Biomimetic nanofiber stem cell composite for skin graft application

BIOMIMETIC NANOFIBER/STEM CELL COMPOSITE FOR SKIN GRAFT APPLICATION MA KUN (Bachelor of Medicine, Shanxi Medical University, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences and Engineering NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgement First of all, I would like to give my sincere thanks to Prof. Seeram Ramakrishna, for his tremendous encouragement and excellent supervision during my PhD study. His foresight of frontier science, his wisdom and enthusiasm, his positive attitude and hard-working spirit always inspired and encouraged me throughout my four-year research life. I am also deeply indebted to Prof. Casey Chan, my co-supervisor, who has given me invaluable advice and constant encouragement, which kept me confident in times of doubt and led me to success from places of difficulties. His passion, intelligence and persistence in scientific research greatly encourage me to explore more in the research field. My special appreciation is to Dr. Thomas Yong and Dr. Susan Liao, who have provided many valuable suggestions and patient guidance during my PhD study. Dr. Susan helped me not only in the academic research, but also in many other aspects of my daily life whenever I needed. My thanks are extended to all other members in the Healthcare and Energy Materials Laboratory: to Ms. Saw Seow Hoon and Mr. Teo Wee Eong who introduced me the basic methods and techniques in cell culture and electrospinning; to Mr. Dong Yixiang, Ms. Koh Huishan, Ms. Michelle, Ms. Fengyu and Dr. Gopal for their invaluable advices, discussions and suggestions; to all the lab officers: Ms. Karen Wang, Ms. Cheng Ziyuan, Mr. Teo Wee Eong, Mr. Yang Fengyi, Mr. Ramakrishna Ramaseshan, Ms. Satinderpal Kaur, Mr. Lenni Teng and Ms. Charlene Wang for their good maintenance and great contribution to the laboratory and their timely help throughout the course of my PhD study. And especially, I would like to express my gratitude to Mr. Filip and Dr. He Liumin who have input brilliant insights and provided great assistance in my stem cell experiments and animal study. And many thanks to Dr. Hewei, who has helped me to grow not only in the academic research, but also in other dimensions of my life. Besides, I would like to extend my thanks to all my other friends in the lab: Li Bojun, Ma Zuwei, Liu Yingjun, Rama, Ahbi, Susi, Anitha, Karen Teo and Rajes et al. for their precious friendship and help during my PhD studies. I am grateful to NUS Graduate School for Integrative Sciences and Engineering for providing the funding for my studies at National University of Singapore. I also would like to thank Prof. Teoh Swee Hin, Prof. Hanry Yu and Prof. Michael Raghunath, previous chairmen in Graduate Program in Bioengineering (GPBE) for their great effort in making GPBE an enriched and warm ‘family’. My special appreciation is to Mr. Steffen Ng, Ms. Pang Soo Hoon, Ms. Chuan Irene and Mr. Marcus Chan, for their great assistance in all the administrative work. Last, I would like to give the highest thanks to my parents, sister and parents-in-law for their constant love, care and support during my study. Special thanks to my dear husband, Tommy, who has accompanied and given me great support and encouragement throughout my studies. Publications Journal Paper 1. Ma K, Chan KC, Liao S, Hwang WYK, Feng Q, Ramakrishna S. Electrospun nanofiber scaffolds for rapid and rich capture of bone marrow-derived hematopoietic stem cells. Biomaterials. 2008, 29: 2096-2103. 2. Ma K, Laco F, Ramakrishna S, Weber HJ, Liao S, Chan KC. Differentiation of bone marrow-derived mesenchymal stem cells into multi-layered epidermis-like cells in 3D organotypic coculture. Biomaterials. 2009, 30: 3251-3258. 