臺灣博碩士論文加值系統

齒槽骨可能會因為嚴重的牙周病、骨癌或是嚼食檳榔等因素造到破壞，在施行清創術(debridement)後，會進行牙周組織重建，所以牙周再生是近年來牙醫師或研究人員重視的研究方向之一，有愈來愈多的研究報告發現添加生長因子(Growth Factor)於齒槽骨缺陷中，除了可刺激牙周周圍細胞的活化、生長及分裂，也提高組織增生的速度與縮短缺損癒合的時間。並於患處填入具有骨引導或是骨誘導的材料，來促進齒槽骨再生。本論文的目的即是以幾丁聚醣以及硫酸鈣製備微粒作為骨填補材料，並添加生長因子，期有助於促進骨組織的癒合的。
血小板膠是ㄧ種由富血小板血漿與凝血酶混合而成的產物，血小板膠內富含有大量且多樣的生長因子。在臨床的應用上，常使用自體血小板膠，除可避免因使用異體血液而傳染疾病外，血小板膠內含的生長因子亦可加速組織的癒合。
在本論文中，係利用高壓電場系統製備幾丁聚醣/硫酸鈣/富血小板血漿微粒，再將製備完成的微粒與凝血酶產生器所製備出的凝血酶相互混合作用，並藉由微粒為載體達到控制生長因子釋放的目的。初步結果顯示所製備之微粒粒徑介於322.2±40.76μm~471.55 ± 57.9μm，具有通透性且可持續釋放生長因子，將材料植入豬齒槽骨中，由組織切片觀察骨新生情況。

Periodontal disease and bone cancer might cause severe alveolus bone loss. One common used therapy to treat this disease is so-called debridement. After that, a bone conductive material is put into defect of alveolus bone to improve the regeneration of bony tissues. Thus, the purpose of the thesis is to prepare chitosan/CaSO4 microspheres, by using a high voltage electrostatic system, to act as dental implant (filling) materials. Besides, platelet gel that enriched growth factors are also used an ingredient to prepare chitosan/CaSO4/PRP microspheres which probably can serve as growth factors slow release carrier (after activated with thrombin).
The preliminary results revealed that the chitosan/CaSO4/PRP microspheres are in the range of 322.2 ± 40.8μm ~ 471.6 ± 57.9μm in diameter. The in-trapped growth factors are able to sustained release at 24-hour period test. Three groups of microspheres, chitosan/CaSO4 microspheres (Group A) , chitosan/CaSO4 microspheres mixed with thrombin (Group B), chitosan/CaSO4/PRP microspheres mixed with thrombin (Group C) are prepared and the efficacy of these microspheres in bone regeneration are evaluated in pig oral model. The animal implantation study will be observed by HE stain and Trichrome stain.

