Nanofabrication, architecture control, and crosslinking of collagen scaffolds and the potential in corneal tissue engineering application

NANOFABRICATION, ARCHITECTURE CONTROL, AND CROSSLINKING OF COLLAGEN SCAFFOLDS AND THE POTENTIAL IN CORNEAL TISSUE ENGINEERING APPLICATION ZHONG SHAOPING NATIONAL UNIVERSITY OF SINGAPORE 2007 NANOFABRICATION, ARCHITECTURE CONTROL, AND CROSSLINKING OF COLLAGEN SCAFFOLDS AND THE POTENTIAL IN CORNEAL TISSUE ENGINEERING APPLICATION ZHONG SHAOPING (M.ENG. INSTITUTE OF PROCESS ENGINEERING, CHINESE ACADEMIC SCIENCES, BEIJING, PRC) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements Acknowledgements In retrospect of my past few years, I have always realized that there are many people who have directed, assisted and supported me during my Ph.D. study. Without them I would never be able to make to this point. Although it would be impossible to name each of them, I would like to express my deep gratitude to all of them. First, I would like to thank my advisor, Dr. Lin-Yue Lanry Yung, for his tremendous effort in guiding, helping and encouraging me during the years. His broad knowledge, insightful thoughts and sparkling ideas have significantly widened my horizons and inspired my research. Personally, I have also greatly benefited from his devoted, energetic and enthusiastic manner towards knowledge. I also thank the members of my oral qualifying exam committees, Dr. Yen Wah Tong, Prof Michael Raghunath, and Prof. En-Tang Kang, for their time and valuable suggestions. Special thanks are due to three them for insightful discussions on many topics, including my oral proposal and research collaboration. I wish to express my heartfelt thanks to all members in this research group, Weijie Qin, Haizheng Zhao, Weiling Tan, Deny Hartono and the staff of the Department of chemical and biomolecular engineering, especially, Fengmei Li, Xiang Li, Koh Hong Boey, and Guangjun Han. Special appreciations are also given to National University of Singapore for its financial support and all teaching programs provided. I am grateful to my family members including my parents, Mr. and Mrs. Guangyao Zhong and Zhulan Huang, siblings, Wanchun Zhong, Qiulan Zhong, Lanxiu Zhong, I Acknowledgements Wanhui Zhong for their continuous encouragement and unconditional love. Finally, I want to thank my wife Xiange Yang for her continuous encouragement, unconditional love and always patience throughout. Her love and support help me to concentrate on this research work without hesitation in the past years. I shall be indebted to all of them forever. II Table of Contents Table of Contents Acknowledgements I Table of Contents III Summary . VI List of Tables IX List of Figures X Chapter Introduction Chapter Background and literature Review . 2.1 Overview of tissue engineering 2.2 Scaffold Materials . 10 2.2.1 Natural materials 10 2.2.2 Synthetic materials . 15 2.3 Scaffold fabrication techniques 17 2.3.1 Phase separation . 18 2.3.2 Solvent casting/particulate leaching 20 2.3.3 Gas foaming . 20 2.3.4 Electrospinning 21 2.4 Tissue engineered cornea 26 2.4.1 Overview of corneal structure, diseases and replacements 26 2.4.2 Cellular selection . 30 2.4.3 Recent progress on corneal tissue engineering 31 2.5 The potential of electrospinning in corneal scaffolding . 40 Chapter Non-aqueous crosslinking of electrospun collagen nanofibers: the effects on physical properties of nanofibers and in vitro fibroblast culture 44 3.1 Introduction . 44 3.2 Experimental section . 49 3.2.1 Materials and reagents . 49 3.2.2 Electrospinning of collagen nanofibers . 49 3.2.3 Crosslinking of collagen nanofibers 50 3.2.4 Surface characterization . 51 3.2.5 Collagenase digestion test 51 3.2.6 In vitro cell culture . 51 3.2.7 Statistical analysis 53 3.3 Results and discussion 54 3.3.1 Surface morphology of collagen nanofibers before and after crosslinking . 54 3.3.2 Collagenase digestion 58 3.3.3 Cell culture in vitro 59 3.4 Conclusions . 64 Chapter Formation of collagen-GAG blend nanofibrous scaffolds and their biological properties 65 4.1 Introduction . 65 4.2 Experimental Section 69 4.2.1 Materials 69 4.2.3 Preparation of nanofibrous collagen-GAG scaffolds . 69 4.2.4 Crosslinking of collagen-GAG scaffolds . 70 4.2.5 Characterization of collagen-GAG scaffolds . 71 4.2.6 Enzymatic stability of nanofibrous collagen-GAG scaffolds 71 III Table of Contents 4.2.7 In vitro evaluation with rabbit conjunctiva fibroblasts 72 4.2.8 Statistical analysis 72 4.3 Results and discussion 73 4.3.1 Electrospinning and characterization of collagen-GAG nanofibers 73 4.3.2 Collagenase degradation 76 4.3.3 XPS analysis 78 4.3.4 Cell culture in vitro 79 4.4 Conclusions . 