3. Filip L, Ma K, Hans JW, Ramakrishna S, Chan KC. The dose effect of human bone marrow-derived mesenchymal stem cells during epidermal development in organotypic co-culture. Journal of Dermatological Science. 2009, 55(3):150160. 4. Ma K, Chan KC, Liao S, Ramakrishna S. Biomimetic nanofiber/stem cell composite for skin graft application in a rat model. Science Translational Medicine. 2009. (submitted) 5. He LM, Liao S, Quan D, Ma K, Chan KC, Ramakrishna S. Topographical effects of electrospun PLLA fibers on viability, proliferation and differentiation of neonatal mouse cerebellum C17.2 stem cells. Biomaterials. 2009. (submitted) Book Chapter 1. Ma K, Chan KC, Ramakrishna S. Textile-based scaffolds for tissue engineering. In: Rajendran S, editor. Advanced textiles for wound care. Woodhead Publishing Limited. UK. 2009, 289-321. Patent 1. Chan KC, Larrick JW, Raghunath M, Ramakrishna S, Liao S, Ma K. Method and construction of a cell composite for tissue repair. US Provisional Patent Application Number: 61/133,298. 27 June 2008. Conferences 1. Ma K, Yong T, Chan KC, Ramakrishna S. Electrospun nanofiber fabrication as synthetic extracellular mesh for E-ROSH cells and its potential for vascular tissue engineering. Proceedings of 3rd Graduate Students’ Symposium, 22 September 2006, Singapore (Postal presentation). 2. Ma K, Yong T, Chan KC, Ramakrishna S. Collagen-blended biodegradable polymer nanofibers: potential substrates for wound healing in skin tissue engineering. Proceedings of the Fifth IASTED International Conference on Biomedical Engineering, 14-16 February 2007, Austria (Oral presentation). 3. Ma K, Liao S, Chan KC, Ramakrishna S. Biomimetic nanofiber / stem Cell composite for skin graft application. Proceedings of Graduate Student Symposium in Biological and Chemical Engineering, 14 September 2007, Singapore (Poster Presentation). 4. Ma K, Hwang WYK, Feng Q, Chan KC, Ramakrishna S. Modification and characterization of blended nanofiber substrates as skin grafts for the capture of bone marrow-derived hematopoietic stem cells. Proceedings of Materials and Processes for Medical Devices TM (MPMDTM) Conference and Exposition, 23-25 September 2007, California, USA (Oral presentation). 5. Ma K, Chan KC, Ramakrishna S. Biomimetic nanofiber scaffolds for efficient adhesion of mesenchymal stem cells in skin graft application. The TERMIS-NA 2008 Annual Conference & Exposition, 7-10 December 2008, California, USA (Poster Presentation) Awarded TERMIS Student Travel Award. 6. Ma K, Laco F, Ramakrishna S, Liao S, Chan KC. In vitro differentiation of bone marrow-derived mesenchymal stem cells into 3D epidermis-like cells in organotypic coculture. International Conference on Materials for Advanced Technologies 2009, 28 June-3 July 2009, Singapore (Poster Presentation). 7. Laco F, Ma K, Weber HJ, Ramakrishna S, Liao S, Chan KC. Differentiation of human bone marrow-derived mesenchymal stem cells into epidermis-like Cells and their role during epidermal regeneration in 3D organotypic coculture. Proceedings of Tissue & Cell Engineering Society (TCES), 8-10 July 2009, Glascow, UK (Poster Presentation). Table of Contents Acknowledgements Publications Table of Contents Summary List of Tables I VI VIII List of Figures List of Appendices List of Abbreviations X XV XVI Chapter Introduction 1.1 Background 1.2 Motivation 1.3 Hypothesis and objectives 1.4 Research rationale and strategy 1.5 Work scope 11 Chapter Literature Review 2.1 Introduction 14 2.1.1 Skin composition and functions 14 2.1.2 Wound healing in vivo 18 2.1.2.1 Four basic responses to skin injury 18 2.1.2.2 Acute wound healing in vivo 20 2.1.2.3 Regeneration of the epidermis in vivo 30 I 2.2 Full-thickness skin wounds 39 2.2.1 Current treatments for full-thickness skin wounds 41 2.2.1.1 Autografts, allografts