DEVELOPMENT OF BIORESORBABLE POLYCAPROLACTONE COMPOSITE MESH FOR ANTIMICROBIAL CONTROL RELEASE AND HAEMOSTATIC PROPERTIES TEO YILING, ERIN (B. Eng, (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 Preface Preface This thesis is submitted for the degree of Doctor of Philosophy in the Department of Mechanical Engineering at the National University of Singapore under the supervision of Professor Teoh Swee Hin, Associate Professor Shabbir Moochhala and Associate Professor Lu Jia. No part of this thesis has been submitted for other degree at other university or institution. To the author’s best knowledge, all the work presented in this thesis is original unless reference is made to other works. Parts of this thesis have been published or presented in the List of Publications shown in page ii. Page | i Acknowledgements List of Publications International Journals: 1. Erin Y. Teo, S.Y. Ong, Mark S.K. Chong, Z. Zhang, J. Lu, S. Moochhala, B. Ho, S.H Teoh. Polycaprolactone-based fused deposition modeled mesh for delivery of antibacterial agents to infected wounds. Biomaterials (2011), v32, I1, Pg 279-287. . 2. M.S.K Chong, S.H Teoh, Erin Y. Teo, Z. Zhang, C.N. Lee, S. Koh, M. Choolani, J. Chan. Beyond Cell Capture: Antibody-conjugation Improves Haemocompatibitliy for Vascular Tissue Engineering Applications. Tissue Engineering Part A (2010), v16, I8, pg 2485-2495 3. Z. Zhang, S.H Teoh, Erin Y. Teo, M.S.K. Chong, W.S. Chong, T.T. Foo,M. Choolani, J. Chan. A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering. Biomaterials (2010), v31, I33, pg 86848695. 4. JCM Teo, Erin Y. Teo, VPW Shim and SH Teoh. Determination of bone trabeculae modulus-an ultrasonic scanning and microCT imaging combination approach, Experimental Mechanics, 46 (2006):453-461 Book Chapter: 5. S.H Teoh, B Rai, K S Tiaw, S K M Chong, Z Zhang and Erin Y. Teo. Nano-tomacro Architectures Polycaprolactone-based biomaterials in Tissue Engineering”. In Biomaterials in Asia. World Scientific Publishing Co. Ltd, 2008. Page | ii Acknowledgements International Conferences: 6. Erin Y. Teo, C.K. Chui, V.P.W Shim, S.H. Teoh. Methods and Experiments of Ultrasonic Monitoring of Bone Remodeling. 2nd Materials Research Society (MRS-S) Conference on Advanced Materials. 2006. Singapore. Poster Presentation 7. Erin Y. Teo, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala, S.H. Teoh. Development of Antimicrobial Scaffold for Tissue Engineering Applications. International Conference on Materials for Advanced Technologies (ICMAT) 2007. Singapore. Oral Presentation 8. Erin Y. Teo, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala, S.H. Teoh. Development of 3D Bioactive Scaffolds for Hemostatic Applications. World Congress on Bioengineering (WACBE). Thailand, Bangkok. July 2007. Poster Presentation. 9. Erin Y. Teo, I.S.K Tan, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala, S.H. Teoh. Fabrications of Electrospun Chitosan and its blend. International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering. ( - Jan 2008, Marina Mandarin Hotel, Singapore) Poster Presentation. 10. Erin Y. Teo, L.H. Yong, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala, S.H. Teoh. Evaluation of the Effects of Tricalcium Phosphate on Platelet Adhesion onto Polycaprolactone sheets. International Conference on Advances in Bioresorbable Page | iii Acknowledgements Biomaterials for Tissue Engineering. ( - Jan 2008, Marina Mandarin Hotel, Singapore). Poster Presentation. 11. Erin. Y. Teo, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala, S.H. Teoh. Evaluation of In Vitro Antimicrobial Efficacy, Elution Profile and Cytotoxicity of Bioactive Polycaprolactone FDM-formed Scaffold. International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering. (5 - Jan 2008, Marina Mandarin Hotel, Singapore). Oral Presentation 12. Erin. Y. Teo, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala, S.H. Teoh. Evaluation of In Vitro Elution Profile, Antimicrobial Efficacy and Cytotoxicity of 3D Bioactive Polycaprolactone honeycomb scaffold. 