目錄
致謝
中文摘要
Abstract
目錄
圖目錄
表目錄

第一章 研究背景及目的
第二章 材料與方法
2-1儀器設備
2-2藥品
2-3 幾丁質幾丁聚醣
2-4 血小板
2-5 硫酸鈣
2-6 高壓電場系統

第三章 研究方法與結果
3-1微粒製備
3-1-1 幾丁聚醣/硫酸鈣/富血小板血漿配製
3-1-2 高壓電場微粒製備
3-2凝血酶製備
3-3濃縮血小板
3-4材料分析
3-4-1 微粒觀察—光學顯微鏡
3-4-2 微粒觀察—掃描式電子顯微鏡
3-5 通透測試
3-6 生長因子釋放
3-7 動物實驗
第四章 結論
參考文獻
自述
圖目錄
圖1-1 牙齒、牙齦結構
圖2-1 幾丁聚醣
圖2-2 血小板
圖2-3 高壓電場系統
圖3-1 不同幫浦流速下未校正幾丁聚醣/硫酸鈣微粒，100倍
圖3-2 不同幫浦流速下未校正幾丁聚醣/硫酸鈣溶液/富血小板血漿，100倍
圖3-3 幾丁聚醣/硫酸鈣/富血小板血漿配製流程
圖3-4 高壓電場系統示意圖
圖3-5 在交聯劑中添加不同濃度PEG所形成的微粒
圖3-6 凝血酶產生器套組(TGD®，Merries)
圖3-7 凝血酶產生器
圖3-8 富血小板血漿 (a)離心前，(b)離心後
圖3-9 高壓電場系統在不同電壓下的微粒
圖3-10 高壓電場系統在不同電壓下的微粒粒徑分佈
圖3-11 高壓電場系統在不同幫浦速率下的微粒
圖3-12 高壓電場系統在不同幫浦速率下的微粒粒徑分佈
圖3-13 幾丁聚醣、富血小板血漿/硫酸鈣溶液以高壓電場在不同幫浦速率下形態觀察
圖3-14 硫酸鈣、富血小板血漿/幾丁聚醣溶液以高壓電場在不同幫浦速率下形態觀察
圖3-15 幾丁聚醣、富血小板血漿/硫酸鈣(●)以及硫酸鈣、富血小板血漿/幾丁聚醣(▲)在不同幫浦流速下的平均粒徑
圖3-16 幾丁聚醣、富血小板血漿/硫酸鈣溶液在不同幫浦速率下微粒SEM觀察
圖3-17 硫酸鈣、富血小板血漿/幾丁聚醣溶液在不同幫浦速率下微粒SEM觀察
圖3-18 幾丁聚醣、富血小板血漿/硫酸鈣溶液在不同幫浦速率下微粒截面SEM觀察
圖3-19 硫酸鈣、富血小板血漿/幾丁聚醣溶液在不同幫浦速率下微粒截面SEM觀察
圖3-20 通透能力測試
圖3-21 PDGF-AB生長因子釋放曲線
圖3-22 8周大、離乳的小豬
圖3-23 沿牙齦上緣切開
圖3-24 在豬齒槽骨上製造5mm缺損
圖3-25 術後六周組織切片，Masson’s Trichrome stain
圖3-26 術後八周組織切片，Masson’s Trichrome stain
圖3-27 術後六周組織切片，HE stain
圖3-28 術後八周組織切片，HE stain
表目錄
表3-1 多樣溶液配製方式
表3-2 動物實驗條件

