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In Situ Forming Gelatin Hydrogels with Enhanced Tissue Adhesiveness by Additional Supramolecular and Disulfide Linkages

In Situ Forming Gelatin Hydrogels with Enhanced Tissue Adhesiveness by Additional Supramolecular and Disulfide Linkages

초록/요약

Hydrogels are polymeric networks with three-dimensional structure, hydrodynamic properties and low surface tension. They can absorb a large amount of water about thousands of times higher than their dry weight but not dissolved. Due to possessing these properties, the hydrogels are very similar to extracellular matrices (ECMs). Moreover, we can engineer the polymeric materials to form a desired hydrogel with specific requirements, practical shapes and dimensions for clinical demands, such as easy operation, biocompatibility, irregular defect coverage and effective host integration. Thus the hydrogels have become the good candidate for developing an artificial extracellular matrices (ECMs) in tissue engineering, cell therapy, drug-delivery system, medical implants, and biosensors. For serving the variety of these applications, adhesive property is priorly necessary in order to keep the hydrogels at the right treated position within the desired time. More importantly, tissue adhesive is also an attractive field in clinic nowadays. It has been considered as an alternative material for sutures, staples and strips due to an integration of several functions (preventing fluid leakage, faster stop bleeding, therapeutic incorporation to induce a healing process or various other purposes of clinical treatments). Although the commercial bio-glues have already launched into the market, they still remain some limitations. For example, cyanoacrylate has strong adhesion, but cytotoxicity due to the degraded products of aldehyde molecules. Fibrin glue is more biocompatible but it has relatively weak adhesive strength and potentially pro-inflammatory proteins. Taken together, the adhesive has to be further developed from the hydrogels to satisfy all clinical requirements, firstly to improve an adhesive force without compromising its biocompatibility. In the previous research, hydroxyphenyl propionic acid-conjugated gelatin (GH) was developed as the compatible hydrogel showing 2- – 3-fold higher adhesive strength when compared to fibrin glue. However, this value was not enough for widely clinical applications. In this study, improving adhesiveness and maintaining the biocompatibility of GH hydrogels are focused. Introducing more crosslinks between material and tissue are explored. Inspired from nature, there are the versatile molecular processes having the adhesive property based on the dynamic and non-covalent interactions. Hydrogen bonding and host-guest interactions are the first choice to improve GH hydrogel adhesiveness. The hypothesis is that if cyclodextrins (CDs) are presence in GH hydrogel matrix, CDs are able to form the additional non-covalent bond (hydrogen bonding and host-guest complex) to enhance both cohesive and adhesive properties of the hydrogels (Chapter 2). In fact, a remarkable enhancement in the adhesiveness of GH hydrogels can be achieved by simply incorporating CDs. Interestingly, because of γ-CD with higher host-guest complexation ratio (γ-CD:guest 1:2 and α-CD:guest 1:1) inside of the GH hydrogel as well as at the hydrogel-skin interface, GH/γ-CD hydrogels achieve the greater hydrogel-tissue interactions, and substantial adhesion to skin. The commercial fibrin glue is utilized as a control, GH/CD hydrogels get 10-fold higher adhesiveness. The adhesive mechanism is understood through using modified gold substrates as a model study. For the second strategy, the additional covalent bonds are studied to highly improve GH adhesiveness. Exploiting the fact that thiol groups or disulfide bonds of skin compositions can react or exchange with thiomers, GH hydrogels are blended with thiomers to increase their crosslinking ability with tissue (Chapter 3). Moreover, under the gelation condition of GH hydrogels using HRP/H2O2, thiomers are also induced to form disulfide cross-linked networks. As expected, the adhesive force of 5 wt% GH is significantly increased 6.5 times when adding 5 wt% thiolated gelatin (GS), that is 15.8 times higher than the adhesive strength of fibrin glue. On the other hand, the gelation time, the elastic modulus, the swelling ratio, and the degradation of GH/CD and GH/GS hydrogels are also tested to confirm their suitable properties for adhesive applications. Cell viability WST-1 assays and live/dead staining with human dermal fibroblasts on those hydrogel surfaces show their excellent cellular compatibility. Overall, we expect that the significant improvement of adhesiveness of GH hydrogels using additionally physical or chemical bonding may set the new stage of that those hydrogels are more advance in bio-applications. GH/CD hydrogels can be investigated to combine therapy with cells and drugs. GH/GS hydrogels with dynamic covalent interactions and sensitive to pH or redox of disulfide bonds can be very interesting for developing the smart hydrogels in future research.

