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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 郭生興(Sang-Heng Kok) | |
| dc.contributor.author | Shao-Hua Chang | en |
| dc.contributor.author | 張少華 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:32:49Z | - |
| dc.date.available | 2016-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-17 | |
| dc.identifier.citation | 1. Chiapasco, M., M. Zaniboni, and M. Boisco, Augmentation procedures for the rehabilitation of deficient edentulous ridges with oral implants. Clin Oral Implants Res, 2006. 17 Suppl 2: p. 136-59.
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Ji, W., et al., Incorporation of stromal cell-derived factor-1 alpha in PCL/gelatin electrospun membranes for guided bone regeneration. Biomaterials, 2013. 34(3): p. 735-745. 35. Bonadio, J., et al., Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nature Medicine, 1999. 5(7): p. 753-759. 36. Naldini, L., Gene therapy returns to centre stage. Nature, 2015. 526(7573): p. 351-360. 37. Fischer, J., et al., Future of local bone regeneration - Protein versus gene therapy. Journal of Cranio-Maxillofacial Surgery, 2011. 39(1): p. 54-64. 38. Scaduto, A.A. and J.R. Lieberman, Gene therapy for osteoinduction. Orthopedic Clinics of North America, 1999. 30(4): p. 625-+. 39. Zhang, X.J. and W.T. Godbey, Viral vectors for gene delivery in tissue engineering. Advanced Drug Delivery Reviews, 2006. 58(4): p. 515-534. 40. Jenkins, G.M. and E.C. Holmes, The extent of codon usage bias in human RNA viruses and its evolutionary origin. 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Mikos, Poly(ethylenimine) and its role in gene delivery. J Control Release, 1999. 60(2-3): p. 149-60. 48. Freidman, Y.H.A.R.J., Animal Models in Orthopaedic Research. Research. Boca Raton, 1999: p. 241-260. 49. Schmitz, J.P. and J.O. Hollinger, The Critical Size Defect as an Experimental-Model for Craniomandibulofacial Nonunions. Clinical Orthopaedics and Related Research, 1986(205): p. 299-308. 50. Gomes, P.S. and M.H. Fernandes, Rodent models in bone-related research: the relevance of calvarial defects in the assessment of bone regeneration strategies. Laboratory Animals, 2011. 45(1): p. 14-24. 51. Kim, J.B., et al., Non-Invasive Detection of a Small Number of Bioluminescent Cancer Cells In Vivo. Plos One, 2010. 5(2). 52. Kirker-Head, C.A., Potential applications and delivery strategies for bone morphogenetic proteins. Advanced Drug Delivery Reviews, 2000. 43(1): p. 65-92. 53. Oakes, D.A. and J.R. Lieberman, Osteoinductive applications of regional gene therapy - Ex vivo gene transfer. Clinical Orthopaedics and Related Research, 2000(379): p. S101-S112. 54. Boussif, O., et al., A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in-Vivo - Polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(16): p. 7297-7301. 55. Sidi, A.A., et al., Phase I/II Marker Lesion Study of Intravesical BC-819 DNA Plasmid in H19 Over Expressing Superficial Bladder Cancer Refractory to Bacillus Calmette-Guerin. Journal of Urology, 2008. 180(6): p. 2379-2383. 56. Kunath, K., et al., Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine. Journal of Controlled Release, 2003. 89(1): p. 113-125. 57. Hunter, A.C., Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity. Advanced Drug Delivery Reviews, 2006. 58(14): p. 1523-1531. 58. Moghimi, S.M., et al., A two-stage poly(ethylenimine)-mediated cytotoxicity: Implications for gene transfer/therapy. Molecular Therapy, 2005. 11(6): p. 990-995. 59. Amler, M.H., P.L. Johnson, and I. Salman, Histological and histochemical investigation of human alveolar socket healing in undisturbed extraction wounds. J Am Dent Assoc, 1960. 