以靜電紡織製備蠶絲蛋白/明膠多層平行纖維支架應用於椎間盤纖維環修補

Abstract

當椎間盤因退化而逐漸失去吸收外力衝擊的能力，會導致椎間盤的纖維環 (annulus fibrosis) 承受過大的壓力而破損，椎間盤內的膠狀物質本核 (nucleus pulposus) 將會流出椎間盤外，形成所謂的椎間盤突出 (disc herniation)，進而壓迫到周圍的神經組織，使病人感到疼痛。目前臨床上面臨的問題是，骨科醫師在移除椎間盤突出後並無適當的手段或材料修復纖維環的缺損，該缺損有很高的機率再發生椎間盤突出，是潛在的危險區域。本篇研究的目的為開發模擬纖維環組織天然結構的材料，用以修復手術後纖維環的缺損，防止椎間盤突出的再發生。 本篇研究使用兩種天然高分子：蠶絲蛋白以及明膠作為材料，利用靜電紡織技術搭配高轉速收集器以製備出具有順向結構的單層電紡纖維膜，並將單層電紡纖維膜以明膠疊起，交聯後形成多層電紡支架以仿造天然椎間盤纖維環的結構。 本研究以FT-IR分析由天然蠶絲中萃取純化出的蠶絲蛋白，證實本研究能由天然蠶絲中萃取純化出蠶絲蛋白；以SEM觀察製備完成之單層電紡纖維膜與多層電紡支架，確認以該方法可製備出直徑在200 - 300 nm均勻且具方向的電紡纖維結構；拉升試驗的結果顯示本實驗製備的單層電紡纖維膜之楊式係數為3.18 - 6.70 MPa、最大拉升強度為0.96 - 1.81 MPa、伸長量為60 - 90 %、韌性為0.3 - 1.1 MJ/m3，均相似或優於天然的單層AF組織；量測多層薄膜支架的膨脹率的結果顯示，多層電紡支架約有20 - 30 %的膨脹率，可藉由膨脹以達到固定多層電紡支架的效果。 目前在細胞結果中，由WST-1、LDH與Live/dead染色結果可知，本研究所製備出來的蠶絲蛋白/明膠電紡纖維膜具有良好的生物相容性。進一步利用SEM觀察到椎間盤細胞能貼附於電紡纖維膜上，並沿著纖維的順向結構延長、細胞逐漸遷移進入電紡纖維的孔洞。而在動物實驗的MRI的影像訊號上，推測本研究製備的多層電紡支架能阻止high intensity zone (HIZ) 的形成，因而更有利於纖維環之修復；椎間盤攝影術的實驗結果顯示植入多層電紡支架的組別對於纖維環缺損的修復成效明顯優於未經處理的組別。 因此，我們認為本研究所製備的蠶絲蛋白/明膠多層電紡支架具有修復椎間盤纖維環缺損的潛力。

Intervertebral disc degenerates with age and would lose the function of absorbing impact, which lead to the lesions in annulus fibrosis (AF). Then the gelatinous core in intervertebral disc, nucleus pulposus (NP) may leak outside of the disc, forming the so called disc herniation. It would compress the surrounding nerves, causing the suffering of the patients. Currently, the clinical problem is that there is no appropriate approach or material to seal the AF lesions caused by discectomy. The untreated defects in AF are potential sites for recurrent herniation. To solve this problem, we developed a scaffold which mimics the structure of native AF to repair the lesions after discectomy and prevent recurrent herniation. In this study, we chose two nature polymer as scaffold material: silk fibroin and gelatin. To produce the scaffold which mimics the structure of native AF, we use electrospinning technic with high speed rotating collector to produce membranes with preferred orientation fiber. Then gelatin is used as adhesive to stack the membrane layer by layer with specific angle alternatively. After cross-linking, a multilayer scaffold would be produced. In this study, FT-IR is used to confirm the fingerprint region of fibroin extracted from nature silk cocoon. The single layer electrospun membrane and multilayer scaffold were analyzed by SEM to confirm that the fiber diameter is around 200 - 300 nm with preferred orientation. The results of tensile test showed several mechanical properties of the single layer electrospun membrane developed in this study: elastic modulus is around 3.18 - 6.70 MPa, UTS is around 0.96 - 1.81 MPa, elongation is around 60 - 90% and toughness is around 0.3 - 1.1 MJ/m3. And all the mentioned mechanical properties are similar or superior to native AF tissue. The results of swelling test showed that the multilayer scaffold would swell 20 - 30% in aqueous solution, and the swelling would provide better scaffold fixation after surgery. The current results of in vitro study, WST-1, LDH and Live/dead staining were done to verify the biocompatibility of electrospun membrane. The SEM images showed that both AF cell and NP cell can adhere on electrospun membrane and migrate into the pores of electrospun fibers. From MRI images of in vivo study, we suspected that the multilayer scaffold developed in this study may prevent the formation of high intensity zone (HIZ), hence it’s better for annulus repair. The results of discography showed that the effectiveness of annulus repair of implant group was much superior to untreated (injury) group. Hence, we supposed that the fibroin/gelatin multilayer scaffold developed in this study have great potential for annulus fibrosis repair.