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Enhanced therapeutic angiogenesis in mouse hindlimb ischemia model by electrical stimulation and extracellular matrix
전기 자극과 세포외 기질을 이용한 마우스 하지 허혈 모델에서의 혈관 재생 향상

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dc.contributor.advisor김병수-
dc.contributor.author정근재-
dc.date.accessioned2018-05-28T16:30:08Z-
dc.date.available2018-05-28T16:30:08Z-
dc.date.issued2018-02-
dc.identifier.other000000149938-
dc.identifier.urihttps://hdl.handle.net/10371/140750-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 공과대학 화학생물공학부, 2018. 2. 김병수.-
dc.description.abstractThe present study is the report on the enhancing therapeutic angiogenesis in mouse hindlimb ischemia model by usage of electrical stimulation (ES) derived from solar cell and injectable decellularized extracellular matrix (IDM) for stem cell transplantation. In ischemic tissue, most of the transplanted cells and native cells undergo apoptosis and necrosis due to the low oxygen and nutrient delivery. Therefore, improved cell therapy method of acellular therapy method is required for enhancing or replacing previous therapy.
In chapter 3, the solar-cell-based device was designed, which converts light energy to electrical energy, can generate an electrical stimulus that would control cell behavior and stimulate therapeutic angiogenesis in a mouse ischemic hindlimb. For easy utilization of the device in vivo, we designed a solar cell circuit that consisted of an implantable electrode and a solar panel that adhered to the skin. Conventional clinical ES usually involves a large electrical device, which may require patient hospitalization. By contrast, the solar-cell-based wearable device developed in this study overcomes the limitation. In an in vitro experiment, ES applied to various types of cells associated with angiogenesis significantly enhanced cell migration and secretion of angiogenic paracrine factors. To evaluate the therapeutic efficacy of the device in vivo, the electrode of the solar cell device was implanted into the ischemic region of a mouse hindlimb, and the solar panel part of the device was attached to the back of mouse for exposure to light. The device successfully converted light energy into electrical energy and generated ES. ES induced cell migration and promoted the secretion of angiogenic paracrine factors. Furthermore, use of the solar cell device led to significant increase in the number of capillaries and arterioles at the ischemic region, and prevented muscle necrosis and loss of the ischemic limb.
In the chapter 4, IDM was investigated and examined whether the IDM can enhance transplanted cell grafting and therapeutic efficacy. In an in vitro experiment, IDM and ADSC complex (cell-IDM) enhanced cell viability and upregulation of angiogenic paracrine factors. To evaluate the therapeutic efficacy in vivo, cell-IDM was implanted into the ischemic region of a mouse hindlimb. Transplantation of cell-IDM induced significant increase in the number of capillaries and arterioles at the ischemic region, and prevented muscle necrosis.
The result of this study may be applicable for the enhancing and optimizing therapeutic angiogenesis in both cell transplantation model and in vivo implantable device model. Moreover, this study provided a disposable and easily usable and implantable ES-generating solar cell device and easily applicable developed stem cell transplantation method to treat angiogenic disease.
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dc.description.tableofcontentsChapter 1. Research backgrounds and objective 1
1.1. Angiogenesis 2
1.2. Ischemic disease and cell therapy 5
1.3. Electrical stimulation on therapeutic angiogenesis 7
1.4. Extracellular matrix (ECM) 10
1.5. Improvement cell transplantation therapy 12
1.6. Research objective of thesis 14
Chapter 2. Experimental methods 16
2.1. Fabrication and characterization of organic photovoltaic cell 17
2.1.1. Fabrication of organic photovoltaic cell 17
2.1.2. Construction of implantable electrodes 18
2.1.3. Characterization of organic photovoltaic cells 19
2.2. Fabrication and characterization of injectable decellularized matrix 20
2.2.1. Fabrication of injectable decellularized matrix (IDM) 20
2.2.2. IDM characterization 21
2.2.3. DNA content assay 22
2.3. In vitro assays 23
2.3.1. Cell isolation and culture 23
2.3.2. In vitro cell culture for electrical stimulation 26
2.3.3. Scratching wound-healing assay 27
2.3.4. Enzyme-linked immunosorbent assay 28
2.3.5. Apoptosis assay 29
2.3.6. Cell cycle analysis 30
2.3.7. Surface marker expression analysis 31
2.3.8. Cell viability assay 32
2.3.9. In vitro hypoxic condition cell assay 33
2.3.8. IDM cell attachment assay 34
2.4. In vivo assays 35
2.4.1. Modeling of hindlimb ischemia 35
2.4.2. Treatment of mouse hindlimb ischemia for solar cell implant 36
2.4.3. Treatment of mouse hindlimb ischemia for Cell-IDM 37
2.4.4. Laser Doppler imaging analysis 38
2.4.5. Immunohistochemistry 39
2.4.6. Histological examination 40
2.4.7. Western blot analysis 41
2.4.8. Transplantation of PKH26-labeled hMSCs 42
2.4.9. Live imaging 43
2.5. Statistical analysis 44

Chapter 3. Therapeutic angiogenesis via solar cell facilitated electrical stimulation 45
3.1. Introduction 46
3.2. Results 50
3.2.1. In vitro cell migration and angiogenic paracrine factor secretion by various cell types induced by ES 50
3.2.2. Photovoltaic capacity of solar cell device 57
3.2.3. In vivo homing of MSCs 62
3.2.4. Microvessel density in ischemic limbs 67
3.2.5. Limb perfusion and limb salvage of the ischemic limb 70
3.3. Discussion 73
Chapter 4. Injectable decellularized matrix for therapeutic angiogenesis 77
4.1. Introduction 78
4.2. Results 81
4.2.1. Fabrication of Cell-IDM complex . 81
4.2.2. Optimization of incubation time for hADSC attachment to IDM 84
4.2.3. In vitro prevention of hADSC anoikis by IDM 87
4.2.4. In vitro upregulation of angiogenic factor expression 91
4.2.5. In vivo engraftment of hADSCs implanted to ischemic tissue 93
4.2.6. Angiogenic paracrine secretion in ischemic limbs 95
4.2.7. Microvessel density in ischemic limbs 97
4.2.8. Blood perfusion in the ischemic limb 100
4.3. Discussions 103
Chapter 5. Conclusions 106
References 109
요약 (국문초록) 124
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dc.formatapplication/pdf-
dc.format.extent3960709 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectangiogenesis-
dc.subjectischemic disease-
dc.subjectstem cell transplantation-
dc.subjectsolar cell-
dc.subjectdecellularized matrix-
dc.subjectextracellular matrix-
dc.subject.ddc660.6-
dc.titleEnhanced therapeutic angiogenesis in mouse hindlimb ischemia model by electrical stimulation and extracellular matrix-
dc.title.alternative전기 자극과 세포외 기질을 이용한 마우스 하지 허혈 모델에서의 혈관 재생 향상-
dc.typeThesis-
dc.contributor.AlternativeAuthorGun-Jae Jeong-
dc.description.degreeDoctor-
dc.contributor.affiliation공과대학 화학생물공학부-
dc.date.awarded2018-02-
Appears in Collections:
College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Ph.D. / Sc.D._화학생물공학부)
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