利用新穎性生醫材料於再生醫學的應用：在牙組織再生及神經重建之研究

Abstract

再生醫學（regenerative medicine）是結合生物學、材料科學、工程學和醫學等學科以刺激病患自體組織器官再生，或是製造出具有功能性的類器官（organoids），利用移植來修復或替換體內因外傷、疾病或老化而受損的細胞、組織甚至器官。 組織工程（tissue engineering）是實現再生醫學的一種方法，藉由細胞、生醫材料（biomaterials）及適當的生長訊息（signals）相互調控，以促進生物的再生能力。其中，利用生醫材料製成載體（carrier）或支架（scaffold）來調控基因、生長因子及其他能與細胞作用之物質，得以探討幹細胞分化以及組織再生過程之分子作用機制與其未來的醫療應用。 本論文共分成兩個部分進行研究，主要以不同的新穎性生醫材料來進行組織再生，以探討其於再生醫學上的應用。第一部分是利用一種帶正電之膽固醇製成的磁性奈米載體：GCC-Fe3O4，遞送小分子核糖核酸-218（microRNA-218，miR-218）及其抑制劑，以刺激人類牙髓幹細胞（human dental pulp stem cells，hDPSCs）礦化（mineralization）。此奈米載體具有極高的遞送效率，並對細胞不產生明顯的細胞毒性（cytotoxicity），即使在含有血清的培養條件中也能展現出優異的遞送效果。研究結果顯示，小分子核糖核酸-218具有負向調控牙齒礦化之功能，藉由抑制小分子核糖核酸-218，牙髓幹細胞之礦化現象明顯增加。此現象也被證實與MAPK/ ERK（mitogen-activated protein kinases/ extracellular signal-regulated kinases）之訊息傳導路徑有關。由於GCC-Fe3O4奈米載體中含氧化鐵並帶有磁性，未來可進一步利用磁力進行操控，並能以非侵入式顯影系統做即時性的體內追蹤，無論是在生醫研究領域或臨床治療上都能提供更廣泛的應用性。 成人的神經系統再生能力有限，一旦受損，往往是永久而難以復原的。在牙齒幹細胞的分化研究中，我們曾經嘗試以誘導神經分化的培養基（neurodifferentiation medium）進行幹細胞之神經性分化，但其結果並不理想。因此，第二部分的研究專注於神經組織的再生，利用幹細胞及生物相容性支架，進行組織工程，以創造出視網膜神經節細胞（retinal ganglion cells，RGCs）最佳的生長環境。此部分利用聚谷氨酸苄酯（poly-γ-benzyl-L-glutamate，PBG）製成三維立體支架（3D scaffold），將自人類胚胎幹細胞（human embryonic stem cells，hESCs）誘導分化成的視網膜神經節前驅細胞（retinal ganglion cell progenitors，RGCPs）、視網膜神經節細胞及視網膜組織分別培養於其中，以觀察其神經生長與再生之情形。研究結果發現，PBG支架相較於組織工程中常見的聚己內酯（polycaprolactone，PCL）支架，不但能支持神經細胞的生存，更能顯著的促進神經纖維生長（neurite outgrowth）。藉由視神經的重建，不但能讓視覺退化之病患得以重見光明，並為同樣難以修復之中樞神經系統創造再生的可能性。 整體而言，藉由開發新穎性的生醫材料進行組織工程，以促進再生醫學之各種應用，不論是在牙齒再生或神經的重建，都具有極大的臨床實用價值。若能有效減輕病患的痛苦，並改善其生活品質，對病患自身及全體人類的健康都將是一大福祉。

Regenerative medicine combines biology, materials science, engineering, and medicine to replace or regenerate cells, tissues or organs for restoring or establishing their normal functions. It can be achieved by stimulating autologous repair mechanisms for self-regeneration or creating functional organoids for transplantations to repair or replace damaged cells, tissues, or even organs that caused from trauma, diseases or aging in the body. Tissue engineering is considered as one of the major approaches for regenerative medicine, which combining cells, biomaterials, and appropriate signals to promote the regeneration abilities of organisms. Novel biomaterials can be made into carriers or scaffolds to manipulate genes, growth factors and many other cell-reacting substances for exploring the mechanisms of stem cell differentiation and tissue regeneration as well as developing future medical applications. This dissertation includes two parts of research by exploiting different biomaterials for tissue regeneration as applications in regenerative medicine. In the first part, a cationic cholesterol-based magnetic nanocarrier: GCC-Fe3O4 (GCC-Fe) was developed to deliver microRNA-218 (miR-218) and miR-218 inhibitor for promoting mineralization potentials of human dental pulp stem cells (hDPSCs). Results showed that this nanocarrier had an extremely high transfection efficiency with insignificant cytotoxicity. Furthermore, the transfection efficiency was not disturbed under serum-containing culture conditions. On the other hand, it showed that miR-218 had a negative regulation role in hDPSCs mineralization. By inhibiting miR-218, the mineralization degree of hDPSCs was increased significantly. It was also confirmed that this induced mineralization was related to the mitogen-activated protein kinases/ extracellular signal-regulated kinases (MAPK/ ERK) pathway. Since GCC-Fe contains superparamagnetic iron oxide (SPIO) nanoparticles, it can be further manipulated by magnetic force, and tracked by non-invasive imaging systems in vivo. GCC-Fe has great potentials to be widely applied in biomedical researches as well as clinical applications. The regeneration ability of adult nervous system is limited. Once impaired, often lead to permanent damages which were nearly impossible to recover. In the previous study of stem cell differentiation, the attempt of using neurodifferentiation medium for human dental stem cells induction was not satisfying. Therefore, the second part of this dissertation focused on nerve regeneration. In this study, three-dimensional (3D) scaffolds of poly-γ-benzyl-L-glutamate (PBG) and polycaprolactone (PCL) were designed with different alignments. The survival and regeneration abilities of human embryonic stem cell (hESC)-derived retinal ganglion cell progenitors (RGCPs), primary retinal ganglion cells (RGCs), and retinal explants were used for evaluation respectively. Results indicated that PBG scaffolds could support the survival of neurons and promote long and robust neurite outgrowth compared to PCL scaffolds. Through this tissue engineering approach, not only the optic nerve can be reconstructed for vision impaired patients to restore their vision; but also the optic nerve can be served as a model for studying regeneration of the central nervous system (CNS). In conclusion, developing novel biomaterials to fulfill all kinds of applications in regenerative medicine is crucial. No matter for tooth regeneration or nerve reconstruction, they all have great values in clinical practice to alleviate the suffering of patients and improve their quality of life. With the progress of tissue engineering, it will further promote the health of individuals as well as the well-being for all.