Enhanced Tissue Integration and Hemocompatibility of Biomaterials through Tyrosinase-Catalyzed Oxidative Reaction
- 주제(키워드) Biomaterials , injectable hydrogels , tissue adhesiveness , surface modification , antithrombogenic properties , antibacterial properties.
- 발행기관 아주대학교
- 지도교수 Ki Dong Park
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000027789
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Biomaterials have been widely applied in the biomedical fields, including tissue engineering/regeneration to restore, replace, or build up the diseased and damaged tissues/organs. Nowadays, many effort is being made to develop biomaterials that can have the good bulk properties while minimizing the host responses by immune system upon implantation into the body. Also, the great aspiration has been raised to biocatalysts, to produce a “green” chemical industry with high efficiency. In particular, the enzyme-catalyzed reaction is attracting much attention for preparation of injectable scaffolds and modification of biomaterial surfaces due to its biocompatibility, high selectivity and controllable reactions under mild reaction conditions. In this dissertation, we utilized tyrosinase-catalyzed reaction as a simple and effective strategy for preparation of injectable hydrogels and modification of polymeric substrates for biomedical applications. Hydrogels have been used as potential candidates for tissue engineering application because they have unique properties that mimic the extracellular matrix (ECM). Particularly, the in situ forming hydrogels are preferred because they can minimize the invasive surgery and allow easy encapsulation of cells/therapeutic agents in the polymer solutions. Moreover, they can easily fill the damaged tissues with any irregular shape. In this regard, the strong adhesion between materials and surrounding tissues should be optimized to enhance the performance of hydrogels in vivo. The crosslinking reaction catalyzed by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) has proven its efficiency for the rapid formation of hydrogels under physiological conditions. The tissue adhesiveness of hydrogels was enhanced as increasing the mechanical strength (cohesion strength) of hydrogels, which is controllable by varying the concentration of H2O2. In this study, we hypothesized that the addition of tyrosinase (Tyr) would promote the more interactions between hydrogels and surrounding tissues, leading to dual-enzymatically crosslinked hydrogels with superior adhesive strength compared to HRP-crosslinked hydrogels (Chapter 2). As expected, Tyr converted the phenol moieties of polymer derivatives into active o-quinones, followed by rapid reactions with nucleophiles on tissue surfaces (e.g., amines, thiols), thereby enhancing the tissue adhesiveness of hydrogels. The effect of enzyme compositions on physico-chemical properties of dual enzymatically crosslinked hydrogels, such as mechanical strength, gelation rate, swelling degree and adhesive strength was investigated. The optimal concentration of additional Tyr was 0.25 kU/mL, which significantly increased the adhesive strength to 2 times and 5 times higher than that of only HRP-catalyzed hydrogels and commercially fibrin glues, respectively. The in vitro cell study results also demonstrated the enhanced cellular activities of dual enzymatically crosslinked hydrogels. In chapter 3, we introduced a new strategy to simultaneously impart antithrombotic and anti-infective properties to biomaterials surfaces in a one-step process, using Tyr -catalyzed oxidative reaction. Heparin, an anticoagulant agent approved by FDA, was conjugated with tyramine (termed “HT”) to provide the phenol moieties as substrates for enzymatic activities of Tyr. The catechol forms were generated by Tyr were used for two functions: (1) immobilization of HT molecules for antithrombotic and (2) in situ formation of Ag NPs for antibacterial activity. The successful immobilization of both heparin and Ag NPs on surfaces was confirmed by analyses of water contact angles, XPS, TEM and AFM. We found that the amount of heparin and Ag NPs immobilized on surfaces were controllable by varying the reaction time and feed amount of silver ions, respectively. The resulting HT/Ag NPs immobilized surfaces possess high stability for over 30 days. Importantly, the modified surfaces achieved thromboresistant properties by inhibiting the fibrinogen absorption, platelet adhesion and prolonging the blood clotting time. In addition, the integrated HT/Ag NPs immobilized surfaces exhibited excellent antibacterial performances against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. From the obtained results, we expect that the Tyr-catalyzed reactions can be used as a facile and effective approach for enhanced adhesive properties of hydrogels and surface modification for versatile biomedical applications, such as tissue regeneration and implantable biomedical devices.
more목차
Abstract i
Table of Contents iv
List of Figures vii
List of Tables xi
List of Abbreviations xii
Chapter 1. General introduction 1
1. Biomaterials 2
1.1. Natural polymers 3
1.2. Synthetic polymers 4
2. Hydrogel 8
2.1. Enzyme-catalyzed cross-linkable hydrogels 11
2.1.1. Transglutaminase (TGase)-catalyzed cross-linking reaction 11
2.1.2. Horseradish peroxidase (HRP)-catalyzed cross-linking reaction 12
2.1.3. Tyrosinase (Tyr)-catalyzed cross-linking reaction 17
2.1.4. Dual enzymatic cross-linking systems 19
3. Surface modification of biomaterials 21
3.1. Surface modification for antithrombogenic properties 24
3.2. Surface modification for antibacterial properties 27
3.2.1. Silver nanoparticles immobilized surfaces 30
3.3. Mussel-inspired chemistry for hydrogel preparation and surface modification 33
4. Overall objective 37
5. References 39
Chapter 2. In situ forming hydrogels with enhanced tissue adhesiveness by dual enzyme-mediated crosslinking 47
1. Introduction 48
2. Materials and methods 50
2.1. Materials 50
2.2. Synthesis and characterization of gelatin-hydroxyphenyl propionic acid (GH) conjugate 52
2.3. Hydrogel preparation and gelation time 52
2.4. Mechanical strength of hydrogels 53
2.5. Tissue-adhesive strength of hydrogels 54
2.6. Swelling and degradation test 55
2.7. Cytotoxicity test 56
2.8. Statistical analysis 57
3. Results and discussion 57
3.1. Synthesis and characterization of GH conjugates 57
3.2. Hydrogel fabrication and gelation time 59
3.3. Mechanical strength 60
3.4. Effect of Tyr-cross-linking activity on tissue-adhesive strength 62
3.5. Swelling and degradation 68
3.6. In vitro cytocompatibility 69
4. Conclusions 71
5. References 72
Chapter 3. Surface modification with enhanced antithrombotic and antibacterial activities 77
1. Introduction 78
2. Experimental section 80
2.1. Materials 80
2.2. Synthesis and characterization of heparin-tyramine (HT) polymer 81
2.3. Preparation of PU substrates 82
2.4. Co-immobilization of HT and Ag NPs onto PU substrates 84
2.5. Characterization of immobilized substrates 84
2.6. Surface stability and silver release profile 86
2.7. In vitro antithrombogenic evaluation 86
2.7.1. Protein adsorption 86
2.7.2. Platelet adhesion 87
2.7.3. Blood clotting time 88
2.8. Antibacterial activity tests 89
2.9. In vitro cytotoxicity tests 90
2.10. Statistical analyses 91
3. Results and discussions 91
3.1. Synthesis and characterization HT polymer 91
3.2. Surface immobilization and characterization 93
3.3. Surface stability and silver release behavior 98
3.4. In vitro evaluation of antithrombogenicity 99
3.4.1. Fibrinogen adsorption 99
3.4.2. Platelet adhesion 100
3.4.3. Blood clotting time 102
3.5. Antibacterial properties 103
3.6. Cytotoxicity tests 105
4. Conclusions 107
5. References 107
Chapter 4. Overall conclusions 111