and xenografts 41 2.2.1.2 Tissue-engineered skin grafts 42 2.2.1.2.1 Reactants in tissue-engineered skin grafts 43 2.2.1.2.2 Commercialized tissue-engineered skin grafts 48 2.2.1.2.3 Drawbacks of current commercialized skin grafts 51 2.2.2 Potential of human bone marrow-derived mesenchymal stem cells (BMMSCs) for wound healing and skin regeneration 54 2.2.2.1 Characteristics of human BM-MSCs 2.2.2.1.1 Identification of human BM-MSCs 55 55 2.2.2.1.2 Immune escape and suppression by human BM-MSCs 60 2.2.2.2 Contributions of human BM-MSCs in wound healing and skin regeneration 63 2.2.3 Potential of electrospun nanofiber scaffolds (NFS) as tissue-engineered skin grafts 66 2.2.3.1 NFS as tissue-engineered scaffolds 66 2.2.3.2 Fabrication of NFS by electrospinning 68 2.2.3.3 Electrospun NFS as tissue-engineered skin grafts 74 2.2.4 Summary 79 Chapter In vitro Epidermal Differentiation of Human BM-MSCs 3.1 Introduction 80 3.2 Materials and methods 82 II 3.2.1 Reagents 82 3.2.2 Cell culture 83 3.2.3 Viability and proliferation of human BM-MSCs 84 3.2.4 Immunostaining analysis of human BM-MSCs 85 3.2.5 Organotypic coculture 85 3.2.6 Histological and immunological analyses of organotypic coculture 87 3.2.7 Statistical analysis 88 3.3 Results 88 3.3.1 Viability and proliferation of human BM-MSCs cultured in different media 89 3.3.2 Immunostaining analysis of human BM-MSCs cultured in different Media 90 3.3.3 Histological analysis of organotypic coculture 93 3.3.4 Immunostaining analysis of organotypic coculture 95 3.4 Discussion 97 3.5 Conclusion 102 Chapter 4: Fabrication and Modification of Electrospun NFS 4.1 Introduction 104 4.2 Materials and methods 106 4.2.1 Reagents 106 4.2.2 Fabrication of Collagen/PLGA blended NFS 106 4.2.3 Characterization of Collagen/PLGA blended NFS 107 4.2.4 Modification of Collagen/PLGA blended NFS 111 III 4.2.5 Characterization of CD29 Ab-conjugated Collagen/PLGA blended NFS 112 4.2.6 Adhesion efficiency of human BM-MSCs on different substrates 113 4.2.7 Morphology of human BM-MSCs on different substrate 113 4.2.8 Statistical analysis 114 4.3 Results 114 4.3.1 Morphology of Collagen/PLGA blended NFS 114 4.3.2 Porosity and pore size of Collagen/PLGA blended NFS 116 4.3.3 Chemical composition of Collagen/PLGA blended NFS 117 4.3.4 Mechanical properties of Collagen/PLGA blended NFS 117 4.3.5 Hydrophilicity of Collagen/PLGA blended NFS 119 4.3.6 In vitro degradation of Collagen/PLGA blended NFS 120 4.3.7 Characterization of CD29 Ab-conjugated Collagen/PLGA blended NFS 123 4.3.8 Adhesion efficiency of human BM-MSCs on different substrates 123 4.3.9 Morphology of human BM-MSCs on different substrates 126 4.4 Discussion 127 4.5 Conclusion 133 Chapter 5: In vivo Study of Nanofiber/Stem cell Composites as Skin Grafts for Full-thickness Skin Wounds in a Rat Model 5.1 Introduction 135 5.2 Materials and methods 136 5.2.1 Reagents 136 5.2.2 Animals 137 IV 5.2.3 Skin graft preparation 137 5.2.4 Surgical procedures 138 5.2.5 Wound size measurements 140 5.2.6 Histological and immunological analyses 140 5.2.7 Quantification of collagen synthesis 142 5.2.8 Statistical analysis 142 5.3 Results 143 5.3.1 Animals 143 5.3.2 Wound size measurements 143 5.3.3 Histological analyses 147 5.3.4 Immunological analyses 153 5.3.5 Quantification of collagen synthesis 168 5.4 Discussion 170 5.5 Conclusion 184 Chapter Conclusion and Recommendations 6.1 Main conclusions 186 6.2 Recommendations for future work 188 References 193 Appendices 219 V Interactions between human mesenchymal stem cells and natural killer cells. 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Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts. Proc Natl Acad Sci U S A 1998; 95:3908-3913. 