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Phase modification of cartilage extracellular matrix preserving antiangiogenic properties and its clinical application as an antiadhesive agent
- Author(s)
- 윤희웅
- Advisor
- 민병현
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2020-08
- Language
- eng
- Keyword
- Anti-adhesion; Anti-angiogenesis; Cartilage; Decellularization; Extracellular matrix; Extracellular vesicles
- Alternative Abstract
- 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.
- Fulltext
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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