61: p. 32-44. 60. Hassan, M. and B.A. Klaunberg, Biomedical applications of fluorescence imaging in vivo. Comp Med, 2004. 54(6): p. 635-44. 61. Hickson, J., In vivo optical imaging: preclinical applications and considerations. Urol Oncol, 2009. 27(3): p. 295-7. 62. Williams, T.M., et al., Advantages of Firefly Luciferase as a Reporter Gene - Application to the Interleukin-2 Gene Promoter. Analytical Biochemistry, 1989. 176(1): p. 28-32. 63. Piattelli, M., et al., Bone reactions to anorganic bovine bone (Bio-Oss) used in sinus augmentation procedures: a histologic long-term report of 20 cases in humans. Int J Oral Maxillofac Implants, 1999. 14(6): p. 835-40. 64. Thomas, M.V. and D.A. Puleo, Calcium Sulfate: Properties and Clinical Applications. Journal of Biomedical Materials Research Part B-Applied Biomaterials, 2009. 88b(2): p. 597-610. 65. Kim, S.G., et al., Use of particulate dentin-plaster of Paris combination with/without platelet-rich plasma in the treatment of bone defects around implants. International Journal of Oral & Maxillofacial Implants, 2002. 17(1): p. 86-94. 66. Lineaweaver, W.C., A tribute to Richard F. Edlich, M.D., Ph.D., editor-in-chief. Journal of long-term effects of medical implants. J Long Term Eff Med Implants, 2002. 12(1): p. ix-xii. 67. Fitts, D.A., Ethics and animal numbers: informal analyses, uncertain sample sizes, inefficient replications, and type I errors. J Am Assoc Lab Anim Sci, 2011. 50(4): p. 445-53. 68. Faul, F., et al., G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods, 2007. 39(2): p. 175-91. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49522 | - |
| dc.description.abstract | 牙科植體雖已被廣泛應用於缺牙區域的重建，但並非所有病患的條件都適合植牙。對於嚴重骨缺損的區域，引導骨再生術(guided bone regeneration, GBR)是一個常施行的術式，其目的為豐隆齒槽骨以利人工牙根植入，但仍有技術上之困難有待突破，其中包括如何加強缺損區骨生成能力，和確保軟組織傷口穩定癒合。過去文獻對於如何增加骨再生的能力，都是偏向於生長因子的利用，但生長因子要廣泛應用在臨床齒槽骨豐隆手術上，目前仍有其困難。另外，如何促進缺損區上方的軟組織穩定癒合也是增加引導骨再生術成功率的重要因素，但過往對於這部份的相關研究不足。
組織再生需要幹細胞（stem cells）遷移（migration）及分化（differentiation），如何吸引幹細胞以促進組織修復已成為目前再生醫學主要的發展方向。基質細胞衍生因子-1 (stromal cell-derived factor-1α, SDF-1α), 被認為是重要的幹細胞趨化素（chemokine）具有趨化特殊潛能間葉幹細胞(mesenchymal stem cell, MSC)的能力。歷年來的文獻也已證實基質細胞衍生因子-1對軟組織癒合及硬組織再生都有幫助。但過去報告多以蛋白質作為研究對象，蛋白質會有作用效期短、安全性及成本昂貴等問題。因此本研究以基因療法為策略，建構帶有基質細胞衍生因子-1α基因的質體（plasmid），以探討基質細胞衍生因子-1α基因療法的效果。 實驗中包含第一部分的聚乙烯亞胺(polyethylenimine)轉染效率測試，以此決定用於動物實驗的聚乙烯亞胺及質體DNA之最佳比例。第二部分的基質幹細胞遷移測試，檢視此質體經轉染到哺乳類細胞後是否能正常表現基質細胞衍生因子-1α蛋白質及趨化基質幹細胞。第三部分為動物實驗，利用大鼠頭蓋骨缺損模型，植入膠原蛋白膜及補骨材，並加入基因轉染試劑及質體混合液，以觀察骨骼再生與軟組織癒合之情形。 由第一部分的實驗結果得知，當聚乙烯亞胺與DNA之比例為7.5時(3uL聚乙烯亞胺及1ug DNA)，在48小時之後，可達到五成（55.68%）的轉染率。所以聚乙烯亞胺與DNA應該維持在高比例，而兩者皆應維持低濃度，在作用時間充足的條件下，仍可以達到相當的轉染效率。經第二部分的測試，證實重組之質體能在轉染至293FT細胞之後，正常表現出基質細胞衍生因子-1α蛋白質。並且在幹細胞遷移測試中，確認此蛋白質可以趨化基質幹細胞，跟對照組相比多達3.5倍。第三部分以大鼠頭蓋骨缺損為模型來評估基質細胞衍生因子-1α對於傷口癒合的療效，分為軟組織癒合及骨再生的觀察。加入基質細胞衍生因子-1α質體的組別其平均軟組織癒合速度在第14天已達100%，而對照組則只有88.7%；利用微型斷層掃描評估得知，加入基質細胞衍生因子-1α質體的組別，在術後四週其骨再生體積比率為12.9%±3.64 (n=8)比對照組7.07%±4.85 (n=6)多，兩者有統計學上顯著的差異(P<0.05)；而組織切片的結果，也同樣顯示加入基質細胞衍生因子-1α質體的組別其骨新生面積比率，與對照組相比，在四個特定時間點都是比較多的。 本實驗的結果顯示，基質細胞衍生因子-1α基因療法對於骨再生及軟組織癒合有一定的效益，將來可應用以提升齒槽脊豐隆手術之成功率。 | zh_TW |
| dc.description.abstract | Although dental implantation has been widely applied for the reconstruction of edentulous ridge, implant placement may not be feasible in cases with poor bone volume. For the severely atrophic ridge, the technique of guided bone regeneration (GBR), is commonly used for alveolar ridge augmentation. However, there are still technical difficulties remain to be resolved. Such difficulties include how to increase the regenerative potential of bone tissue in defect areas and the assurance of stable soft tissue healing. Past researches mainly focused on the use of growth factors for promoting bone regenerative capacity. However, obstacles still exist in the use of growth factors for alveolar ridge augmentation. How to assure the stable healing of soft tissue overlaying the defect area is another key point for increasing the success rate of GBR, but studies focusing on this subject are rare.
