Anti-angiogenic effect of porcine cartilage derived extra-cellular matrix suspension on experimentally induced rabbit corneal neovascularization model
Anti-angiogenic effect of porcine cartilage derived extra-cellular matrix suspension on experimentally induced rabbit corneal neovascularization model
- 주제(키워드) Extra-cellular matrix(ECM) , cartilage , Bio-material , Anti-angiogenesis , Corneal neovascularization
- 발행기관 아주대학교
- 지도교수 민병현
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000017808
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Object: As an avascular tissue, articular cartilage has anti-angiogenic molecules such as endostatin, chondromodulin-1 and thrombospondin-1. Previously, we have fabricated the suspension form of extra-cellular matrix (ECM) derived from porcine cartilage and confirmed its anti-angiogenic property in-vitro. The purpose of this study was to investigate the anti-angiogenic effect of topical administration of porcine cartilage derived ECM powder suspension (PCP-s) on the experimentally induced rabbit corneal neovascularization model. Materials and Methods: Preparation of porcine cartilage derived ECM powder was performed using a multiple process, including decellularization, pepsinization and sterilization. Lyophilized porcine cartilage ECM powder was stored at -20 oC until use. Ethylene oxide sterilization was performed. Finally, PCP-s powder was dissolved in normal saline to yield the desired concentrations. For pilot study, we optimized rabbit corneal neovascularization model and concentration of each experimental group. Corneal neovascularization was induced by silk 8-0 suture, 3mm distance from the limbus, parallel to the margin of cornea in 20 rabbits (40 eyes). After suturing, rabbits were randomly divided into 4 groups based on treatment materials: group 1, normal saline; group 2, 10 mg/ml of type I collagen suspension; group 3, 10 mg/ml of PCP-s and group 4, 15 mg/ml bevacizumab. All drops were administered three times daily for a week. After one week, the corneal neovascularization score and vessel invasion area were evaluated. Immunohistochemical analysis was performed to evaluate the expression of vascular endothelial growth factor (VEGF) and CD-31. For biochemical analysis, hemoglobin contents in cornea tissue were evaluated. Results: In the microscopic analysis, group treated with PCP-s showed significantly decreased vessel invasion area compared to saline and type I collagen suspension treated group (p<0.001). In the histological evaluation, corneal neovascularization induced in all groups. However hematoxylin and eosin staining indicated that group treated with PCP-s decreased vessel number compared to saline and type I collagen suspension treated group (p<0.01 and p<0.05, respectively). In the immunohistochemistry analysis, there was less expression of VEGF and CD31 in the subepithelial stroma of group treated with PCP-s compared to other control groups. In the biochemical assay, PCP-s treated group showed decreased hemoglobin contents compared to type I collagen suspension treated group (p<0.05). Conclusions: In this study, suspension form of porcine cartilage derived ECM (PCP-s) significantly reduced corneal neovascularization according to microscopical, histological and biochemical evaluations. These results indicate that PCP-s maintains its anti-angiogenic effect on corneal neovascularization in-vivo. Therefore, PCP-s could be a useful therapeutic biomaterial for many anti-angiogenic applications including the prevention of corneal neovascularization.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iv
LIST OF FIGURES v
I. INTRODUCTION 1
II. MATERIALS AND METHODS 5
1. Preparation of porcine cartilage derived ECM powder suspension 5
2. Experimental animals 8
3. Optimization of rabbit corneal NV model 8
4. Efficacy range of PCP-s on the rabbit corneal NV model 9
5. Duration of PCP-s on the cornea 9
6. Study groups and topical administration on the cornea 10
7. Analysis of corneal NV by microscopic examination 11
8. Analysis of corneal NV by histological examination 11
9. Hemoglobin content analysis of corneal NV 13
10. Statistical analysis 13
III. RESULTS 14
IV. DISCUSSION 29
V. CONCLUSION 35
REFERENCES 36
국문요약 42
