Effect of an Epineurial-like Biohybrid Nerve Conduit on Nerve Regeneration

本研究建立一個新方法，藉由植覆脂肪幹細胞(ADSCs)於聚乳酸(PLA)導管外壁製作仿生導管。運用氣泡成形法及相轉移製作多孔性的聚乳酸導管，並測試兩種不同孔洞結構(GS(< 44 m)) and GL(44~125 m))之機械性質、滲透率、含水率及降解特性，分別於導管上植覆脂肪幹細胞(ADSC)進行細胞活性分析及貼附生長情形之觀察。 由測試結果證實GS基材具有較佳的結構穩定性、滲透性、孔隙率，而細胞活性測試4、7、10天結果證實，GS導管具較佳的細胞活性。將植覆脂肪幹細胞(ADSCs)於聚乳酸(PLA)導管外壁培養7天後，植入大鼠周邊神經斷端10 mm及6周的試驗，進行神經再生及神經功能恢復的評估，運用步態分析、電生理及組織學評估。於神經功能試驗結果表明自體神經(Autograft)及植覆ADSCs的AGS導管有較佳的恢復，於電生理測試中，Autograft組及AGS組相較於GS及AGL導管有較佳的振幅及神經傳導速率。組織學分析中，Autograft組及AGS組形成較緻密的神經再生組織及結締組織，而AGS組的神經的再生面積、髓鞘化的神經數目均較GS及AGL組為佳。ADSCs可增加血管增生而增加營養的傳輸，由S-100染色結果可見AGS組再生神經的分佈較GS及AGL組為佳。由此結果猜測ADSCs與內生性的許旺細胞交互作用並產生神經營養因子而促進周邊神經的再生，而導管的設計亦為產生神經外膜狀的生物性神經導管的支持關鍵。A novel approach of making a biomimetic nerve conduit was established by seeding adipose-derived adult stem cells (ADSCs) on the external wall of porous poly(D, L-lactic acid) (PLA) nerve conduits. The PLA conduits were fabricated using gas foaming salt and solvent-nonsolvent phase conversion. We examined the effect of two different porous structures (GS and GL) on ADSC growth and proliferation. The GS conduits had better structural stability,permeability, and porosity, as well as better cell viability at 4, 7, and 10 days. The epineurial-like tissue was grown from ADSC-seeded conduits cultured for 7 days in vitro and then implanted into 10 mm rat sciatic nerve defects for evaluation. The regeneration capacity and functional recovery were evaluated by histological staining, electrophysiology, walking track, and functional gait analysis after 6 weeks of implantation. Experimental data indicated that the autograft and ADSC-seeded GS conduits had better functional recovery than the blank conduits and ADSC-seeded GL conduits. The area of regenerated nerve and number of myelinated axons quantified based on the histology also indicated that the autograft and AGS groups performed better than the other two groups. We suggested that ADSCs may interact with endogenous Schwann cells and release neurotrophic factors to promote peripheral nerve regeneration. The design of the conduit may be critical for producing a biohybrid nerve conduit and to provide an epineurial-like support.Content Abstract…………………………………………………………………………………. I 摘要……………………………………………………………………………………... II Content………………………………………………………………………………….. III List of Tables…………………………………………………………………………… VI List of Figures…………………………………………………………………………... VII Chapter 1 Introduction…………………………….…………………...………...……. 1 1.1 The connective tissue of the peripheral nervous system…………………..….. 1 1.2 Surgical treatments for peripheral nerve injury……………………….……... 1 1.3 Nerve conduit application for peripheral nerve injury………………..……… 2 1.4 Effect of Adipose-derived stem cells (ADSCs) on nerve regeneration……….. 2 1.5 Seeding method of cells…………………………………………………………. 3 1.6 Specific Aim……………………………………………………………………… 3 Chapter 2 Review of literature………………………………………………………... 5 2.1 Anatomy of peripheral nerve…………………………………………………… 5 2.2 Electrophysiology of nervous system…………………………………………... 6 2.2.1 perineurium…………………………..………………………………………. 6 2.2.2 perineurium…………………………………………………………….…..… 7 2.2.3 epineurium……………………………………………………………….…... 8 2.3 Extracellular matrix (ECM) …………………………………………………… 9 2.3.1 Compositions of extracellular matrix (ECM) ………………….……………. 9 2.3.2 The effect of ECMs on tissue engineering nerve conduit………..…………… 11 2.4 Tissue engineering nerve conduits application on peripheral nerve repair..... 14 2.4.1 Functional nerve conduit………………………………………………..…… 15 2.4.2 Fabrication methods for nerve conduit…………………………………...…. 16 2.4.3 The physical properties of the nerve conduit…………………………............ 17 2.4.4 Bioactive nerve conduit…………………………………………………….... 18 2.4.5 The effect of neurotrophic factors on peripheral nerve regeneration………... 20 2.4.6 The effect of mesenchymal stem cells (MSCs) on peripheral nerve generation. ………………………………………………………………….... 22 2.5 Evaluation methods in peripheral nerve regeneration………………………... 24 2.5.1 Assessment of functional recovery…………………………………………… 24 2.5.2 Electrophysiological study………………………………………………..….. 26 2.5.3 Gastrocnemius muscle weigh……………………………………………...…. 28 2.5.4 Nociceptive withdrawal reflex (WRL)……………………………….……….. 29 2.5.5 Histological examination………………………………………...................... 30 2.5.6 Magnetic resonance imaging (MRI)…………………………………………. 31 Chapter 3 Materials and Methods…………………………………………………….. 33 3.1 Fabrication of PLA substrates and conduits…………………………………... 33 3.2 Characterization of the PLA substrates……………………………………….. 33 3.3 Isolation and cell culture of rat ADSCs………………………………………... 34 3.4 Seeding rat ADSCs on the external wall of the conduit………………………. 35 3.5 Gene analysis………………………………………………………………......... 36 3.6 Surgery………………………………………………………………………….. 38 3.7 Functional assessment of nerve regeneration……………………………......... 39 3.8 Electrophysiological study……………………………………………………… 40 3.9 Histological examination………………………………………………………... 40 3.10 Statistical analysis……………………………………………………………… 41 Chapter 4 Results………………………………………………………………………. 42 4.1 The microstructure and characteristics of the PLA substrates………………. 42 4.2 Cell studies in vitro……………………………………………………………… 45 4.3 In vivo animal implantation……………………………………………….......... 48 4.3.1 Gait analysis………………………………………………………….……… 48 4.3.2 Electrophysiological study……………………………………..……………. 48 4.3.3 Tracking of DiI-labeled ADSCs……………………………………………… 51 4.3.4 Histological examination………………………………………………..…… 52 Chapter 5 Discussion………………………………………………............................... 56 Chapter 6 Conclusions…………………………………………………………………. 59 Future work……………………………………………………………………………. 60 References………………………………………………………………………..……. 61 List of publication……………………………………………………………………… 74 Introduction to Author………………………………………………………………… 7