研發新型牙髓幹細胞外泌體仿生支架應用於牙髓再生治療與開發可溶於尤加利油之高分子根管封填材料

Abstract

現代牙髓病學的治療範式正從單純的感染控制，轉向以組織保存與功能再生為核心的治療方式。然而，現行治療面臨兩大材料學瓶頸：一是傳統根管充填材料馬來膠缺乏與齒質的化學鍵結且難以移除，增加再治療的風險與難度；二是再生牙髓療法（REP）缺乏能精準調控微環境並誘導功能性牙本質—牙髓複合體（Dentin-Pulp Complex）再生的生物載體。 本研究旨在建立一套橫跨從傳統封填至再生療法的材料系統，分為兩部分進行探討。 第一部分聚焦於開發具備可再治療性的新型根管封填材料。 研究引入綠色化學概念，以可再生原料異山梨醇（Isosorbide）作為硬鏈段，合成生物基聚氨酯（Bio-PU）。經 FTIR、TGA 與 DSC 分析證實，Bio-PU 具備優異的熱穩定性與符合臨床操作的黏彈性。機械性質測試顯示 Bio-PU 的韌性顯著優於傳統馬來膠。在模擬再治療的溶解度測試中，硬鏈段含量 40%（HS40）的配方展現出最佳平衡，能被臨床常用溶劑尤加利油（Eucalyptol）軟化與溶解。 第二部分旨在開發搭載牙髓幹細胞外泌體（DPSC-Exo）的仿生支架系統。 研究首先利用尺寸排阻層析法（SEC）純化出高純度 DPSC-Exo，經次世代定序（NGS）證實其富含 miR-136，生物資訊分析顯示該分子具備調控血管生成與骨生成偶聯（Angiogenic-Osteogenic Coupling）的潛力。接著，本研究分別開發光固化水膠（γPGCL）與氫氧基磷灰石/明膠仿生支架（HGel）兩種載體，並比較其負載外泌體後的再生效能。大鼠顱骨缺損與牙髓暴露模型顯示，HGel 仿生支架結合 DPSC-Exo 能發揮最佳協同效應，其新生骨體積比（BV/TV）達 46.0%，並成功誘導緻密且連續的修復性牙本質橋形成，證實仿生礦化微環境對於外泌體誘導硬組織再生至關重要。 總結而言，本研究提出了一項整合性策略：在封填端，以 HS40 Bio-PU 提供高韌性且易於再治療的安全封填選擇；在再生端，利用 HGel 搭載 DPSC-Exo 透過 miR-136 機制重建牙髓功能。 此雙軌材料平台有望解決當前牙髓治療的關鍵臨床缺口。

Modern endodontics is shifting from a paradigm of infection control toward one focused on tissue preservation and functional regeneration. However, current therapies face two critical material limitations: first, traditional gutta-percha obturation lacks chemical bonding to dentin and is difficult to remove, increasing the risks and complexity of retreatment; and second, regenerative endodontic procedures lack bioactive carriers capable of precisely modulating the microenvironment to induce functional dentin–pulp complex regeneration. This study aims to establish an integrated material system spanning from retreatment to regeneration, divided into two parts. The first part focuses on developing a novel, retreatable root canal obturation material. Incorporating green chemistry concepts, a bio-based polyurethane was synthesized using renewable isosorbide as the hard segment. FTIR, TGA, and DSC analyses confirmed the material's excellent thermal stability and viscoelasticity suitable for clinical manipulation. Mechanical testing revealed that Bio-PU possesses significantly superior toughness compared to traditional gutta-percha. In solubility tests simulating retreatment, the formulation with 40% hard segment content exhibited the optimal balance, showing the capability to be effectively softened and dissolved by eucalyptol, a common clinical solvent. The second part focuses on developing a novel, retreatable root canal obturation material. Incorporating green chemistry concepts, a bio-based polyurethane was synthesized using renewable isosorbide as the hard segment. FTIR, TGA, and DSC analyses confirmed the material's excellent thermal stability and viscoelasticity suitable for clinical manipulation. Mechanical testing revealed that Bio-PU possesses significantly superior toughness compared to traditional gutta-percha. In solubility tests simulating retreatment, the formulation with 40% hard segment content exhibited the optimal balance, showing the capability to be effectively softened and dissolved by eucalyptol, a common clinical solvent. The second part focuses on the development of novel exosome-loaded biomimetic scaffolds. High-purity exosomes derived from dental pulp stem cells were isolated using size-exclusion chromatography. Next-generation sequencing revealed that these exosomes are enriched in various bioactive molecules. Bioinformatics analysis confirmed their potential to regulate both angiogenesis and the coupling of bone formation. Subsequently, two carrier systems were developed: a photocurable hydrogel and a hydroxyapatite/gelatin biomimetic scaffold. In rat calvarial defect and pulp exposure models, the HGel scaffold combined with DPSC-Exos demonstrated the best synergistic effect, achieving a bone volume fraction of 46.0% and inducing the formation of dense, continuous reparative dentin bridges. These results confirm that a biomimetic mineralized microenvironment is crucial for exosome-mediated hard tissue regeneration. In conclusion, this study establishes an integrated strategy: for obturation, the HS40 Bio-PU provides a tough, sealable, and easily retreatable option; for regeneration, the HGel system delivers DPSC-Exo to reconstruct pulp function. This dual-track material platform addresses critical clinical gaps in modern endodontic therapy.