利用水性聚胺酯與白芨多醣體製成奈米纖維薄膜以減少創傷後之神經肌腱沾粘並促進神經修復

Abstract

肢體損傷常合併肌腱與周邊神經的斷裂，然而肌腱與神經手術後，經常遭遇沾粘及功能恢復不佳等問題。故本研究之目的有二：1. 利用生醫材料，減少肌腱與神經手術後之沾粘；2. 促進周邊神經軸突生長。因此研究將涵蓋肌腱與神經之細胞實驗與動物實驗，並分述如下。 在美國，有三分之一的創傷是跟上肢有關，其中多合併肌腱與血管神經損傷。由於肌腱癒合緩慢，故術後配戴副木固定時間往往長達四周，也因而帶來沾粘等問題，延遲了患者恢復手部活動度之時間，更引起工時的損失，實帶來顯著之社會經濟負擔。故如何減少肌腱損傷後、手術重建後之沾粘，實是重要之課題。 而周邊神經損傷的問題在於，其軸突再生速度每日小於1 mm，即便手術接合後，其功能需極長時間回復。因軸突生長緩慢，該神經支配之肌肉多因長時間的神經失養而萎縮，即便軸突最後成功再生，肌肉運動功能仍無法完整復原。又四肢神經損傷多伴隨周圍軟組織傷害，導致後續疤痕增生嚴重，令神經被壓迫、沾粘；即便顯微手術及器械的進步，神經接合後之功能回復仍難預料。故減低神經沾粘與加速軸突再生有其必要性。周邊神經損傷相當常見，多因車禍與工傷；其中上肢神經損傷佔75%，所致手部失能及復健、醫療費用極為可觀。據美、歐統計上肢神經損傷，僅尺神經損傷，每位患者醫療費用及工傷損失達42,000美金。若涵蓋所有上肢神經損傷，美國一年損失一千萬日的工時。將上下肢神經損傷合併計算，美國2008年耗費其保險系統約1500億美元，故知其市場之大。此外神經損傷導致患者難以拿筆、使用筷子等，亦影響其生活品質。 目前市售產品較多針對腹腔抗沾粘，如Seprafilm、Surgiwrap，然這些產品接觸體液極易黏著或破裂，因此肢體手術時，欲包裹肌腱或神經等長柱狀組織並不易施展。且這些產品降解速率快，肌腱、神經尚未恢復前即已消失。最後，這些產品並非針對周邊神經所設計，無法促進神經軸突再生。 因此本研究利用電紡製備奈米纖維薄膜，材質方便手術施展不易破裂，容易包覆於長柱狀結構的組織（例如肌腱、神經）。此電紡膜以水性PU(water-borne Polyurethane, WPU)為基材，以電紡混紡織成，孔徑為0.95~1.29 µm之間，可有效避免fibroblast浸潤，故可減少肌腱或神經受疤痕沾粘、壓迫。本研究第一階段將此材料應用於兔子肌腱再接模型，成功減少了肌腱沾粘，提昇關節活動度，且不影響肌腱癒合的強度。 本研究第二階段則將WPU與白芨多醣體 (Bletilla Striata polysaccharide, BSP)結合並進行電紡以製備薄膜（WPU/BSP薄膜）。第二階段的研究，我們將觀察以下重點：1.嘗試以水為溶劑將WPU混合BSP做為神經薄膜的內膜基材，不使用有機溶劑，以降低BSP被破壞及有機溶劑之毒性疑慮； 2. WPU/BSP薄膜於許旺氏細胞遷移試驗、增生試驗，及神經元軸突外長試驗是否顯示正向促進神經修復的效果；3. 純WPU薄膜於動物實驗及動物神經傳導實驗是否能顯示其抗沾粘之效果，包括肌肉電位、神經傳導速率、肌肉收縮力量是否有較對照組為佳; 4. WPU/BSP薄膜於動物實驗中，效果是否比純WPU薄膜好； 5. WPU膜於兔子神經再接模型中，是否可維持數個月才被生物體完全降解。

