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Laser-Activated Biomaterials for Tissue Repair

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Laser-Activated Biomaterials for Tissue Repair THE UNIVERSITY OF NEW SOUTH WALES Faculty of Engineering Laser-Activated Biomaterials for Tissue Repair Antonio Lauto Thesis Submitted for the Degree of Doctor of Philosophy, 2005 Supervisor: A/Professor Albert Avolio Co-Supervisor: Dr John Foster 1 Declaration I hereby declare that this thesis submission is my own work and that to the best of my knowledge, it contains no material previously published or written by another person nor material which to substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgment is made in the text. Antonio Lauto 2 Acknowledgments I would like to thank my supervisors Albert Avolio and John Foster for their precious help and suggestions throughout my investigations. I am also indebt to Drs Stoodley, Liao, Esposito, Mingin, Sarris and McKenzie for their collaborative effort in the surgical procedures and tissue histology preparation. A special thank to Michael Doran and Lynn Ferris for their help during the cytotoxic tests of the solders and adhesives. I am grateful to Fernando Camacho, Nick Zweneveld, Katie Levick and Jenny Norman for revising part of the manuscript and processing samples for AFM and SEM analysis. I would like also to thank Dr Poole-Warren for the initial collaboration and supervision of the project. Finally, a cheerful thanks to Damia for her patience and verve, Elda for her striking style, Dr James Hook for his friendship and Dr Bruce Lachter for his invaluable advises. This work was partially funded by the ARC discovery grant # DPO345899 and partially by an Engineering UNSW Faculty Grant (2004). 3 To Jeroslav Seifert, …. waiting for the greatest miracle: criminals, politicians, capitalists, soldiers and priests turned into poets! If You Call Poetry... If you call poetry a song - and people often do - then I've sung all my life. And I marched with those who had nothing, who lived from hand to mouth, I was one of them. I sang of their sufferings, their faith, their hopes, and I lived with them through whatever they had to live through. Through their anguish, weakness and fear and courage and poverty's grief. And their blood, whenever it flowed, spattered me. Always it flowed in plenty in this land of sweet rivers, grass and butterflies and passionate women. Of women, too, I sang. Blinded by love I staggered through my life, tripping over dropped blossoms or a cathedral step. Jeroslav Seifert 4 Abstract Background. Laser tissue repair usually relies on haemoderivate solders, based on serum albumin. These solders have intrinsic limitations that impair their widespread use, such as limited repair strength, solubility and brittleness. Furthermore, the solder activation temperature (65-70 0C) can induce significant damage to tissue. In this study, new laser-activated biomaterials for tissue repair were developed to overcome some of the shortcomings of traditional solders. Materials and Methods. Solder strips were welded onto sheep intestine using a diode laser. The laser delivered continuously a power of 170 ± 10 mW at l = 808 nm, through a multimode optical fiber (core size = 200 mm) to achieve a dose of 10.8 ± 0.5 J/mg. The solder thickness and surface area were 0.15 ± 0.01mm and 12.6 ± 1.0 mm2 respectively. The solder contained albumin, indocyanine green, water and a natural crosslinker for amino groups: genipin. Flexible and insoluble strips of chitosan adhesive (surface area ~34 mm2, thickness ~20 µm) were also developed and bonded on sheep intestine with a laser fluence and irradiance of 52 ± 2 J/cm2 and ~15 W/cm2 respectively. The temperature between tissue and adhesive was measured using small thermocouples. The strength of repaired tissue was tested by a calibrated tensiometer. The adhesive was also bonded in vivo to the sciatic nerve of rats to assess the thermal damage induced by the laser (fluence = 65 ± 11 J/cm2, irradiance = 15 W/cm2) four days post-operatively. Finally, fibroblasts were cultured in extracted media from chitosan adhesive to assess cytotoxicity. 5 Results. The repair strength of the genipin-albumin solder was double that of traditional albumin solders (0.21 ± 0.04 vs. 0.11 ± 0.04 N, n=30). Chitosan adhesives successfully repaired intestine tissue, achieving a repair strength of 0.50 ± 0.15 N (shear stress = 14.7 ± 4.7 KPa, n=30) at a temperature of 60-65 0C. The laser caused demyelination of axons at the operated site; nevertheless, the myelinated axons retained their normal morphology proximally and distally. Media extracted from chitosan adhesive showed negligible toxicity to fibroblasts. Conclusion. A novel chitosan-based adhesive has been developed, which is insoluble, flexible and adheres firmly to tissue upon infrared laser activation. Further research is needed to reduce the thermal damage to the tissue. 