探討鹼化改質3D列印聚己內酯之材料於神經分化以做為治療脊髓損傷之應用

Abstract

脊髓損傷(Spinal cord injury, SCI)是一種既嚴重又複雜的神經系統病症， 會造成無法恢復的神經嚴重損壞，導致身體主要的運動、感覺和自主神經功能障礙，嚴重一點的脊髓損傷可能還會影響四肢的活動甚至癱瘓，一輩子都需要別人照顧。以往針對脊髓損傷較常見的治療方法有藥物治療、關節固定、外科手術、復健等，但這些都無法完全治癒脊髓損傷。現今醫療發達，已有一些很有前景的新興治療方法正在被研究中，例如 : 神經再生，而讓神經再生的方法有幹細胞移植、奈米技術、生物材料、組織工程等。 本論文是利用3D列印做出聚己內酯(Polycaprolactone, PCL)支架，再將其表面透過氫氧化鈉做不同程度的鹼化，使PCL支架的親水性增加，讓細胞可以在濕潤的環境生長；使PCL支架表面更加粗糙，讓神經細胞可以更容易攀附生長。 材料分析藉由水接觸角(Contact angle)分析材料親疏水性；微細形狀測定機 (surface profiler)分析材料的粗糙度；傅立葉轉換紅外線光譜儀(FTIR)分析材料表面官能基；差示掃描量熱法(DSC)測量材料隨溫度範圍的熱性質，以及利用光學顯微鏡(Optical Microscope, OM)、金相顯微鏡(Metallographic Microscope)、掃描式電子顯微鏡(Scanning Electron Microscope, SEM)觀察材料表面的結構和形貌。PCL支架之生物相容性分析選用PC12細胞進行測試，用Presto Blue測試細胞在材料上的活性以及用SEM觀察細胞附著在支架上的生長情形。並且用神經生長因子(Nerve Growth Factor, NGF)誘導PC12細胞神經分化1、4、7天後用免疫螢光染色觀察分化出的神經數量、長度差異以及細胞群分布狀況。觀察鹼化不同濃度與時間對材料及神經細胞的影響。 實驗結果顯示，鹼化PCL支架會增加材料表面親水性及粗糙度，支架表面結構會從光滑、平整變為粗糙、凌亂，也多了許多孔洞，且鹼化時間越久或鹼化濃度越高，這些現象會更顯著；FTIR結果顯示鹼化PCL支架會增加水解的極性官能基，使其酸含量變多；DSC結果顯示在鹼化過程中產生結晶的主要原因是溫度的變化；cell viability實驗結果顯示鹼化處理過之PCL材料較適合細胞生長，此外，鹼化濃度越高，作用時間越久的材料更適合細胞生長及貼附；由掃描式電子顯微鏡實驗結果得知細胞隨培養天數增加會從單顆或小群聚的狀態變成大群聚的狀態，且形狀會從一顆一顆粒粒分明的形狀變成細長並且延伸，鹼化濃度高的材料其細胞質具有絲狀延伸物，此延伸物有助於細胞固定在材料表面；神經細胞分化免疫螢光染色結果顯示鹼化處理時間越久或濃度越高之PCL材料使細胞更趨近群聚生長狀態，且也更利於細胞神經分化以及交感神經元(類神經元)長度也會長得較長。 從實驗結果可得結論，鹼化PCL支架會增加材料表面親水性、粗糙度、極性官能基、孔洞等，使表面結構改變，且鹼化時間越久或鹼化濃度越高，這些現象更為顯著；然而，這些改變會使材料更適合神經細胞生長、貼附及分化。本實驗鹼化濃度最高且鹼化時間最久之PCL為PCL-3M-24hr，此材料的親水性是所有材料中最為親水的；粗糙度是所有材料中最為粗糙的，細胞實驗結果也顯示此材料是所有材料中最利於細胞生長、貼附以及最有利於神經分化的。

Spinal cord injury(SCI)is a serious and complex neurological condition that causes severe and irreparable damage to nerves, resulting in major motor, sensory and autonomic dysfunction of the body. If it is more serious, it may affect the activities of the limbs or even paralyze, and it will need others to take care of it for the rest of its life. In the past, the more common treatment methods for spinal cord injury included drug therapy, joint fixation, surgery, rehabilitation, etc., but none of these could completely cure the spinal cord injury. With the development of medical treatment nowaDays, some promising emerging treatment methods are being studied, such as: nerve regeneration. Methods for nerve regeneration include stem cell transplantation, nanotechnology, biomaterials, tissue engineering, etc. This study mainly that we used 3D printing to make Polycaprolactone(PCL)scaffolds, and to modify the surface of the scaffolds to help the nerves have a better growth environment. The surface of PCL scaffolds were alkalized to varying degrees with sodium hydroxide to increase the hydrophilicity of the PCL scaffolds, allowing cells to grow in a humid environment and making the surface of the PCL scaffolds rougher, allowing nerve cells to grow more easily. The hyphophilicity and hydrophobicity properties of PCL materials were analyzed with contact angle measurement. The surface roughness of the PCL materials were analyzed by Surface profiler. The functional groups on the surface of the PCL materials were analyzed by Fourier Transform Infrared Spectrometer(FTIR). Measuring thermal properties of materials over temperature range with Differential Scanning Calorimeter(DSC), and observing the structure and morphology of the PCLmaterials surface with Optical Microscope(OM), Metallographic Microscope, and Scanning Electron Microscope(SEM). PC12 cells were used to analyze the biocompatibility of PCL scaffolds, and test cell viability with Presto Blue. The growth of cells attached to the scaffold was observed by SEM. Nerve growth factor(NGF)was used to induce PC12 cell neural differentiation for 1, 4, and 7 Days, and immunofluorescent staining was used to observe the number and length of differentiated nerves and the distribution of cell populations. The results showed that the alkalized PCL scaffold would increase the hydrophilicity and roughness of the materials. The surface structure of the scaffolds would change from smooth and flat to rough and messy, and there would be many holes, and the longer the alkalization time or the higher the alkalinization concentration, the phenomenon would be more pronounced . FTIR results showed that alkalized PCL scaffolds would increase the hydrolyzed polar functional groups and increase the acid content. DSC results showed that the main cause of crystallization during alkalization was the change of temperature. Cell viability experiments showed that alkalized PCL materials is more suitable for cell growth. In addition, materials with higher alkalization solution concentration or longer alkalization times would be more suitable for cell growth and attachment. Results of the scanning electron microscope experiment showed that the cells would change from single or small clusters to large clusters with the increase in the number of Days of culture, and the shape would change from a single granular shape to a slender and elongated shape. Materials with a high concentration of alkalization, its cytoplasm has filamentous extensions, which help cells to fix on the surface of the material. Results of immunofluorescent staining on nerve cell differentiation showed that the materials with higher alkalization solution concentration or longer alkalization times, its cells would closer to the cluster growth state, and it was also more conducive to the neural differentiation of cells and the length of sympathetic neurons would be longer. According to the results, it can be concluded that alkalization of PCL scaffolds would increase the surface structure of the material such as hydrophilicity, roughness, polarity functional groups, and pores, and these phenomena would be more significant when the longer of the alkalization time or the higher the alkalization concentration. However, these changes would make the materials more suitable for the growth, attachment and differentiation of nerve cells. The PCL with the highest alkalization concentration and the longest alkalization time in this experiment is PCL-3M-24hr. The hydrophilicity of this material is the most hydrophilic among all materials; the roughness is the roughest among all materials, and the cell experiment also shows that this material is the most conducive to cell growth, attachment and neural differentiation among all materials