Trauma of extremities are commonly associated with tendon and nerve injuries, and reconstruction of tendons or nerves are commonly followed by scarring and adhesion with unsatisfactory outcome. The purpose of this study is to design a biomaterial to: 1. apply around tendons and nerves after repair or reconstruction to reduce adhesion; 2. promote axon outgrowth of peripheral nerves. Therefore, in the thesis, there will be in vitro and in vivo studies related to tendons and peripheral nerves. In the US, about one third of trauma affect upper extremities and commonly cause tendon, muscle, and neurovascular damage. Because tendons heal slowly, thus postoperative splinting often lasts for more than 4 weeks, which also brings complications such as tendon adhesion and limited range of motion, which further compensates the working ability of patients and causes socioeconomic problems. Peripheral nerve injury (PNI) is often encountered because of the relatively superficial location of the nerves in the upper extremity, even a minor injury can cause impairment of sensory and motor functions, resulting in a nonfunctional hand. Because axons of peripheral nerves regenerate slowly (< 1 mm/day) even after meticulous repair, PNI especially in upper extremity trauma have been an important cause of morbidity and disability in both the working and nonworking population, with its true impact greatly underestimated. In the United States, 18 million acute upper extremity injuries resulted in 32 million days of restricted activity and 10 million lost working days over a period of 1 year, and the medical expanse and work loss related to just ulnar nerve injury reached 42000 USD per patient. In 2008, all PNIs resulted in approximately $150 billion spent in annual health-care dollars in the US. While the axons of peripheral nervous system have some capacity for regeneration after repair, the results are often unsatisfactory and only partially complete despite of advancement of microsurgery. In addition, the reconstruction options for those with poor functional return remain limited and prognosis remains disappointing. If axonal regeneration could be promoted, the consequences of sensory loss and weakness could be ameliorated in cases of severe PNI. However, current products on the market (eg. Seprafilm, Surgiwrap etc.) focus mainly on antiadhesion and are not designed for tendons or peripheral nerves. Those products do not promote axon regeneration and are relatively difficult to wrap around tendons or nerves during the surgery because they either breakdown or get attached to surrounding tissue easily. In addition, those products degrades within one to two weeks in vivo and could not offer long-term antiadhesion effect before the tendons and nerves heal. The present study is divided into two parts. Firstly, electrospun water-borne polyurethane (WPU) nanofibrous membranes (NFMs) were created for use after the repair or reconstruction of tendons to reduce adhesion. In the electrospinning process, water was employed as the solvent for WPU, and this solvent was ecofriendly and nontoxic. The nanofibrous architecture and pore size of the WPU NFMs were analyzed. Their microporosity (0.78–1.05 µm) blocked the penetration of fibroblasts, and therefore reduced the scarring and adhesion around the tendon during healing. The release of WPU mimicked the lubrication effect of the synovial fluid produced by the synovium around the tendon. In vitro cell studies revealed that the WPU NFMs effectively reduced the number of fibroblasts that became attached and that there was no significant cytotoxicity. In vivo studies with the rabbit flexor tendon repair model revealed that WPU NFMs reduced the degree of peritendinous adhesion, as determined using a gross examination; a histological cross section evaluation; and measurements of the range of motion of interphalangeal joints (97.1 ± 14.7 and 79.0 ± 12.4 degrees in proximal and distal interphalangeal joints respectively), of the length of tendon excursion (11.6 ± 1.9 cm), and of the biomechanical properties. In the first part of the study, WPU NFM was confirmed as a good carrier and offers antiadhesion effect. In the second part of the study, we integrated the Bletilla Striata polysaccharide (BSP) with the WPU to create NFMs (BSP/WPU NFM). This part of study would focus on: (1) the use of water instead of organic solvent as the major solvent for the process of electrospinning of WPU and BSP, in order to avoid the toxicity from organic solvent and to prevent destruction of BSP; (2) the observation of the BSP released from the BSP/WPU NFM and its effect on Schwann cell migration, Schwann cell proliferation, and in neurite outgrowth assays; (3) the effect of pure WPU NFM membrane in anti-adhesion and in functional outcome in animal studies, including compound motor action potential, nerve conduction velocity, and muscle contraction force; (4) comparison between BSP/WPU NFM and pure WPU NFM in cell and animal studies; (5) the duration before the BSP/WPU NFM is degraded in animal study.