以不同電紡參數製備有方向性的蠶絲蛋白奈米纖維在韌帶組織工程上的應用

Abstract

韌帶損傷是常見的運動傷害，韌帶一旦受損就很難自行痊癒，需要手術重建， 但目前沒有一條人工韌帶可以完全取代天然韌帶。韌帶組織工程建立一個模仿韌帶生物結構的三維支架，以提供組織受損期間的機械支撐並促進組織再生。而順向性的纖維可以模仿韌帶細胞的生長環境，使細胞分泌較多的細胞外間質。 本研究利用靜電紡絲法製備蠶絲蛋白奈米纖維，透過參數調控在12 %、20 kv及1.2 ml/h下可以收集到平均直徑在500-600 nm的蠶絲奈米纖維，且在2800 rpm下可收集到最有方向性的纖維，並添加PEO增加在高轉速下的穩定性。另外我們也發展一個柱狀收集法，電紡時搭配柱狀收集板，可製備出一個三維、多孔及有方向性的鬆散支架，可改善傳統電紡製備的纖維太過緻密以致細胞不易進入支架的問題。 在SEM圖顯示具方向性的支架與韌帶細胞共培養7天後，可以看到細胞沿著纖維方向生長並分泌胞外基質；在LIVE/DEAD螢光染色顯示韌帶細胞在第七天時仍有很高的活性；利用SEM與組織切片觀察後，發現不但有細胞浸潤的情形，且細胞能在內部增殖及分泌胞外基質；另外膠原蛋白含量測定中，在7與14天膠原蛋白皆有增加的趨勢。 本研究證實我們發展的柱狀收集法製備的支架，確實可提供韌帶細胞良好的立體生長環境並且分泌胞外基質，達到修復效果，在韌帶再生上是非常有潛力的。

Ligament injuries are a common sports injury. Once the ligament is injured, it is difficult to heal by itself and needs surgery reconstruction. In fact, there is no clinical artificial ligament to replace the native ligament currently. Ligament tissue engineering is capable of constructing a biomimetic 3¬D scaffold for ligament, providing mechanical support in the period of injury and promoting tissue regeneration. Moreover, the aligned fibers are able to mimic the proliferating environment of ligament cells, making the cells to secrete more ECM. During the experiment, we fabricated silk fibroin nanofibers by using electrospinning and by controlling process parameters. The silk fibroin nanofibers , ranging between 500-600 nm, were collected with 12% in solution concentration、20 kV in electrical field and 1.2 ml/h flow rate directional fibers can be obtained at 2800 rpm of rotating wheel . In addition, we also added PEO to make electrospinning process more stable. Furthermore, we used a novel column collection to fabricate 3D, porous, aligned, and loose scaffolds. It changes the structure of fibers and improves the cell infiltrating into scaffolds. In our research, SEM images show that the ligament cells grow along with the direction of fibers and secret ECM after they are cultured with aligned scaffolds for 7 day. LIVE/DEAD fluorescence images show that ligament cells still have high activity after 7 days of culturing. SEM and histological images prove not only cell infiltration, but also cell proliferation and ECM secretion. In addition, collagen content increase after 7 and 14 day of culturing. This study demonstrates that the new scaffolds fabricated by column collection. can provides good environment for ligament cells adhesion, proliferation and further more secrete ECM. These scaffolds provide a potentiality in ligament regeneration.