218 Appendix A BM-MSCs Labeling by Qtracker655 Cell Labeling Kit The nanocrystals in Invitrogen Q-Tracker 655 cell labeling kit are nanometer-scale atom clusters composed of a cadmium-selenium core and an inner zinc sulphide shell (CdSe/ZnS) and an outer polyethylenglycol shell. A custom peptide bonded to the outer shell allows the quantum dots to be endocytosed by cells. This kit is effective for labeling a variety of cell types and exhibits no significant leaking or transfer from cell to cell. It provides long-term labeling of live cells with no apparent toxic effects. Other advantages include photostability and resistance to chemical and metabolically degeneration. BM-MSCs labeling protocol: 1. Take 10µl component A and 10µl component B and mix them, incubate for at room temperature. 2. Add 2ml of fresh culture medium, shake or votex it for 30 seconds. 3. Add 2ml of the above solution to one T-75 flask with BM-MSCs at 90% confluence. 4. Incubate for h at 37ºC incubator with 5% CO2. 5. Wash the cells with medium or PBS (depending on continuous culture or subculture or transplantation) 219 Appendix B Hematoxylin & Eosin (H&E) Staining Method This section is for samples that had undergone Paraffin Wax Embedding: 1. Dewax sections in Xylene X OR until the wax are gone. Blot excess xylene before going into ethanol. Note: DO NOT touch the timer if there’s Xylene on your gloves. 2. Clear sections in Ethanol 100% X 95% X 70% X 3min 3. Wash in deionized water for Blot excess water before going into hematoxylin. 4. Fix in 100% Ethanol X (For Cryo-sections only) 5. Stain in Mayer’s Hematoxylin for 10 OR Harris’ Hematoxylin for 6. Rinse in water. 7. Optional: X 5min in tap water. 8. Optional: Dip in 1% acid alcohol for second, rinse in water till water is clear. 9. Dip in Alkaline solution for a few seconds. 10. Rinse in water. Blot excess water before going into eosin. 11. Counter-stain in Eosin for 2-3 seconds ONLY. 12. Rinse in water. 13. Optional: View under microscope to check staining. If unsatisfactory, destain in 1% acid alcohol. Rinse in water. 14. Dehydrate in Ethanol 70% X 95% X 100% X 220 15. Clear in Xylene X 16. Mount sections in DePex mounting medium. 17. Allow drying before viewing. Results: Nuclei--------blue to blue black (stained by Hematoxylin) Cytoplasm---pink (stained by Eosin) 221 Appendix C Sample Preparation for Scanning Electron Microscopy Observation Scanning electron microscopy (SEM) is used to image nanofiber scaffolds with/without cells. 1. Wash the samples times with PBS (5 each wash). 2. Immerse samples in 2.5% glutaraldehyde. Incubate for h. 3. Wash times with DI water (5 each wash). 4. Immerse samples in 50% ethanol and incubate for 15 min. 5. Repeat step with 75% ethanol. 6. Repeat step twice with 95% ethanol. 7. Repeat step twice with 100% ethanol, incubating for 30 each time. 8. Aspirate the solution from the culture wells and allow the samples to dry overnight. 9. Gold-coat samples prior to SEM oberservation. 222 [...]... microenvironment Therefore, this study suggests a great potential of nanofiber/ stem cell composites as efficient skin grafts for the treatment of acute full-thickness skin wounds However, such parameters as the minimum therapeutic dosage of BM-MSCs, functions of differentiated BM-MSCs and healing properties on a large tight-skinned animal model must all be ascertained before clinical application is attempted... research to produce bioengineered skin grafts is being pursued with the aim to replace the use of autologous skin grafts In this project, it is hypothesized that tissue-engineered skin grafts composed of biodegradable nanofiber scaffolds (NFS) enriched with bone marrow-derived mesenchymal stem cells (BM-MSCs) can promote wound healing and skin regeneration in acute full-thickness skin wounds The differentiation... antibody grafting and BM-MSCs