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|標題:||Effects of Ginkgo biloba extract EGb 761 and ultrasound on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid) conduits for peripheral nerve regeneration
|關鍵字:||銀杏;Ginkgo biloba;低強度超音波;聚乳酸聚甘醇酸共聚物;矽膠管;史旺氏細胞;神經再生;組織工程;low-intensity ultrasound;poly(DL-lactic acid-co-glycolic acid);silicone conduit;Schwann cells;nerve regeneration;tissue engineering||出版社:||化學工程學系||摘要:||
在組織工程神經導管的研究中，導管內植覆的史旺細胞（Schwann cells）是否能恢復功能是一個極大的挑戰。本研究分別應用銀杏葉粹取物(EGb 761)與低強度超音波刺激(low-intensity ultrasound)來保護與活化植入聚乳酸聚甘醇酸共聚物(PLGA)神經導管內之Schwann cells。研究首先利用無血清之培養液來模擬周邊神經外傷之環境，以培養Schwann cells，槽中再加入不同濃度之EGb 761 (0, 1, 10, 20, 50, 100, 200 g/ml)。結果發現加入EGb 761後，細胞貼附與存活率增加、LDH減少、MTT增加，其中又以10-100 g/ml最明顯。動物實驗方面;6×103 個Schwann cells植入神經導管中，管內並填入含有不同濃度EGb 761(0, 10, 50, 100 g/ml)之明膠 (gelatin)以防滲出。導管植入10 mm之老鼠坐骨神經缺陷中。自體移植(autograft)當作另一控制組以進行比較。在肌電圖評估方面：運動神經元動作電位(MUAP)及顫動電位(Fib)於第2,4及6週測量。實驗動物於手術後6週犧牲，結果發現Fib隨著時間訊號減弱，此代表肌肉漸漸恢復；MUAP則於4週後才出現，並且實驗組皆明顯優於控制組(0 g/ml)及自體移植組，尤其是 10 g/ml組，另外於組織切片中，10 g/ml髓鞘數目(axon)亦為最多。
另一方面在進行低強度超音波實驗時，導管是否具有生物降解性(biodegradation)亦列入考慮。我們利用PLGA與silicone作為神經導管材料，將。細胞植入兩種導管後於無血清培養液中進行培養，超音波於12, 24 及 48小時後以強度0.05 W/cm2，刺激時間每次3 分鐘，進行刺激。結果發現PLGA導管中，有經過超音波刺激組的LDH下降，MTT明顯上升，尤其與未刺激的比較更明顯。在動物實驗方面，9 × 103 Schwann cells植入PLGA 與Silicone 神經導管中，導管兩端並縫入老鼠坐骨神經15 mm之缺陷上，每隻老鼠在2週中接受12次治療，每次5分鐘，頻率 1MHz、強度0.3 W/cm2 (SATP)。手術8週後犧牲並取出植入之導管。
研究發現添加適當濃度的EGb 761(10-50 g/ml)於組織工程神經導管中，可增加植覆的Schwann cells的效率，並增加再生神經髓鞘數目與面積，加速功能性之恢復。另一方面有超音波刺激之PLGA神經導管其髓鞘數目與面積皆比未刺激的大很多。另外超音波刺激Silicone導管，會引發大量纖維組織包覆植入的導管，並延遲髓鞘再生。
Functional restitution of seeded Schwann cells in artificial nerve channels following trauma or disease is a challenge for developing a tissue-engineering conduit for peripheral nerve regeneration. This study both investigated the effects of Ginkgo biloba (EGb 761) extract and low-intensity ultrasound on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid)(PLGA) conduits by in vitro and in vivo trials for peripheral nerve regeneration. At first, the seeding efficiency of Schwann cells in serum-deprived culture medium, which simulated the environment of mechanical trauma on an injured nerve site, was improved by adding different dosages of EGb 761 (0, 1, 10, 20, 50, 100, 200 g/ml). The analytical results showed enhanced cell attachment and survival, reduced LDH release and increased MTT values, particularly in the range 10-100 g/ml. The PLGA nerve conduits seeded with Schwann cells (6×103 cells) and filled with gelatin containing EGb 761 (0, 10, 50, 100 g/ml) were implanted to 10 mm right sciatic nerve defects in rats. Autograft was performed as another control. Electromyography was assessed based on the motor unit action potential (MUAP) and fibrillation potential (Fib) at 2, 4 and 6 weeks during all periods. The specimens of the experimental and control groups were harvested for histological analysis at 6 weeks after surgery. The Fib was found to gradually decay, and the MUAP was found to not be present until 4 weeks after surgery. Meanwhile, the experimental groups were all statically better than the control group (without EGb 761) and autografts were observed at 6 weeks, especially at the concentration of 10 g/ml, where there was higher amplitude of MUAP and a significantly larger number of myelinated axons.
In addition, low intensity ultrasound was utilized on seeded Schwann cells within PLGA conduits by in vitro and in vivo trials as well. The possible differences in the ultrasonic effects between biodegradable and non-biodegradable materials as the conduits were also studied, by using silicone rubber tubes as comparisons. The application of 0.05 W/cm2, 3 min/treatment of ultrasound was applied on the Schwann cells in serum-deprived culture medium. After 12, 24 and 48 hr, only PLGA conduit groups had shown the release of LDH decreased and the MTT values increased, as compared to the sham groups. The PLGA nerve conduits seeded with Schwann cells (9 × 103 cells) were implanted to 15 mm right sciatic nerve defects in rats. Each conduit received 12 ultrasonic treatment sessions over 2 weeks after one day of resting. Ultrasound was applied as follows: frequency 1MHz, intensity 0.3 W/cm2 (SATP), treatment 5 min/day. The sham groups were utilized under the same condition for comparison. The specimens of implanted grafts were harvested for histological analysis 8 weeks after surgery.
It was concluded that a proper concentration of EGb 761 (10-50 g/ml) promoted seeding efficiency of Schwann cells in a tissue engineered PLGA conduit. Addition of EGb 761 in Schwann cells-seeded conduit could increase the total number of myelinated axons in nerve regeneration and improve peripheral nerve functional recovery. We also found that PLGA groups (with and without Schwann cells) with pulsed ultrasonic stimulation resulted in a significant acceleration in number and area of regenerated axons at the midconduit of implanted grafts, as compared to sham groups. The ultrasonic stimulation activated the seeded Schwann cells in MTT testing. And in the vivo trial, the ultrasonic application on silicone groups induced mass fibrous tissues that covered the nerve conduits and retarded the axon regeneration.
KEY WORDS: Ginkgo biloba, low-intensity ultrasound, poly(DL-lactic acid-co-glycolic acid), silicone conduit, Schwann cells, nerve regeneration, tissue engineering.
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