심혈관계응용을 위한 헤파린이 고정화된 생체재료에 관한 연구
Heparin-Immobilized Nanobiomaterials for Cardiovascular Applications
- 주제(키워드) heparin , bovine pericardium , small intestinal submucosa , polylactide , acellular matrix , blood compatibility , calcification , cardiovascular application
- 발행기관 아주대학교 대학원
- 지도교수 박기동
- 발행년도 2008
- 학위수여년월 2008. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 본문언어 영어
초록/요약
Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities that are associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Therefore, the incorporation of heparin to biomaterials has been studied by dispersing heparin within the biomaterial (Kito and Matsuda 1996), by ionic binding or covalent immobilization of heparin (Bourin and Lindahl 1993). The biological activities of heparin primarily result from their interaction with hundreds of different proteins including enzymes (i.e., thrombin), lipoproteins, growth factors, chemokines, viral coat proteins, extracellular matrix proteins, etc (Linhardt 2003). Specific recognition between heparin and proteins also requires defined sequences within the heparin chain. Heparin predominantly exhibits linear helical secondary structures with sulfo and carboxyl groups displayed at the defined intervals and in defined orientations along the polysaccharide backbone. Heparin-binding domain on a protein would, therefore, require a minimum number of saccharide residues within the heparin chain to appropriately display these charged groups to facilitate a tight and specific interaction (Capila and Linhardt 2002). The incorporation of heparin to biomaterials has been widely studied to improve the biocompatibility (blood and cell) of biomaterials. In particular, heparinized surfaces play crucial roles in biological phenomena as interfaces between biological environments (i.e., blood, tissues and etc.) and biomaterials for a variety of biomedical applications. Such surfaces have been mainly prepared and investigated by coating, immobilization on certain natural (or synthetic) surfaces, and so on. As advancing in biomedical fields, however, bioactive surfaces with ‘nano’ size have lately attracted considerable remark, which can designate as a novel term ‘nanointerface’. The nanointerface has many advantages with various functionalities such as the immobilization of bioactive molecules to utilize it for biomedical applications. In this dissetation, some examples of heparin-conjugated surfaces have been introduced in terms of material for cardiovascular application. The use of biodegradable polymers has been recently proposed for several applications including tissue engineering, gene therapy, novel drug delivery systems and implantable devices. Further applications of biodegradable polymers have been desired in blood-contacting situations such as vascular prosthesis where temporary reconstruction and/or stabilization of tissue and organs is needed, and intravascular stents that should maintain blood vessels open after balloon dilatation (Agrawal et al. 1992). However, thrombus formation and cell compatibility are still serious problems in the use of biodegradable polymers like polylactide and investigations on properties of the materials that contact tissue are rarely found. Therefore, heparin-conjugated linear PLA (PLA-hep) to improve blood compatibility and its related biocompatibility of degradable polymers were investigated and introduced in this dissertation. Since artificial prosthetics such as synthetic polymers and metals were used in the field of cardiovascular surgery, biomaterials possessing essential characteristics of native tissues and organs which are to be replaced have been constantly required for the development of an ideal artificial substitute. Consequently, the use of xenograft and allograft tissues has been in the center of cardiovascular research (Hilbert et al. 1998). Small intestinal submucosa (SIS), a relatively acellular collagen-based matrix derived from porcine small intestine, has been extensively used as a cardiovascular bioprosthesis such as heart valve (Matheny et al. 2000), vascular graft (Roeder et al. 1999), and tissue patch for the repair of myocardial infarction (Badylak et al. 2003). Collagen-based biomaterial such as SIS has typically required chemical or physical pretreatment to enhance in vivo biochemical and mechanical properties. The heparin binding which led to the formation of bridge between adjacent fibrils reduced the calcium accumulation site. In this dissertation, The SIS and BP that is chemically modified with heparin and the heparin-immobilized SIS and BP were studied to investigate the effect of heparin immobilization on blood compatibility, in vitro fibroblast attachment, and in vivo calcification of SIS and BP. An outline of each chapter is as follows. Chapter I : introduced the general bacground for understanding of the objects. this chapter involves the introduction of each materials and objects. Capter II : acellular matrix of bovine pericardium (ABP) was chemically modified by the direct coupling of heparin (Hep) after glutaraldehyde (GA) cross-linking. The effects of Hep coupling on durability and calcification were investigated and the biocompatibility was evaluated in vitro and in vivo. Obtained results attest to the usefulness of Hep treated ABP for cardiovascular bioprostheses. Chapter III : chemical modification of biological tissue was developed by the immobilization of heparin to small intestinal submucosa (SIS) using glutaraldehyde (GA). Heparinized SIS tissue was characterized. heparin immobilized SIS can be applied as a novel bioprosthesis for a variety of cardiovascular applications. Chapter IV : A heparin conjugated biodegradable polymer was developed by the direct coupling of heparin to polylactide (PLA). PLA was covalently coupled with heparin by DMAP / DCC. Heparin conjugated PLA was characterized.This novel polylactide (PLA)-heparin conjugate could be applied as blood/tissue compatible biodegradable materials for implantable medical devices and tissue engineering.