Tissue regeneration requires the migration and differentiation of stem cells. Hence, recruitment of stem cells has become the main direction of development in the field of regenerative medicine. Stromal cell-derived factor-1α (SDF-1α), considered as an important stem cell chemokine, has been reported to have the capacity of attracting multipotent mesenchymal stem cells (MSCs). Studies have shown that SDF-1α is beneficial for soft tissue healing and bone regeneration, and most of the studies used SDF-1α protein as the research subject. However, protein has its limitations, such as rapid degradation leading to short lasting duration, safety concern and poor cost efficiency. Consequently, gene therapy has been the main strategy in the present study. We constructed a plasmid encoding mSDF-1α and investigated the therapeutic effect of mSDF-1α gene therapy. First, in our experiment, in-vitro transfection efficiency with polyethylenimine(PEI) was tested in order to determine the optimal ratio of PEI and plasmid DNA, which was adopted in the following animal study. Second, MSC transwell migration assay was performed to ascertain the production of SDF-1α protein by transfected mammalian cells and the protein can promote MSC migration. The third part of the experiment employed the rat calvarial defect model. Application of PEI/DNA condensates in combination with the use of collagen barrier membrane and bone substitutes was performed. The effects on soft tissue healing and bone regeneration were evaluated. Based on the results of the first part of experiment, when the ratio of PEI and DNA was 7.5(3uL PEI and 1ug DNA), the transfection efficiency can reached 55.68% after 48 hours. The data indicated that PEI and DNA should be maintained at a high ratio with low concentration. Therefore, an acceptable transfection rate can be obtained after sufficient time. The second part of experiment verified that the SDF-1α protein can be expressed normally by mammalian cell 293FT after gene transfection. In the MSC transwell migration assay, the SDF-1α protein-added group promoted 3.5 times more MSCs migration compared to the control group. In the third part of experiment, the rat calvarial defect model was used to evaluate the therapeutic effect of SDF-1α gene therapy on wound healing, including soft tissue healing and bone regeneration. In the aspect of assessing soft tissue healing, the SDF-1α-treated group showed an average 100% healed tissue after 14 days, while the control group was only 88.7%; In the use of micro-tomography, the data showed that SDF-1α-treated group had more new bone formation (12.9% ± 3.64) than the PEI-only group (7.07% ± 4.85) with statistically significant differences (P <0.05) ; In the histomorphometric analysis, the results demonstrated that more newly-formed bone can be detected in SDF-1α-treated group compared with the control group at four different time points. Results of the study suggest that SDF-1α gene therapy is beneficial for bone regeneration and soft tissue healing and may have the potential to improve the success rate of alveolar ridge augmentation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:32:49Z (GMT). No. of bitstreams: 1 ntu-105-R03422015-1.pdf: 4520215 bytes, checksum: b7db3e6cd04ad28257ace8d3f4aa8a3d (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書........................................................................................................... i