參考文獻
[1] Available: http://scienceblogs.com/afarensis/2006/03/14/what_teeth_can_tell_us_about_h/
[2] D. L. Steed, “Debridement,” The American Journal of Surgery 187, 71S–74S
[3] H. Devlin, P. Sloan, “Early bone healing events in the human extraction socket,” Int. J.Oral Maxillofac. Surg. 31: 641–645, 2002
[4] P. L. M. Tomson, C. J. Butterworth, and A. D. Walmsley, “Management of peri-implant bone loss using guided bone regeneration: A clinical report,”
The Journal Of Prosthetic Dentistry, Volume 92, 92:12-16 2004
[5] T. W. Chen, S. J. Chang, Y. T. Hsu and S. M. Kuo, "Alginate-coated Chitosan Membrane for Guided Tissue Regeneration," Journal of Applied Polymer Science, vol.102, no.5, pp.4528-4534, 2006.12
[6] S. M. Kuo, S. J. Chang, T. W. Chen, T. C. Kuan, "Guided Tissue Regeneration for using a Chitosan Membrane: An Experimental study in Rats," Journal of Biomedical Materials Research Part A., vol.76A, pp.408-415, 2006.02
[7] F. Rose and R. Oreffo, “BREAKTHROUGHS AND VIEWS Bone Tissue Engineering: Hope vs Hype,” Biochemical and Biophysical Research Communications 292, 1–7, 2002
[8] T. Kawata, A. Matsuki, S. Kohno, T. Fujita, H. Sugiyama, C. Tokimasa, M. Kaku, K. Tsutsui, H.B. Moon, K. Tanne, “A new transplant bone for maxillary alveolar cleft,” Journal of Experimental Animal Science 43 19–28, 2004
[9] J. H. Lenz, K. O. Henkel, V. Hingst, R.udiger von Versen, K. H. Gundlach, “Reconstruction of the frontal calvarian continuity in a child using a freezepreserved autogenous bone graft.” Journal of Cranio-Maxillofacial Surgery , 31, 154–158, 2003
[10] P. V. Giannoudis, H. Dinopoulos, E. Tsiridis, “Bone substitutes: An update,” Injury, Int. J. Care Injured 36S, S20-S27, 2005
[11] D. Stubbsa, M. Deakina, P. Chapman-Sheatha, W. Bruceb, J. Debesc, R.M. Gillies, W.R. Walsha, “In vivo evaluation of resorbable bone graft substitutes in a rabbit tibial defect model,” Biomaterials ,25: 5037–5044,2004
[12] X. Huangfu, and J. Zhao, “Tendon-Bone Healing Enhancement Using Injectable Tricalcium Phosphate in a Dog Anterior Cruciate Ligament Reconstruction Model,” The Journal of Arthroscopic and Related Surgery, 23:5: 455-462, 2007
[13] R. V. Silva, J. A. Camilli, C. A. Bertran, N. H. Moreira, “The use of hydroxyapatite and autogenous cancellous bone grafts to repair bone defects in rats,” Int. J. Oral Maxillofac. Surg., 34: 178–184, 2005
[14] W. F. Mousa, M. Kobayashi, S. Shinzato, M. Kamimura, M. Neo, S. Yoshihara, T. Nakamura, “Biological and mechanical properties of PMMA-based bioactive bone cements,” Biomaterials, 21:2137-2146, 2000
[15] K. Anselme, “Review : Osteoblast adhesion on biomaterials,” Biomaterials, 21: 667-681,2000
[16] T. W. Axelrad, S. Kakar, T. A. Einhorn, “New technologies for the enhancement of skeletal repair,” Injury, Int. J. Care Injured, 38S1:S49-S62,2007
[17]W. V. Giannobile, “Peridental tissue engineering by growth factors,” Bone, 19:1:23S-37S, 1996
[18]T. A. Linkhart, S. Mohan and D. J. Baylink, “Growth factors for bone growth and repair : IGF, TGF and BMP,” Bone, 19:1:1S-12S, 1996
[19] Y. Wan, K. Creber, B. Peppley, V. Tam Bui, “Ionic conductivity of chitosan membranes,” Polymer, 44:1057-1065, 2003
[20] C. Chatelet, O. Damour, A. Domard, “Infuence of the degree of acetylation on some biological properties of chitosan flms,” Biomaterials, 22:261-268,2001
[21] X.Z. Shu, K.J. Zhu, “Controlled drug release properties of ionically cross-linked chitosan beads: the influence of anion structure,” International Journal of Pharmaceutics, 233:217-225, 2002
[22] E. Guibal, “Heterogeneous catalysis on chitosan-based materials: a review,” Prog. Polym. Sci.,30:71–109, 2005
[23] K Miyatake, Y. Okamoto, Y. Shigemasa, S. Tokura, S. Minami, “Anti-inflammatory effect of chemically modified chitin,” Carbohydrate Polymers, 53:417-423, 2003
[24] J. Benesch, P. Tengvall, “Blood protein adsorption onto chitosan,” Biomaterials 23:2561-2568, 2002
[25] Y. Okamoto, R. Yano, K. Miyatake, I. Tomohiro, Y. Shigemasa, S. Minami, “Effects of chitin and chitosan on blood coagulation,” Carbohydrate Polymers, 53: 337-342, 2003
[26] T. C. Chou, E. Fu, C. J. Wu and J. H. Yeh, „Chitosan enhances platelet adhesion and aggregation,” Biochemical and Biophysical Research Communications, 302: 480-483, 2003
[27] Y. C. Chung, H. L. Wang, Y. M. Chen , S. L. Li, „Effect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens,” Bioresource Technology 88:179–184, 2003
[28] A. H. Lazarus, S. Song, and A. R. Crow, “Understanding Platelet Function Through Signal Transduction,” Transfusion Medicine Reviews, 17:1:45-56, 2003
[29] M. Schmugge, M. L. Rand, J. Freedman, “Platelets and von Willebrand factor,” Transfusion and Apheresis Science 28:269-277, 2003
[30] I. Ramasamy, “Inherited bleeding disorders: disorders of platelet adhesion and aggregation,” Critical Reviews in Oncology/Hematology 49:1–35, 2004
[31] H. Xiao, C. J. Jepkorir, K. Harvey, and D. G. Remicka, “Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets,” Cryobiology 44:179-188, 2002
[32] K. WAHNG, E. ROMAS, L. DONNAN, D. M. FINDLAY, “Bone biology,” Baillidre''s Clinical Endocrinology and Metabolism, l:1-22, April 1997
[33] D. Donati, C. D. Bella, M. Colangeli, G. Bianchi, M. Mercuri, “The use of massive bone allografts in bone tumour surgery of the limb,” Current Orthopaedics, 19:393-399, 2005