2008 Annual Tissue Engineering and Regenerative Medicine International Society- TERMIS-EU. Tissue Engineering Part A. 2008, 14(5): 691-943. Portugal. June 2008. Poster Presentation. 13. Erin Y. Teo, S.H. Teoh, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala. Evaluation of In vivo Antimicrobial Properties of 3D Bioactive Polycaprolactone Honeycomb Scaffold. 2008 Annual Tissue Engineering and Regenerative Medicine International Society-Asia Pacific Region (TERMIS-AP). Taipei, Taiwan. 6-8 November 2008. Oral Presentation. Awarded Best Oral Presentation Award. 14. Mark Chong, Erin Y. Teo, Jerry Chan, Mahesh Choolani, Chuen Neng Lee, Swee Hin Teoh. Generation of Biphasic Constructs from Microthin Biaxially Stretched Polycaprolactone Films. 2008 Annual Tissue Engineering and Regenerative Page | iv Acknowledgements Medicine International Society-Asia Pacific Region (TERMIS-AP). Taipei, Taiwan. 6-8 November 2008. Poster Presentation. Awarded Best Travel Award. 15. S.H. Teoh, Erin Y. Teo, Jia Lu, Shabbir Moochhala. Development of Antimicrobial 3D Polycaprolactone Honeycomb Scaffold. International BoneTissue-Engineering Congress (Bone-tec 2008). Hannover, Germany. 7-9 Novermber 2008. Oral Presentation. 16. Erin Y. Teo, G.Y Tay, S.H Teoh. Drug Elution Characteristics Study of Gentamicin Sulphate Incorporated Polycaprolactone-Tricalcium Phosphate Scaffold. 2nd Asian Biomaterials Congress. Singapore. 26 June-27 June 2009. Oral Presentation. 17. Erin Y. Teo, C.Q Lai, Mark Chong, Jerry Chan, S.H. Teoh. Evaluation of Platelet Adhesion Properties on Polycaprolactone-tricalcium Phosphate film. International Conference on Materials for Advanced Technologies 2009. Singapore. 28 June-3 July 2009. Poster Presentation. 18. Erin Y. Teo, S.H. Teoh, Shin-Yeu Ong, Jia Lu, Shabbir Moochhala. Evaluation of In vivo Antimicrobial Properties of 3D Bioactive Polycaprolactone Honeycomb Scaffold. International Conference on Materials for Advanced Technologies 2009. Singapore. 28 June-3 July 2009. Oral Presentation. Page | v Acknowledgements Acknowledgements I would like to thank my supervisors Professor Teoh Swee Hin, A/Prof Shabbir Moochhala and A/Prof Lu Jia for their teaching, guidance, support and trust after these years of study. This work would not have been possible without them. I would also like to thank my family, my parents, and my beloved brother. Thanks for believing in me, supporting me in whatever I decide to do, caring and worrying for me when I had my accident in the last year of my PhD study. My greatest appreciations would also go to the staff and students of BIOMAT Lab, specifically Dr Mark Chong, Dr Zhang Zhiyong, Dr Tiaw Kay Siang, Asst Prof Chui Chee Kong, Dr Bina Rai, Dr Jeremy Teo, Dr Wen Feng, Puay Siang, all my FYP students and many, many others who have made my life as a PhD student colourful and enjoyable. Thanks for all the help which you have given to me throughout these years. Special thanks also goes to the lab officers of materials lab (Mechanical Engineering), Thomas Tan, Khalim, Aye Thien, Hong Wei, Mdm Zhong; staff of Coagulation lab (Obstetrics and gynaecology), A/Prof Stephen Koh, Bee Lian, Raymond and A/Prof Jerry Chan and Lay Geok from the Experimental Fetal Medicine group (obstetrics and gynaecology). Really appreciate all the help be it in terms of procurement, machine handling, drawing blood etc. Page | vi Acknowledgements I would also like to especially thank the staff of DSO-DMERI Combat Care lab, specifically Shin Yeu, Li Li, Mui Hong, Dr Wu Jian, Cecilia and all others who have helped with my experiments in DSO. My gratitude also goes to A/Prof Ho Bow and Mr Ng Han Chong from microbiology for teaching me so patiently and guiding me through the totally unfamiliar grounds of microbiology. I would also like to thank all staff from Osteopore International