2014-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655854摘要：牙周炎（Periodontitis）是由牙周致病菌誘發之發炎反應所造成之牙周組織破壞的結果，根據衛生署的統計，牙周炎侵犯超過60%的台灣成年人，並已成為成人牙齒脫落的主要原因之一。要修復受到破壞牙周組織，除了控制牙周發炎之外，亦必須阻隔不利於牙周再生細胞之長入、及藉由提供生物活性因子以增加組織之再生能力。然而，即使生物活性因子、如血小板衍生生長因子（PDGF）和牙釉基質衍生物（EMD）已被證實具有促進再生功效，目前臨床療法仍無法有效重建牙周軟組織（如牙周韌帶）；另一方面，對於組織再生過程中之發炎反應也缺乏有效的控制。基於我們過去透過動物實驗證明生物活性因子之再生促進功效，以及抗糖化劑（anti-glycation agent）的抗發炎作用之成果，在本計畫中我們提出四組元載體（four-component scaffold）的設計以其能更有效率、更精確地完成牙周組織再生。我們研究方向將分為三個重點主題一、製作符合牙周再生需求的四組元載體載體將分為硬組織組元、軟組織組元、屏障組元、以及發炎偵測組元四個部分。硬組織組元將由經鍛燒並保留柱狀骨結構之牛骨，並攜帶促進骨生成活性因子，用以促進齒槽骨再生；軟組織組元則使用gelatin/chondroitin-6-sulfate/hyaluronic acid (G-C6S-HA) 交互鍵結之纖維結構攜帶活性因子以促進牙周韌帶再生；屏障組元位於載體表面，使用gelatin 或G-C6S-HA 製成小於10 um 孔隙之緻密纖維結構以阻隔牙齦軟組織之滲透增生；發炎偵測組元則以氫氧磷酸鈣為主體攜帶抗糖化劑製成pH-response 水膠，以注射方式填充至載體內部。載體之物理性質、攜帶藥物或因子之效率、因子體外釋放狀況及生物相容性將會分別進行評估。二、確立牙周韌帶再生之較佳活性因子規劃將使用第一部份製作、不含發炎偵測組元之三組元載體，在軟組織組元培養牙周韌帶細胞，攜帶PDGF、EMD、或是PDGF+EMD，並於其上分別覆蓋象牙質（dentin）薄片或是鈦金屬薄盤後，植入SCID mice 背部皮下組織，對於牙周韌帶再生型態及排列進行組織學、組織化學、及免疫組織化學染色觀察。三、評估四組元載體攜帶活性因子之活體生物相容性及牙周再生之效能將使用第一部份製作之四組元載體，於軟組織、硬組織、及發炎偵測組元中攜帶不同組成之活性因子及抗糖化劑，植入Sprague-Dawley Rat 之背部皮下組織及大型牙周骨缺損區以觀查體內生物相容性及牙周再生效能。生物相容性將透過IVIS Specturm 對caspase activity 進行及時評估，並觀察周圍組織反應。牙周再生則透過微電腦斷層影像、組織切片、及免疫組織染色進行質化及量化評估。藉由完成這個研究計畫，可對於經由結合抗發炎及活性因子而驅使之牙周再生有進一步的認識；而針對牙周組織個別需求創造之四組元載體，透過生物活體實驗所得到之資訊，更可提供建立新一代牙周治療術式之理論基礎。<br> Abstract: As a consequence of extreme inflammation, periodontitis is characterized by the damage of multicompartmental tooth-supporting structure. Periodontitis has affected more than 60% population and become one of the major causes of tooth loss in Taiwan. To restore the damaged tissue, it is essential to control inflammation, exclude unwanted tissue, and promote regenerative capability. Although someinvestigations had implicated that platelet-derived growth factor (PDGF) and enamel matrix derivatives(EMD) was able to regenerate the whole periodontal apparatus, regeneration of periodontal ligament (PDL), the soft tissue compartment of periodontium, was still clinically unpredictable. On the other hand, since the anti-inflammatory effect of anti-glycation agents and capability of growth factors as well as bioactive molecules had been confirmed in our previous studies, we propose to develop a four-component scaffold to effectively regenerate periodontal tissues, and the following three specific aims will be specifically investigated in this study,1. To fabricate a four-component scaffold fulfilling the requirement of periodontal regeneration.The scaffold will consist of a soft tissue compartment by gelatin/chondroitin-6-sulfate/hyaluronic acid (G-C6S-HA) meshwork encapsulating bioactive molecules for regenerating PDL, and hard tissue compartment by calcined bone matrix encapsulating osteo-promtive factor for alveolar bone. The occlusive surface of scaffold will be covered by a dense gelatin or G-C6S-HA layer with the pore size less than 10 um to prevent the downgrowth of unwanted soft tissue, and the whole scaffold will be occupied with pH-sensitive hydrogel containing anti-glycation agents in order to control inflammation. The physical properties, controlled release profiles and in vitro biocompatibility of scaffold with encapsulating molecules will be evaluated.2. To evaluate the most appropriate formulation to regenerate periodontal ligament (PDL).By utilizing the scaffold without adding pH-sensitive hydrogel in SA1, PDGF, EMD, or PDGF/EMD combination will be applied in the soft tissue component of scaffold with the pre-loading of PDL cells and covered by a dentin slice or titianium disc. By delivering this composite to the subcutaneous pouch of SCID mice, the formation and alignment of PDL and cementum will be evaluated by histology, histochemistry, and immunohistochemistry.3. To characterize the biocompatibility and efficiency of periodontal regeneration from different formulations of anti-glycation agents and bioactive molecules carried in the scaffold.The four-component scaffold fabricated in SA1 will be delivered to the subcutaneous tissue and periodontal osseous defects of Sprague-Dawley rats under different formulations of bioactive molecules and anti-glycation agents to assess in vivo biocompatibility and regeneration efficiency. Biocompatibility will be evaluated by realtime caspase activity using the IVIS spectrum and histology, and the regeneration efficiency in periodontal regeneration will be evaluated by micro-computed tomography, histology, and immunohistochemistry.Accomplishing this study will aid better understanding of periodontal regeneration driven by the coordination of anti-inflammatory and bioactive molecules. On the other hand, the results from the in vivo examinations of the four-component scaffold will enable us to establish the fundamental of a novel modality for treating periodontitis.牙周炎組織工程生長因子載體periodontitistissue engineeringgrowth factorscaffold