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Phase modification of cartilage extracellular matrix preserving antiangiogenic properties and its clinical application as an antiadhesive agent
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 민병현 | - |
| dc.contributor.author | 윤희웅 | - |
| dc.date.accessioned | 2022-11-29T02:32:14Z | - |
| dc.date.available | 2022-11-29T02:32:14Z | - |
| dc.date.issued | 2020-08 | - |
| dc.identifier.other | 30267 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/19736 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2020. 8 | - |
| dc.description.tableofcontents | BACKGROUND 1 1.1. Decellularized extracellular matrix (dECM) as a functional biomaterial 2 1.2. Bio-inductive components of dECM 2 1.2.1. Collagen 2 1.2.2. Glycosaminoglycan 3 1.2.3. Fibronectin 3 1.2.4. Laminin 3 1.2.5. Growth factors 4 1.2.6. Cryptic peptide 4 1.2.7. Extracellular vesicles 5 1.3. Clinical applications of dECM-based biomaterials 5 1.4. Considerations for dECM-based biomaterials research 7 1.5. Articular cartilage ECM 7 1.6. Thesis overview 8 CHAPTER I: Preparation of cartilage acellular matrix powder suspension for anti-angiogenic application 10 2.1. Introduction 11 2.2. Materials and methods 13 2.2.1. Chemicals, reagents, and tissues 13 2.2.2. Production of dg-CAM powder 13 2.2.3. Physical characterization of the dg-CAM powder 14 2.2.4. Biochemical characterization of the dg-CAM powder 14 2.2.5. Quantification of anti-angiogenic factors in the dg-CAM powder 15 2.2.6. Cell culture 16 2.2.7. Assessment of cell viability and proliferation 16 2.2.8. Cell migration assay 16 2.2.9. Cell adhesion assay 17 2.2.10. Tube formation assay 17 2.2.11.Statistical analysis 17 2.3. Results 18 2.3.1. Optimization of pepsin digestion condition of dg-CAM powder 18 2.3.2. Physical properties of the dg-CAM powder 20 2.3.3. Biochemical characterization of the dg-CAM powder 22 2.3.4. The amount of anti-angiogenic factors in the dg-CAM powder 24 2.3.5. Inhibitory effect of the dg-CAM powder on the proliferation, migration, and adhesion of endothelial cells 26 2.3.6. Inhibitory effect of the dg-CAM powder on tube formation of endothelial cells 31 2.4. Discussion 33 CHAPTER II: Identification and characterization of cartilage acellular matrix derived extracellular vesicles as a potential anti-angiogenic molecule 37 3.1. Introduction 38 3.2. Materials and methods 39 3.2.1. Chemicals, reagents, and tissues 39 3.2.2. Preparation of decellularized ECM (dECM) 39 3.2.3. Solubilization of dECM samples 39 3.2.4. Tissue-derived EV (tEV) isolation 40 3.2.5. tEV imaging 41 3.2.6. tEV size determination 41 3.2.7 Western blot analysis 41 3.2.8. Selection of candidate microRNA (miRNA) 41 3.2.9. Analysis of miRNA using qRT-PCR 42 3.2.10. Cell culture 42 3.2.11. tEVs fluorescent labeling 43 3.2.12. Cell viability and proliferation assay 43 3.2.13. Cell migration assay 43 3.2.14. Cell adhesion assay 44 3.2.15. Tube formation assay 44 3.2.16. Matrigel plug assay 44 3.2.17. Rabbit corneal neovascularization (NV) 45 3.2.18. Histological analysis of rabbit cornea 45 3.2.19.Statistical analysis 46 3.3.Results 47 3.3.1. Optimization of CEVs isolation 47 3.3.2. Characterization of tEVs 49 3.3.3. Differences in angiogenesis-related miRNA embedded in CEVs and SEVs 51 3.3.4. Effect of the tEVs on the viability of HUVECs and hMSCs in vitro 53 3.3.5. Effect of the tEVs on the proliferation, adhesion, migration of HUVECs and hMSCs in vitro 55 3.3.6. Effect of the tEVs on the tube formation of HUVECs in vitro 57 3.3.7. Effect of the tEVs on the vessel invasion in Matrigel plug assay in vivo 59 3.3.8. Effect of tEVs on the suture-induced corneal neovascularization (NV) in the rabbit model 61 3.4. Discussion 65 CHAPTER III: Preparation of a cross-linked cartilage acellular matrix film as an anti-adhesive agent for the prevention of post-surgical peritendinous adhesions 69 4.1. Introduction 70 4.2. Materials and methods 72 4.2.1. Preparation of CX-CAM film 72 4.2.2. Biochemical characterization of UX-CAM film 72 4.2.3. Physical characterization of UX- and CX-CAM film 73 4.2.4. Degradation profile of CX-CAM film 73 4.2.5. Cell culture 74 4.2.6. Cell viability and proliferation assay 74 4.2.7. Cell migration assay 74 4.2.8. Cell adhesion assay 74 4.2.9. Rabbit Achilles tendon injury model 75 4.2.10. Gross analysis 75 4.2.11. Histologic analysis 76 4.2.12. Immunohistochemical analysis 77 4.2.13. Ultrasound evaluation 77 4.2.14. Biomechanical evaluation of adhesion 77 4.2.15.Statistical analysis 77 4.3. Results 78 4.3.1. Biochemical characterization of UX-CAM film 78 4.3.2. Optimization of cross-linker concentration 80 4.3.3. Physical changes in CAM film after cross-linking 83 4.3.4. Degradation profile of CX-CAM film 85 4.3.5. Cell viability, cell migration and cell adhesion 87 4.3.6. In vivo efficacy of CX-CAM film in a rabbit tendon repair model 89 4.4. Discussion 96 CONCLUSIONS 100 REFERENCES 102 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Phase modification of cartilage extracellular matrix preserving antiangiogenic properties and its clinical application as an antiadhesive agent | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2020. 8 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 1151647 | - |
| dc.identifier.uci | I804:41038-000000030267 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000030267 | - |
| dc.subject.keyword | Anti-adhesion | - |