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목차

Chapter 1. General introduction 1
1.Brief overview of skin architecture and compositions 3
2.Brief introduction of wound management and potential care 8
3.Conventional methods in wound management 11
4.Introduction of tissue adhesives 13
4.1. Roles of tissue adhesive in biomedical applications 14
4.2. Mechanism of tissue adhesive 16
4.2.1 Mechanical interlocking 16
4.2.2 Intermolecular bonding 17
4.2.3 Diffusion theory 19
4.2.4 Electrostatic bonding 20
4.3.Requirements and challenges of tissue adhesive in biomedical applications 20
4.4.Current adhesives and their limitations 22
4.4.1. Synthetic adhesive 22
4.4.1.1 Cyanoacrylate adhesive 22
4.4.1.2 Polyethylene glycol (PEG)-based adhesive 23
4.4.2. Natural adhesive 24
4.4.2.1 Fibrin-based adhesive 24
4.4.2.2 Collagen/gelatin-based adhesive 25
4.4.2.3 Albumin-based adhesive 27
4.4.3.Hybrid adhesive 27
4.4.4.Mussel-mimicked adhesive 29
5.Hydrogels 32
6.Overall objective 39

Chapter 2. In situ forming gelatin hydrogels with enhanced adhesiveness using the additional supramolecular interactions 43
1.Introduction 44
2.Materials and methods 49
2.1. Materials 49
2.2. Synthesis and characterization of hydroxyphenyl propionic acid conjugated gelatin (GH) 51
2.2.1. Synthesis procedure of GH 51
2.2.2. UV-Vis spectroscopy and phenol content calculation 52
2.2.3. 1H NMR technique 54
2.3. Synthesis and characterization of modified gold substrate for adhesive mechanism study 55
2.4. High-resolution ESI-MS of CDs/Tyramine complexes 56
2.5. 2D-ROESY (Rotating frame nuclear Overhauser Effect Spectroscopy) 56
2.6. Contact angle measurement of hydrogels 57
2.7. Formation and gelation time of GH and GH/CD hydrogels 57
2.8. Quantitative peroxide assay 58
2.9. Mechanical strength 60
2.10. Tissue adhesive strength 60
2.11. In vitro proteolytic degradation 61
2.12. Cytotoxicity evaluation 62
2.13. Statistical analysis 63
3. Results and discussion 63
3.1. Synthesis and characterization of GH conjugates 63
3.2. Formation of in situ cross-linkable GH and GH/CD hydrogels 66
3.3. Impact of CD on physico-chemical properties of gelatin-base hydrogels 68
3.4. Adhesive force of GH/CD hydrogels and investigating the role of CDs on improvement of adhesive strength of gelatin hydrogels 71
3.5. Understanding hydrogel-skin interactions 77
3.6. Cytotoxicity evaluation 81
4.Conclusions 83

Chapter 3. In situ forming gelatin hydrogels with enhanced adhesiveness by the combination of disulfide and phenol-phenol crosslinks 84
1. Introduction 85
2. Experimental section 90
2.1. Materials 90
2.2. Polymer synthesis and characterization 92
2.2.1. Synthesis of gelatin-hydroxyphenyl propionic acid (GH) 92
2.2.2. Synthesis of thiolated gelatin (GS) 92
2.2.3. Synthesis of thiolated gelatin-hydroxylphenyl propionic acid (GHS) 93
2.2.4. Thiol measurements 95
2.2.5. Attenuated total reflectance – infrared reflection (ATR-IR) spectroscopy 96
2.3. Preparation of precursor polymer mixtures 97
2.4. Hydrogel formation and gelation time 98
2.5. Mechanical strength 99
2.6. Tissue adhesive test 100
2.7. Swelling ratio 101
2.8. Enzymatic degradation test 102
2.9. Cytotoxicity test 103
2.10. In vivo hemostatic ability test 104
2.11. Statistical analysis 105
3. Results and discussions 106
3.1 Synthesis and characterization of GH, GS, and GHS polymer 106
3.2 GH/GS hydrogel formation 108
3.3 Gelation time 109
3.4 Mechanical strength, swelling ratio and degradation test 112
3.5 Adhesive strength 115
3.6 In vitro cytotoxicity of GH/GS hydrogels 119
3.7 In vivo hemostatic test 120
4. Conclusions 121

Chapter 4. Overall conclusions and future works 123
1. Overall conclusions 124
2. Future works 126

References 129

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