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Growth Factor-Releasing Nanofibrous Patches for the Regeneration of Chronic Tympanic Membrane Perforation
- 주제(키워드) Chronic Otitis Media , Electrospinning , Radial alignment , Tissue engineering , Growth Factor
- 발행기관 아주대학교
- 지도교수 정연훈
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028459
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Chronic tympanic membrane (TM) perforation is a tubotympanic disease caused by traumatic or inflammatory factors. To date, various types of tissue engineered techniques have been applied for the regeneration of chronic TM perforations. However, the application of nanofibers with radially aligned nanostructures and release of growth factors simultaneously has never been applied in the regeneration of chronic TM perforations. In this study, Growth factor-releasing radially aligned nanofibrous patches were developed and applied for the regeneration of chronic perforated TMs. In the first purpose, the insulin-like growth factor-binding protein 2 (IGFBP2) were incorporated to radially aligned nanofibrous patches. At first, radially aligned nanofibrous polycaprolactone (PCL) patches were developed using electrospinning. Random nanofibers (NFs) were deposited onto a planar plate, and aligned nanofibers (AFs) were deposited onto custom-made round shape collector. For the experimental groups, patches were classified by alignments and the presence of IGFBP2: random fibers without IGFBP2 (RF-w/oIGFBP2), aligned fibers without IGFBP2 (AF-w/oIGFBP2), random fibers with IGFBP2 (RF-wIGFBP2), and aligned fibers with IGFBP2 (AF-wIGFBP2). To confirm the efficiency of each patch, in vitro and in vivo studies were performed. In the second purpose, epidermal growth factor (EGF) was incorporated to radially aligned nanofibrous patches. These patches were fabricated by the same methods which used for the IGFBP2-incoporated patches. And also these patches were classified as follows; random fibers without EGF (RF-w/oEGF), aligned fibers without EGF (AF-w/oEGF), random fibers with EGF (RF-wEGF), and aligned fibers with EGF (AF-wEGF). The effectiveness of EGF-releasing nanofibrous patches were confirmed by cell viability, cell morphology, wound healing assay, and animal studies. In addition, the mechanism of TM regeneration also was studied by mRNA and protein level studies. In the first experiment, the presence of IGFBP2 was analyzed by fourier-transform infrared spectroscopy (FTIR). In addition, the releasing of IGFBP2 was sustained until 20 days. In the cell viability test, there was no significant difference among all patches on day 1, 3, and 5. By the animal studies, 96 ears were applied, and both IGFBP-2 incorporated patches showed the efficiency for regeneration of chronic TM perforations. However, there were no significant difference between RF-wIGFBP2 and AF-wIGFBP2. From the second experiment, radial alignments and the presence of EGF in the patches were analyzed. EGF was confirmed to be released from the patches until 8 weeks. In an in vitro study, cell viability, immunocytochemistry, and wound healing assay were rationally enhanced by the combination of radial alignments and EGF release ability. An in vivo animal study showed that the EGF-releasing nanofibrous patches effectively stimulated the healing of the chronic TM perforations. The TMs healed by AF-wEGF showed histological properties similar to those of normal TMs. The markers of proliferation and migration were confirmed by quantitative real-time polymerase chain reaction, which enhanced by the combination of radial alignments and EGF. Hence, the EGF-releasing radially aligned nanofibrous patches will be an efficient platform for the regeneration of chronic TM perforations, opening the gate for nonsurgical treatments of chronic otitis media.
more목차
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 8
1. Fabrication of growth factor-releasing nanofibrous patches 8
2. Characterization of growth factor-releasing nanofibrous patches 10
3. Cell culture 10
4. Cell viability test 10
5. Immunocytochemistry 11
6. Wound healing assay 11
7. In vivo animal study 11
8. Histological study 12
9. Quantitative real-time PCR 12
10. Statistical analysis 14
Ⅲ. RESULTS 16
1. Fabrication of radially aligned nanofibrous patches 16
2. IGFBP2-releasing radially aligned nanofibrous patches for chronic TM perforations 19
2.1. Characterization of IGFBP2-releasing nanofibrous patches 19
2.2. In vitro properties of IGFBP2-releasing nanofibrous patches 21
2.3. Development of chronic TM perforation animal model 23
2.4. Efficacy of IGFBP2-releasing nanofibrous patches for chronic TM perforation 25
3. EGF-releasing radially aligned nanofibrous patches for chronic TM perforations 29
3.1. Characterization of EGF-releasing nanofibrous patches 29
3.2. In vitro properties of EGF-releasing nanofibrous patches 32
3.3. Wound Healing assay 35
3.4. Development of chronic TM perforation animal model 37
3.5. Efficacy of EGF-releasing nanofibrous patches for chronic TM perforation 39
3.6. Regulation of mRNA expression by EGF-releasing nanofibrous patches 45
Ⅳ. DISCUSSION 47
Ⅴ. CONCLUSION 55
REFERENCES 56
국문요약 61
