林立德臺灣大學:臨床牙醫學研究所蕭仰嶔Siaw, Yang-KhimYang-KhimSiaw2010-05-262018-07-092010-05-262018-07-092009U0001-1808200913282100http://ntur.lib.ntu.edu.tw//handle/246246/184178自Branemark等人於1977年提出人工植體的骨整合理論及植體長期使用的臨床結果以來,人工牙根的發展至今已相當成熟.且可以達到九成以上的成功率及存活率.植體的表面處理已從機械切割的光滑表面進展到各種粗糙度的表面處理,植體的外型也向圓錐螺紋的設計. 然而臨床上為了要突破那一成不到的失敗及併發症,甚至是要更快速且穩定形成植體骨整合,也由於對骨癒合過程中分子生物機轉的瞭解,目前的研究是致力於植體表面找出有效黏附並可以促進植體周圍組織癒合的分子接合,以期達到更快速達到骨整合的目的. 先前研究指出，利用噬菌體顯現法技術可以篩選出與鈦合金表面專一性結合的標的胜肽，在本實驗中利用此方法,找出一株對鈦合金表面有高度親和力的噬菌體—TBP-E, 進一步將TBP-E對應的十二個氨基酸胜肽TBP-E2合成後發現相對的具有相對高度的親和力,未來則希望可以在本胜肽截取更小的功能性胜肽或結合上已知功能的生長因子,活化鈦合金表面並可提供未來一個植體表面處理中置入有利於骨整合及組織癒合新穎的治療方式。Since Prof. Branemark introduced the methodology of osseointegrated dental implantology in 1960s, the development of dental implantation technology has become mature and the success rate of treatment has reached an incredible level of more than 90 percent in average. Titanium is the most widely used metal in orthopedic joint replacement, fixation screws and plates, and dental implants. Titanium is preferred over other metal materials because it provides good biocompatibility and corrosion resistance with very low allergenicity. Even though titanium owns such superiority, some problems and limitations of its application in dental implantation still remain to be improved. For instance, when titanium dental implants are used, patients are generally obliged to endure restricted mastication for several weeks until the tight adhesion and osseointegration between the implant and local tissues is established. In addition, dentists also face the failure of implantation and other complications, such as progressive marginal bone loss or recession of marginal mucosa, in spite of its high success rate reported in the literature. Therefore, shortening the time required for osseointegration and enhancing the success rate of implantation through appropriate implant modification are major objectives to substantially improve patients’ life quality. To achieve those objectives, a variety of methods for surface modification of titanium implants have been proposed, including plasma flame spraying, sandblasting, acid-etching, and coating with ceramic. In this study, we screened a phage displayed random peptide library for searching peptides that can specifically bind onto titanium surface and then we identified the binding properties of those titanium binding peptides. We identified two phage clones named TBP-A and TBP-E showing different binding affinities after the biopanning procedures. The corresponding peptides were then synthesized and studied with the following competitive binding inhibition with original phage clone and synthetic peptide binding experiments. Among all the synthetic peptides, TBP-E2 has dominant binding affinity compared to others The novel peptide that can specifically bind to titanium may be good a candidate to immobilize growth factors onto implant surface to achieve the goal of improving titanium surface.口試委員會審定書 Ⅰ要 ⅡBSTRACT ⅢNTRODUCTION 1 Current Limitation of Dental Implant 1 Appropriate strategies for implant surface modification 2 Biomimetics: Future trends in Implant Dentistry 3 Challenges of biomimetic implant surface modification 5 Using peptide binders to link the implant surface and bioactive gents 6hage Display 8tudy Objective 12ATERIALS AND METHODS 13 Phage-Display Chemical-panning/Bio-panning Procedures 13reparation of Phage Stock 14hage Titering 15laques Amplification for ELISA or Sequencing 16dentification of Titanium-binding Phage Clones by LISA 16NA Sequencing and Computer Analysis 18LISA-based Binding Assays of Phage Particles 18eptide Synthesis 19n vitro Peptide Competitive Binding Inhibition of Phage lone TBP-A and E 20LISA-based Binding Assays of Synthetic Peptides 21tatistical analysis 21ESULTS 22hage-Display Chemical-panning/Bio-panning 22dentification of Titanium-binding Phage Clones 23LISA-based Binding Assays of TBP-A and TBP-E hage Clones (Fig. 3) 24ompetitive Inhibition of Synthetic Peptide to original hage clone 25inding of the synthetic peptide to Ti-6Al-4V disc 27ISCUSSION 29hage-Display Chemical-panning/Bio-panning 29dentification of Titanium-binding Phage Clones 30LISA-based Binding Assays of TBP-A and TBP-Ehage Clones 31ompetitive Inhibition of Synthetic Peptide to Original hage Clone 32inding of the Synthetic Peptide to Ti-6Al-4V Disc 33eptide sequence analysis and functional motif characterization 34roposed experimental focus in the future 35uture perspectives 37EFERENCES 38IGURES 41ig. 1A. Five rounds of biopanning were done on Ti-6Al-4Vsing phage-displayed random peptide library by acid lution technique 41ig. 1B. Four rounds of biopanning were done on Ti-6Al-4V sing phage-displayed random peptide library by E.coli lution technique 41ig.2. Elisa of selected Clones Binding to Ti-6Al-4VDiscs. 42ig.3A-C. Dose-dependent Binding of TBP-A and TBP-E hages to Ti -6Al-4V Discs. 43ig.4A~C. In vitro Peptide Competitive Binding Inhibition toriginal Phage Clone TBP-A and E. 45ig.4D. In vitro Peptide Competitive Binding Inhibition to original Phage Clone TBP-A and E. (IC 50 concentration) 47ig.5A. In vitro Binding of Synthetic peptide to Ti-6Al-4V discs. 48ig.5B. In vitro Binding of Synthetic peptides to Blank wells. 49application/pdf489002 bytesapplication/pdfen-US噬菌體顯現法表面處理,鈦合金植體Phage DisplaySurface TreatmentTitamium Implanthttp://ntur.lib.ntu.edu.tw/bitstream/246246/184178/1/ntu-98-R95422012-1.pdf