6 Table of Contents Abbreviations 10 List of Figures 11 List of Tables 14 Chapter 1. Background 15 1.1 Introduction 15 1.2 Fibrin Sealant 17 1.3 Cyanoacrylate Glues 21 1.4 Laser-Activated Glues 23 1.5 PEG Glues 37 1.6 Chitosan Glues 38 1.7 Genepin 48 1.8 Objectives 49 Chapter 2. Sutureless Laser-Soldering Technique for Reversal Vasectomy 50 2.1 Introduction 50 2.2 Materials and Methods 54 2.2.1 The Protein Solder 54 2.2.2 The Laser System 55 2.2.3 Chitosan Film 55 2.2.4 Thermogravimetric Analysis (TGA) 55 2.2.5 Stent Preparation 56 2.2.6 Elasticity Test 57 2.2.7 Stent Self-Expansion 58 2.2.8 Laser Tissue Soldering 59 2.2.9 Statistical Analysis 61 2.3 Results 62 2.3.1 Thermogravimetric Analysis 62 2.3.2 Stent Preparation 62 2.3.3 Elasticity Test 64 2.3.4 Stent Expansion 64 7 2.3.5 Laser Tissue Soldering 65 2.4 Discussion 67 Chapter 3. Albumin-Genipin Solder for Laser Tissue Repair 72 3.1 Introduction 72 3.2 Materials and Methods 76 3.2.1 The Laser System 76 3.2.2 Solder Preparation 76 3.2.3 Tissue Soldering 77 3.2.4 Tensile Strength 79 3.2.5 Solder Attenuation 79 3.2.6 Cytotoxic Assay 80 3.3 Results 82 3.3.1 Tensile Strength 82 3.3.2 Solder Attenuation 83 3.3.3 Cytotoxicity Assay 84 3.4 Discussion 85 Chapter 4. Chitosan Adhesive for Laser Tissue Repair 91 4.1 Introduction 91 4.2 Materials and Methods 93 4.2.1 Chitosan Adhesive Films 93 4.2.2 Adhesive Attenuation 95 4.2.3 Laser Tissue Repair (LTR) 95 4.2.4 Tensiometer Measurements 97 4.2.5 13C-NMR 98 4.2.6 Thermogravimetric Analysis (TGA) 99 4.2.7 Differential Scanning Calorimetry (DSC) 99 4.2.8 Contact Angle 100 4.2.9 Atomic Force Microscopy (AFM) 100 4.2.10 Young’s Modulus 101 4.2.11 Temperature Measurements 101 4.2.12 Ex-Vivo Histology and Scanning Electron Microscopy (SEM) 102 4.2.13 Cytotoxic Assay 103 8 4.2.14 In Vivo Thermal Damage 104 4.2.15 Statistical Analysis 106 4.3 Results 106 4.3.1 Adhesive Attenuation 106 4.3.2 Tensiometer Measurements 109 4.3.3 13C-NMR 111 4.3.4 Thermogravimetric Analysis and Contact Angle 113 4.3.5 Differential Scanning Calorimeter 114 4.3.6 Atomic Force Microscopy 115 4.3.7 Young’s Modulus 116 4.3.8 Temperature Measurements 117 4.3.9 Ex-Vivo Histology and SEM 118 4.3.10 Cytotoxicity Assay 122 4.3.11 In Vivo Thermal Damage 125 4.4 Discussion 128 Chapter 5. Conclusions 134 References 138 Publications, Patents and Presentations Arising from This Thesis 167 Appendix 168 9 Abbreviations AFM Atomic Force Microscopy BSA Bovine Serum Albumin CB Carbon black CMAP Compound muscle action potential CW Continuous wave DSC Differential Scanning Calorimetry H&E Hematoxylin and Eosin IBC Isobutyl cyanoacrylate IG Indocyanine green KS Kologorov-Smirnov LTS Laser tissue soldering LTW Laser tissue welding MPC Methoxypropyl cyanoacrylates PBS Phosphate buffer solution PEG Polyethylene glycol PLGA Poly(lactic-co-glycolic acid) SEM Scanning electron microscopy TGA Thermogravimetric Analysis 10 List of Figures Chapter 1 Figure 1. Different approaches for wound closure and sealing in existence today 16 Figure 2. Sutures, staples and clips in today clinical practice 17 Figure 3. Diagram of the two-component fibrin glue and its clot formation 18 Figure 4. Histological characterization of thermally damaged bladder 27 Figure 5. Albumin inside carotid artery 28 Figure 6. Nerve repair with solid albumin solder 30 Figure 7. Distal longitudinal neurorraphy immediately after laser-solder repair 30 Figure 8. Transverse section of solder strips bonded to the serosa layer 33 Figure 9. A solder strip is laser bonded to the intimal layer of a rat aorta 34 Figure 10. Two-layer strip after laser welding with the serosa layer 37 Chapter 2 Figure 1. Diagram of reanastomosing the vasa with sutures 50 Figure 2. Intraluminal sutures are applied to align the vasa 52 Figure 3. Schematic drawing of the stent preparation 56 Figure 4. Diagram of the surgical procedure 60 Figure 5. The weight loss of chitosan films given as a function of temperature 62 Figure 6. SEM view of a 3 mm long chitosan stent 63 Figure 7. Strain-stress diagram of a chitosan strip 65 Figure 8. A vas stenosis in the proximity of the anastomotic site 66 Figure 9. Longitudinal section of a sperm granuloma at the anastomotic site 68 11 Figure 10. Longitudinal section of the proximal site of a sutured vas deferens 69 Chapter 3 Figure 1. Molecular structure of genipin 74 Figure 2.
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