30, 100, 180μm. The 180μm mesh owns the lowest surface to volume ratio. We have observed that the cell would gradually aggregate in the wells of 100 and 180μm. Nylon mesh coated with chitosan even in basal medium. GAG staining as well as SOX9, Aggrecan, and Col2a1 gene expression indicate chondrogenesis. Results indicate that the 180μm mesh is the best for MSC chondrogenesis suggesting the importance of cell-cell contact. Interestingly, chondrogenic medium does not promote chondrogenesis when MSCs are cultivated in chitosan coated meshes.In Nylon mesh coated with CP modified chitosan, we observe that cells aggregate within 6 hours in the wells of 100 and 180μm mesh. Much higher SOX9, Aggrecan, and Col2a1 expression are observed when MSCs are cultivated in Nylon mesh coated with CP modified than with virgin chitosan. From these results, we find that slightly reduction of surface adhesivity enhances cell-cell contact. And the enhancement of cell-cell contact is critical to chondrogenesis.