摘要…………................................................................................................................ii Abstract................................................................................................................iv Contents...........................................................................................................................vii Chapter 1 Introduction 1 1.1 Guided bone regeneration (GBR) and dental implant treatment 1 1.2 Difficulties in GBR 2 1.3 Growth factors and GBR 4 1.4 Stromal cell-derived factor-1α 6 1.4.1 SDF-1 and soft tissue healing 8 1.4.2 SDF-1 and bone regeneration 9 1.5 Gene therapy 10 1.5.1 Types of vectors 11 1.5.2 Polyethylenimine(PEI) and transfection efficiency 13 1.6 Rat calvarial defect model 15 1.6.1 Critical sized defect (CSD) 17 1.7 In-vivo imaging system (IVIS) 18 Chapter 2 Research Goal 20 Chapter 3 Materials and Methods 21 3.1 Recombinant mSDF-1α-T2A-mCherry/pcDNA3.1 plasmid 21 3.2 Plasmid amplification 22 3.3 Transfection efficiency assay 24 3.3.1 Reporter gene of red fluorescence mCherry 25 3.3.2 Cell counting 26 3.3.3 Transfection with jetPEI/DNA condensates 27 3.4 Mesenchymal stem cell migration assay 28 3.4.1 Collection οf concentrated media 29 3.4.2 MSC transwell migration assay 31 3.5 Animal model 32 3.5.1 Recombinant mSDF-1-T2A-Luc/pcDNA3.1 plasmid 33 3.5.2 Surgical procedures 34 3.5.3 Observation of luciferase activity by in-vivo imaging system 36 3.5.4 Clinical assessment of soft tissue healing 38 3.5.5 Sacrifice of animals and specimen preparation 39 3.5.6 Micro-CT analysis 41 3.5.7 Histomorphometric assessment of bone regeneration 42 Chapter 4 Results 43 4.1 PEI transfection efficiency assay 43 4.2 Promotion of stem cell migration by mSDF-1α 45 4.3 Rat calvarial defect model 47 4.3.1 Gross evaluation of soft tissue healing 48 4.3.2 Evaluation of gene expression by in-vivo imaging system 49 4.3.3 Micro-CT analysis 51 4.3.4 Histomorphometrical evaluation of hard tissue regeneration 53 Chapter 5 Discussion 55 5.1 Summary of findings 55 5.2 Comparison between gene therapy and protein therapy 58 5.3 PEI 60 5.4 Timepoints for observation of new bone regeneration 63 5.5 In-vivo imaging system 64 5.6 Bone graft materials in the defect area 67 5.7 Future perspectives 70 Chapter 6 Conclusions 72 References 74 Appendix 81 | |
| dc.language.iso | en | |
| dc.subject | 間質幹細胞 | zh_TW |
| dc.subject | 基質細胞衍生因子-1 | zh_TW |
| dc.subject | 引導骨再生術 | zh_TW |
| dc.subject | 基因療法 | zh_TW |
| dc.subject | 聚乙烯亞胺 | zh_TW |
| dc.subject | mesenchymal stem cell(MSC) | en |
| dc.subject | gene therapy | en |
| dc.subject | polyethylenimine(PEI) | en |
| dc.subject | stromal cell-derived factor-1(SDF-1) | en |
| dc.subject | guided bone regeneration(GBR) | en |
| dc.title | 基質細胞衍生因子-1alpha基因療法對骨再生及傷口癒合的作用 | zh_TW |
| dc.title | Effects of stromal cell-derived factor-1 alpha gene therapy on bone regeneration and wound healing | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 林思洸(Sze-Kwan Lin) | |
| dc.contributor.oralexamcommittee | 洪志遠 | |
| dc.subject.keyword | 基質細胞衍生因子-1,間質幹細胞,引導骨再生術,基因療法,聚乙烯亞胺, | zh_TW |
| dc.subject.keyword | stromal cell-derived factor-1(SDF-1),mesenchymal stem cell(MSC),guided bone regeneration(GBR),gene therapy,polyethylenimine(PEI), | en |
| dc.relation.page | 112 | |
| dc.identifier.doi | 10.6342/NTU201601815 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-17 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
| 顯示於系所單位： | 臨床牙醫學研究所 | |
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