Pte Ltd and especially Mr Yew Soi Khoon for helping me with troubleshooting the mesh fabrication. And to all that were not mentioned due to space constraints, thanks for helping me in one way or another. I am deeply grateful. Last but not least, I would like to extend my heartfelt thanks to my husband, Thomas, for standing by me always, tolerating my nonsense when I am stressed out and for simply loving me. Page | vii Summary Summary Tissue traumas are often prone to complications such as haemorrhages and inflammation, which can hinder healing. The challenge is to develop a system that curbs these problems by providing haemostatic and localized controlled antimicrobial effects. The strategy developed in this study involves using a haemostatic fused deposition modeled (FDM)-formed mesh composed of Polycaprolactone (PCL) and 20wt% tricalcium phosphate (TCP), loaded with a model antimicrobial drug, gentamicin sulfate (GS; 15wt%), that will be released within a short duration to inhibit bacterial activity without affecting subsequent tissue regeneration. This platform technology can be applied to a variety of applications requiring interventions of haemostasis and drug elution. A series of in vitro and in vivo experiments were conducted to optimize and characterize this drug eluting haemostatic mesh’s drug eluting and haemostatic aspect. Haemostatic effect was preliminarily evaluated through optimizing the FDM-formed PCL-TCP mesh (85% porosity, 1mm thickness) for blood absorption. PCL was observed to promote platelet adhesion and activation, more prominently than glass positive control. Although in terms of contact activation, PCL performed poorer than glass, it was still regarded as a relevant haemostat as it showed significant haemostatic properties comparing to negative control. Upon adding TCP, both surface chemistry and topography were altered. To isolate surface topography effect, experimentations on gold sputtered specimens showed a decrease in platelet adhesion with increased surface roughness due to increasing TCP Page | viii Summary incorporation (0-25wt%). When evaluated with surface chemistry effect, it yielded a general increase in platelet coverage with increasing TCP content. In the balance of the two aspects, PCL with 20wt% TCP was selected for its optimal platelet adhesion. Architectural influence to haemostasis was also studied by comparing micro-scaled (FDM), sub-micron (nanofibers) and macro-scaled (film) architectures. It was shown that FDM-formed PCL structures had comparable blood contact activation and platelet adhesion to glass, justifying its use for this system. For the antimicrobial aspect, the incorporation of GS was optimised. 15wt% GS incorporation rendered most efficient even with bacterial reinoculations. 93% of total GS was released within 168 hours and was found to be non-cytotoxic to human dermal fibroblast. The burst release can be attributed to the hydrophilic surfaces caused by high TCP content in the FDM-formed mesh, with direct relation found between GS release rate and TCP content. Therefore, TCP content of 20wt% was once again chosen for optimum GS release during the critical infectious period while allowing subsequent tissue regeneration. 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Page | 138 [...]... inoculations of bacteria The eluted amounts of gentamicin sulfate are also hypothesized to be non-cytotoxic b To evaluate the effect of TCP incorporation in platform mesh on drug elution Hypothesis: With the addition of TCP, the rate of release of drug will be increased over a period of time due to the lowering of hydrophobicity of the platform mesh c To demonstrate that the antimicrobial incorporated mesh. .. the haemostatic properties of the mesh, individual component material and the influence of mesh architecture on the blood clotting using various assays Chapter 5 is on various antibacterial analyses done to evaluate the elution