臺灣博碩士論文加值系統

自六O年代以來，鈦金屬根狀人工植體已廣泛應用於全口或局部缺牙病人之重建治療。即使文獻報告顯示出植牙治療可達到相當高的成功率及存活率，然而臨床上仍然會遭遇到各種不同的失敗及併發症，包括植體周圍骨質的吸收及軟組織的萎縮，甚至是完全不形成骨整合。因此，許多現階段研究工作致力於人工植體表面特性的改造，藉以促進形成穩定的植體骨整合。主要的研究方向是針對植體表面形態的改變，例如螺紋的設計、表面的粗糙度、甚至是奈米結構的應用。而在九O年代的晚期，由於對骨癒合過程中分子生物機轉的了解，擬生物策略開始被應用於植體表面特性的改造。然而，關於這方面的研究，目前的進展仍相當有限。其中主要原因之一，是缺乏有效的黏附技術將可以促進植體周圍組織癒合所必需之各種分子接合於植體表面，使其具有各種不同的生物活性與功能。在本研究中，利用噬菌體顯現法篩選具有鈦金屬結合能力的小分子蛋白，作為將具有生物活性之分子接合於植體表面的中介分子。經由此技術，我們成功地分離出一株對鈦金屬表面具有高度親和力的噬菌體-PT3，並利用酵素免疫吸附法確定其結合能力，再將其接合於人類第二型骨形成蛋白（hBMP-2) 之前端並利用原核生物表現系統製造重組蛋白，以應用於日後探討固著於植體表面之生長因子對於周圍組織癒合過程的影響。這個嶄新的策略建立一個有效率而簡易的植體表面黏附技術，提供臨床醫師於植牙手術中，直接置入生長因子於植體表面的可能性。

Since the introduction of titanium root-form dental implant in 60’s, it has become a widely used treatment modality in rehabilitation of completely or partially edentulous patients. However, clinicians still face the failure of implant treatment or its complication, such as progressive marginal bone loss, recession of marginal mucosa, or absence of osseointegration, inspite of its reported high success and survival rates in the literatures. In late 90’s, the application of biomimetic modification has been introduced to overcome the problems because of the advances in the understanding of the biology of bone healing process at molecular level. However, there is only marginal success in this field of research after a decade of efforts. One of the obstacles to the success is lack of an efficient and controllable coating methodology to achieve an biologically active surface with diverse function required for the healing process of surrounding tissues. In this study, we identified small adaptor peptides with titanium-binding property with a powerful screening methodology, phage display system. By five rounds of biopanning on Ti-6Al-4V discs, we isolated a clone of phage PT3 with high affinity to the surface of titanium, as verified by ELISA. We also conjugated PT3 at the amino terminus of hBMP-2 and expressed the fusion protein in E.coli. The identity of expressed protein was confirmed with mass spectrophotometry. It was then refolded and purified for further application in evaluation of the effect of immobilized growth factors on the healing process. This novel strategy may offer us a clinically efficient and easy-to-apply coating method in implant treatment.

口試委員會審定書 i

誌謝 ii

摘要 iii

ABSTRACT iv

INTRODUCTION 1
Current Challenges in Implant Treatment 1
Current Challenges in Implant Treatment 3
Physical Modification of Implant Surface 3
Biomimetic Modication of Implant Surface 7
Healing of Peri-implant Bone 8
Rationale of Biomimetic Modification 11
Modification with Biomimetic Peptides 12
In vitro Study of Using RGD-peptides in Surface Modification 14
In vivo Study of Using RGD-peptides in Surface Modification 17
Current Problems in Functionalization of Implant Surface 19
Using Peptidyl Adaptor to Connect Biological Factor onto Implant Surface 20
Using Phage Display to Identify Material-binding Peptides 21
pIII Protein of Filamentous Phage 23
Using pIII to Display Random Peptide Library 24
Various Libraries Available for Biopanning 25
Steps in Phage Display 26
Study Purpose 28

MATERIALS & METHODS 29
Preparation of Phage Stock 29
Biopanning of T-6Al-4V Discs 30
Phage Titering 32
Characterization of Binding Clones 33
ELISA of PT-3 and PT-7 Phage Clones on Ti-6Al-4V Discs and Titanium Particles 36
ELISA of PT-3 and PT-7 Phage Clones on Various Cell Lines 37
ELISA of PT-3 and PT-7 Phage Clones on U2OS cell line 39
Cloning of human Bone Morphogenetic Protein-2 (hBMP-2) cDNA into Expression Vector 40
Construction of PT3-hBMP-2 for Expression of Fusion Protein 41
Expression of rhBMP-2 and rPT3-hBMP-2 in Prokaryotic System 42
Isolation and Solution of Inclusion Body of rhBMP-2 and
rPT3-hBMP-2 43

RESULTS 44
Biopanning on Ti-6Al-4V Discs 44
Characterization of Recovered Clones 46
Binding of PT3 and PT7 Phages to Ti-6Al-4V Discs 48
Binding of PT3 and PT7 to Titanium Particles 49
Binding of PT3 and PT7 phages to Various Cell Lines 51
Expression of rhBMP-2 and rPT3-hBMP-2 in E.coli (BL21) 53
Isolation, Purification, and Renaturation of rhBMP-2 and rPT3-hBMP-2 from the Inclusion Body 54

DISCUSSION 56

CONCLUSION 64

REFERENCES 65

TABLE & FIGURES 78
Table. Summary of Selected Clones 79
Fig. 1. Strategy of Using Phage Display to Functionalize the Surface of Titanium Surface 80
Fig. 2. Biopanning on Ti-6Al-4V Discs 81
Fig. 3. ELISA of Selected Clones Binding to Ti-6Al-4V Discs 82
Fig. 4. Dose-dependent Binding of PT3 and PT7 Phages to Ti-6Al-4V Discs 83
Fig. 5. Dose-dependent Binding of PT3 and PT7 Phages to Titanium Particles 84
Fig. 6. Temporal Change in Binding of PT3 Phages to Titanium Particles 85
Fig. 7. Wide Spectrum of Affinity of PT3 and PT7 Phages to Various Cell Lines 86
Fig. 8. Dose-dependent Binding of PT3 and PT7 Phages to U2-OS Cell Line 87
Fig. 9. Strategy of Construction of PT3-hBMP2 for Expression of Fusion Protein 88
Fig. 10. Cloning of BMP-2 into pET-21a(+) Expression Vector 89
Fig. 11. Construction of PT3-hBMP-2 90
Fig. 12. Induction of rhBMP-2 Expression in BL21 by IPTG 91
Fig. 13. Induction of rPT3-hBMP-2 Expression in BL21 by IPTG 92
Fig. 14. Confirmation of The Identity of Expressed Protein with Mass Spectrophotometry 93

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