| dc.subject.keyword | Anti-angiogenesis | - |
| dc.subject.keyword | Cartilage | - |
| dc.subject.keyword | Decellularization | - |
| dc.subject.keyword | Extracellular matrix | - |
| dc.subject.keyword | Extracellular vesicles | - |
| dc.description.alternativeAbstract | Cartilage is a naturally occurring avascular tissue that provides fundamental anatomical properties for its structure and function. Understanding of the avascular nature of cartilage has led to attractive strategies that anti-angiogenic application of cartilage dECM on an angiogenesis-related disease. However, in the field of dECM-based biomaterials, further advanced development is required for proper clinical applications. First, for the clinical use of dECM, it is necessary to optimize the stable manufacturing process and establish its evaluation methods for safety and efficacy. Secondly, modification of dECM to various formulations should be possible for the proper application of diverse anatomical location. Thirdly, dECM should be able to control the degradation rate according to the pathophysiological mechanism of the desired indications. Fourthly, it is important to understand the biological mechanism of tissue-specific dECM. As dECM has different biological functions and roles depending on the source tissue, identifying differences in bioactivity between tissue-specific dECM can be a significant research area. Also, the study for the biological properties of individual components can help to understand the overall biological mechanisms of the entire dECM. Thus, the purpose of this study was to develop a formulation of cartilage acellular matrix (CAM) for clinical application, analyze molecular components within CAM, and verify its anti-angiogenic therapeutic efficacy. In chapter I, we present a fabrication technology to produce a water-dispersible and biologically functional dECM powder suspension from porcine articular cartilage. The digested-cartilage acellular matrix (dg-CAM) powder was prepared by sequential processes of decellularization, enzymatic digestion, and pulverization. The dg-CAM contained a significantly larger amount of soluble proteins than that of the native cartilage tissue (NCT) and showed an improved dispersion property. It also retained anti-angiogenic molecules, such as thrombospondin-1 and endostatin, as much as the NCT. The inhibitory effect on angiogenesis of the dg-CAM was more prominent than that of type I collagen in inhibiting the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Thus, we suggest that dg-CAM suspension could be a potential anti-angiogenic agent for various neovascularization-related disorders. In chapter II, we investigated whether cartilage-derived extracellular vesicles (CEVs) could recapitulate the anti-angiogenic effects of parent dECM. The CEVs were isolated from the collagenase treated CAM solution, followed by removing fibrous molecules by microfiltration. Finally, CEVs were collected using chemical precipitation reagent. Small intestinal submucosa (SIS), which have shown the pro-angiogenic effects, was used as control. Furthermore, SIS-derived extracellular vesicles (SEVs) were harvested in the same way as above. After isolating bote tissue-derived extracellular vesicles (tEVs), angiogenic regulatory miRNAs were investigated using a quantitative polymerase chain reaction (qPCR). In the CEVs, anti-angiogenic miRNAs (miR-15a, miR-26a, miR-125b, miR-221, miR-222) were relatively enriched, whereas in SEVs, pro-angiogenic miRNAs (miR-10b, miR-17, miR-31, miR-126, miR-146a) were highly expressed. Matrigel plugs containing CEV inhibited vascular invasion, whereas the SEV group promoted it compared to the control group. Furthermore, in the corneal NV assay, CEV effectively inhibited neovessel formation from the conjunctiva to the cornea, but the SEV group showed severe angiogenesis. The present study demonstrated that the tissue-specific tEVs could recapitulate the differential angiogenic regulatory effects. These biological functions of tEV on angiogenesis may play an important role in understanding the overall biological mechanism of dECM-based products. In chapter III, as an extended clinical application of CAM, we hypothesized that anti-angiogenic and anti-adhesive effects of CAM could be suitable properties for post-surgical adhesion. We have aimed to develop a cross-linked CAM film to prevent peritendinous adhesion after surgery. The CAM-film was prepared by casting a dg-CAM suspension on the silicon mold followed by glutaraldehyde cross-linking. Physical characterization of the CAM-film showed denser collagen microstructure, decreased hydrophilicity, and higher tensile strength after cross-linking. The biodegradation profile in vivo was 14 days after cross-linking. Application of the CAM-film after suture repair resulted in significantly less peritendinous adhesions in the rabbit Achilles tendon injury model, evaluated by histology, ultrasonography, and biomechanical analysis. Moreover, the anti-angiogenic effect of the CAM-film was also observed by efficiently inhibiting the expression of angiogenic markers (VEGF, CD31) on the injured site. In conclusion, the current study developed a CAM-film having the anti-adhesive and anti-angiogenic properties, together with biomechanical properties and biodegradation profile suitable for the prevention of peritendinous adhesions. | - |
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