profile, antimicrobial efficacy and cytotoxicity of the various concentrations of GS incorporated The influence of TCP incorporation and surface area to volume ratio of platform... with the best blood absorption capacity for use throughout this work b To study the haemostatic effect of PCL before and after the incorporation of TCP Hypothesis: With the incorporation of TCP into the PCL mesh, haemostatic properties will be improved c To study the effect of different scale of mesh architecture (nano, micro and macro) on blood clotting Hypothesis: Meshes with micro-scaled topographies,... stages and morphology description of platelet activation for the detection of extent in activation of adhered platelets [Ko and Cooper 1993] 48 Figure 4-2 Schematic representation of TEG plot and derivation of the parameters 49 Figure 4-3 SEM micrographs of A) Glass slide after 60 min of immersion in PRP at magnification of 500x, B) PCL after 60 min of immersion in PRP at magnification of 500x... appropriate ones for our application Finally, the principles of biomaterials engineering and the status of the current drug eluting and haemostatic meshes and technologies are reviewed in this chapter 2.2 Haemostasis and blood coagulation In common cases, bleeding occur once there is a disruption of blood vessels and without the timely arrest of the bleeding, hemorrhage and large volume of blood loss... architecture The antimicrobial drugs will then be incorporated into the synthetic polymeric mesh for the localized release at wound or implant sites, with the TCP component and the general mesh FDM architecture to serve the haemostatic function Page | 3 Chapter 1: Introduction 1.2 Research aim and proposal outline The primary goal of this study was to develop and evaluate haemostatic PCL-TCP meshes for the... the delivery of antimicrobial drugs to wound or implant sites and sustain the elution concentrations above the minimum inhibitory concentration for a period of time This was tested out in both in vitro and in vivo environments To achieve this, the specific aims for this study were as follows: 1 For the haemostatic aspect of the study: a To ascertain the configuration and architecture of the mesh with... amplitude of TEG plot, that is indicative of the ultimate strength of the fibrin clot as an indication of surface consumption of clotting proteins 73 Figure 4-21 Blood clotting index measurement for comparison of different architectures 74 Page | xxii List of Figures Figure 5-1 Cumulative Elution Profile of 5wt%, 15wt% and 25wt% GS incorporated PCL-TCP mesh (in terms of percentage... List of Figures Figure 4-15 Graph of percentage area covered by platelets onto PCL-TCP specimens with different percentages of TCP 64 Figure 4-16 Representative SEM micrographs of platelets attached on (a) glass (b) nanofibers (c) FDM-formed mesh (d) film at magnification of 2500x 68 Figure 4-17 Reaction time for fibrin formation Comparing PCL with different scales of architecture and. .. improved haemostatic properties Page | 4 Chapter 1: Introduction 2 For the antimicrobial aspect of the study: a To evaluate the antimicrobial elution profile and its efficacy to eliminate bacteria over time and with repeated inoculation of bacteria while monitoring its cytotoxic effect Hypothesis: The incorporated model drug, gentamicin sulfate, will be eluted over the critical infectious period and retain . DEVELOPMENT OF BIORESORBABLE POLYCAPROLACTONE COMPOSITE MESH FOR ANTIMICROBIAL CONTROL RELEASE AND HAEMOSTATIC PROPERTIES TEO YILING, ERIN. interventions of haemostasis and drug elution. A series of in vitro and in vivo experiments were conducted to optimize and characterize this drug eluting haemostatic mesh s drug eluting and haemostatic. In Vitro Antimicrobial Efficacy, Elution Profile and Cytotoxicity of Bioactive Polycaprolactone FDM-formed Scaffold. International Conference on Advances in Bioresorbable Biomaterials for Tissue