attachment on modified NFS were characterized Demonstrate the efficiency and efficacy of NFS/BM-MSCs composites as skin grafts to promote skin regeneration and wound healing in a rat model with acute fullthickness skin wounds Animal studies on rat Chapter 5 has been done to show the capacity of NFS/BM-MSCs composites as skin grafts to improve wound healing and skin regeneration... amounts of skin are injured, tissue harvest will be circumscribed by the available surface area of unaffected skin and creation of an additional injury [7] Therefore, allogeneic (from a non-genetically identical individual of the same species) 2 Chapter 1 or xenogeneic (from a different species) skin grafts were developed For example, allografts of cadaver skin are commonly used as a temporary cover for full-thickness... deposition (Apligraf®) etc However, none of the current commercialized skin grafts can successfully fulfill the criteria for developing skin substitutes There are three factors that are paramount in the development of tissue-engineered skin grafts: the safety of patient, clinical efficacy and convenience of use [6] Acelluar skin grafts like PermacolTM results in insufficient revascularization They can... are used [20] Therefore, the incorporation of BM-MSCs in skin grafts holds a great potential in promoting wound healing and skin regeneration in full-thickness skin defect Recent advances in tissue engineering techniques have sparked interests in making scaffolds with biocompatible and/or biodegradable polymer nanofibers The rationale for using nanofibers is based on the theory that cells attach and... architectures that promote skin regeneration In this project, we utilized electrospinning technique to fabricate nanofibrous scaffolds that were further modified for fast and rich capture of BM-MSCs and studied the effectiveness of the resultant NFS /stem cell composites as skin grafts to promote wound healing and skin regeneration in a rat model with acute full-thickness skin wounds 1.3 Hypothesis... by grafting CD29 antibody on its surface for rich and fast capture of BM-MSCs within 30 minutes The resultant NFS with attached BM-MSCs were used as skin grafts to directly apply onto the full-thickness skin wounds to promote wound healing and skin regeneration in a rat model 9 Chapter 1 Fig 1.1 The schematic illustration of research strategy The rationale of using NFS/BM-MSCs composites as skin grafts... epidermal and dermal layers to their full depth in full-thickness skin wounds, the epidermal stem cells in hair follicles for epidermal renewal, the basement membrane for cell migration and the blood vessels in dermis for nutrition supply are all destroyed, which create great difficulties in effective clinical treatment for improving skin regeneration Without an adequate blood supply, the repair is... than the diameter of the cells [21] More importantly, the non-woven polymeric architecture of nanofiber scaffolds (NFS) mimics the nanoscale protein fiber meshwork in native ECM The large surface area-to-volume ratio of NFS favors cell adhesion, proliferation, migration and differentiation [22] For skin graft application, the high porosity of NFS provides more structural space for accommodation of migrating . BIOMIMETIC NANOFIBER/STEM CELL COMPOSITE FOR SKIN GRAFT APPLICATION MA KUN ( Bachelor of Medicine, Shanxi Medical University, China) A THESIS SUBMITTED FOR THE. Full-thickness skin wounds 39 2.2.1 Current treatments for full-thickness skin wounds 41 2.2.1.1 Autografts, allografts and xenografts 41 2.2.1.2 Tissue-engineered skin grafts 42 2.2.1.2.1. September 2007, California, USA (Oral presentation). 5. Ma K, Chan KC, Ramakrishna S. Biomimetic nanofiber scaffolds for efficient adhesion of mesenchymal stem cells in skin graft application. The