more초록/요약
본 논문은 심혈관계 응용을 위한 헤파린이 화학적으로 도입된 나노바이오계면에 관한 연구 결과들을 정리하였다. 구체적으로 본 논문의 연구결과들은 인공혈관, 화상치료, 흉부외과 수술용 차폐막, 심혈관계 코팅용 재료로서 응용이 가능한 천연조직과 폴리락타이드 (polylactide)와 같은 생분해성의 합성고분자에 생리활성물질인 헤파린(Heparin)이 도입된 생체재료를 제작하고 이들의 생체적합성, 항 칼슘성과 같은 특성에 관하여 연구하였다. 본 논문은 총 4 Chapters로 구성되었다. Chapter1은 본 연구의 관련분야에 대한 개괄적인 소개와 연구에 사용된 주재료들에 관한 설명으로 정리하였다. Chapter2와 4는 천연조직인 소심막 (bovine pericadium, BP)과 소장내막(small intestinal submucosa, SIS)에 헤파린이 도입된 생체재료에 관한 개발 및 특성연구에 관한 부분과 Chapter 3에서 헤파린이 도입된 생분해성 고분자의 개발 및 특성연구에 관한 부분으로 나누어 설명하였다 각 부분의 연구 내용을 간단히 설명하면 다음과 같다. Chapter1은 본 논문의 목적과 중요성을 이해하는데 필요한 개괄적인 소개 및 각 재료들에 관하여 정리하였다. Chapter 2는 acellular matrix bovine pericardium에 헤파린을 도입한 재료를 개발하고 이 재료의 향상된 생체적합성과 항칼슘성 등에 관한 연구결과들을 정리하였다. Chapter 3은 PLA와 같은 생분해성 합성고분자에 헤파린을 도입하고 혈액적합성 등에 관한 연구내용에 대하여 기술하였다. Chapter 4는 천연조직인 porcine small intestinal submucosa (SIS)에 헤파린이 도입된 생체재료를 제조하고 이에 대한 특성을 평가한 연구내용을 정리하였다. 헤파린이 도입된 소심막과 소장내막에서 모두 향상된 항 캴슘성과 내구성을 보였고 폴리락타이드에서도 항 응혈성 등의 생체적합성이 향상된 결과를 보였다. 따라서 헤파린이 도입된 모든 재료에서 생체적합성이 향상되었고 모든 재료들이 심혈관계용 재료로서 응용이 가능하다 할 수 있겠다.
more목차
Chapter I General Introduction = 1
1.Biomaterials = 2
1.1 Polymeric biomaterials = 2
1.2 classifications of polymeric biomaterials = 3
2. Collagen-based biomaterials = 6
3. Acellular matrix = 10
4. Cross-linking of collagen = 11
4.1Chemical cross-linking = 12
4.2Physical cross-linking = 15
5.Parhologic calcification of biomaterials = 15
6.Prevention of calcification = 17
7.Biodegradable polymer = 19
7.1. Poly(lactide)(PLA) = 21
8. Heparin = 23
8.1 Structure of heparin = 23
8.2 Medical importance of heparin = 25
8.3 Interaction of heparin with protein = 26
9. Biocompatibility and its Evalutions = 32
9.1 Biocompatibilty = 32
9.2 Evalution of biocompatibilty = 33
10. Surface modification for Enhancing Biocapatibility = 35
10.1 surface function in biomaterials = 35
10.2 Bulk functions in biomaterials = 37
11. Objectives = 38
Chapter II Heparin-immobilized ABP = 44
1.Summary = 45
2. Introduction = 46
3. Materials and Methods = 51
3.1. Materials = 51
3.2. Experiment methods = 52
3.2.1. Cell extraction = 52
3.2.2. Light microscopy = 53
3.2.3. Chemical modification of acellular bovine pericardium = 53
3.2.4. Thermal and mechanical properies = 54
3.2.5. Resistance to enzyme digestion = 55
3.2.6. In vivo calcification : rat model = 56
3.2.7.In vitro culture of human dermal fibroblasts = 57
4. Results and Discussions = 58
4.1. Light microscopy of BP and ABP = 58
4.2. Chemical modification of ABP = 59
4.3. Thermal and Mechanical properties = 60
4.4. Resistance to collagenase digestion = 64
4.5. In vivo calcification: rat model = 65
4.6. In vitro culture of HDFs = 66
5. Conclusions = 68
References = 70
Chapter III Heparin immobilized SIS = 73
1. Summary = 74
2. Introduction = 74
3. Materials and Methods = 76
3.1. Materials = 76
3.2. Experiment methods = 76
3.2.1. Preperation of heparin immobilized SIS = 76
3.2.2. Amount of immobilized heparin and its anticoagulant activity = 78
3.2.3. In vitro protein adsorption and platelet adhesion = 78
3.2.4. Invitro fibroblast attachment = 78
3.2.5. In Vivo calcification = 79
4. Results and Discussions = 79
5. Conclusions = 83
References = 84
Chapter IV Heparin-immobilized PLA1 = 86
1. Summary = 87
2. Introduction = 88
3. Materials and Methods = 90
3.1. Materials = 90
3.2. Synthesis of PLA-heparin = 91
3.3. Characterization of PLA-heparine = 92
3.4. Quantitative analysis of heparin = 93
3.5. Activated partial thromboplastin time(APTT) assay = 93
3.6. In vitro protein adsorption = 94
3.7. In vitro platelet adhesion = 94
4. Results and Discussion = 95
4.1. Characterizations of PLA-heparin = 95
4.2. Quantitative analysis of heparin = 98
4.3. Activated partial thromboplastin time(APTT) assay = 98
4.4. In vitro protein adsorption and platelet adhesion = 99
5. Conclusions = 102
References = 104
Publication list = 107
Abstract(in Korean) = 115