83 Chapter Aligned architecture of electrospun collagen scaffolds for in vitro application . 84 5.1 Introduction . 84 5.2 Materials and Methods 87 5.2.1 Materials 87 5.2.2 Preparation of aligned collagen nanofibrous scaffolds 87 5.2.3 Surface characterization . 89 5.2.4 In vitro culture . 90 5.2.5 Statistical analysis 91 5.3 Results and discussion 93 5.3.1 Morphology of aligned nanofibrous scaffold 93 5.3.2 Surface Properties 97 5.3.3 Cell adhesion and proliferation 99 5.3.4 Cell morphology and cell-scaffold interaction 102 5.4 Conclusions . 105 Chapter Electrospinning of collagen and blended collagen-GAG nanofibers using acetic acid as solvent . 106 6.1 Introduction . 106 6.2 Experimental section . 109 6.2.1 Materials 109 6.2.2 Properties of collagen solutions for electrospinning 109 6.2.3 Electrospinning and characterization of collagen nanofibers 109 6.2.4 Cell culture . 110 6.2.5 Statistical analysis 111 6.3 Results and discussion 112 6.3.1 Electrospinnability of collagen in HAc 112 6.3.2 Electrospinnability of blended collagen-GAG in HAc 119 6.3.3 In vitro test . 120 6.4 Conclusions . 125 Chapter Enhanced biological stability of collagen with incorporation of PAMAM dendrimer 126 7.1 Introduction . 126 7.2 Materials and methods 129 7.2.1 Materials 129 7.2.2 Crosslinking of collagen scaffolds . 129 7.2.3 Collagenase digestion 130 7.2.4 Differential Scanning Calorimetry . 131 7.2.5 In vitro cellular test 131 7.2.6 Statistical analysis 132 7.3 Results and discussion 133 7.3.1 Shrinkage temperature and biostability of crosslinked collagen scaffolds 133 7.3.2 Collagenase assay of crosslinked collagen scaffolds . 135 IV Table of Contents 7.3.4 SEM morphology . 136 7.3.5 In vitro cellular testing . 138 7.4 Conclusions . 145 Chapter Conclusions and recommendations 146 8.1 Conclusions . 146 8.2 Recommendations . 148 Publications List . 151 References . 153 V Summary Summary Nanobiotechnology is emerging as a new interdisciplinary field studying and applying the nano-sciences into biotechnology and has gained increasing attraction and importance during the last 10 years. This field could potentially make a major impact on human health by revolutionizing medicine, drug delivery or tissue engineering applications. In particular, nanofibers have attracted the attention of biologists and engineers due to their resemblance to native extracellular matrix (ECM) to meet the demand for fabricating ideal scaffolds for tissue engineering field. Electrospinning technique to produce high functional nanofibers has stimulated researchers to explore the application of nanofiber matrix as a tissue-engineering scaffold. My PhD project was to investigate the nanofabrication, architecture controlling and chemical modification of collagen scaffolds and to evaluate the first use as substrates for in vitro culturing human corneal cells and the subsequent potential in constructing corneal equivalents. In this project, electrospinning technique was adapted to create collagen nanofibrous scaffolds using both the reported solvent of 1,1,1,3,3,3 hexafluoro-2propanol (HFP) and one safer solvent of acetic acid (HAc), which was used as an alternative electrospinning solvents compared with HFP. One major work of this project was performed to develop the electrospinning collagen and improve the quality of collagen nanofibers. The capacity to produce collagen nanofibers more effectively and safely could lead to the generation of ECM-based fabrics with applications in the VI Summary fields of corneal or other soft tissue engineering. Considering high degradation and low mechanical strength of collagen materials, a variety of crosslinking methods, such as glutaraldehyde (GTA), dehydrothermal treatment (DHT) and UV irradiation were performed to increase the biostability and mechanical strength of collagen nanofibers, and their effects on the physical and biological properties of nanofibers were investigated by in vitro culturing of corneal fibroblasts. The electrospun collagen scaffolds exhibited similar chemical composition and physical structure present in native ECM. This study showed that aqueous crosslinking brought great damage on the structural integrity of collagen nanofibers, while the GTA vapor, DHT or UV treatment could increase the biostability of the nanofibers and preserve the porous structure. A novel nanofibrous collagen-GAG scaffold was constructed by electrospinning using a mixture of TFE and water as the dissolving solvent. The potential of applying the nano-scale collagen-GAG scaffolds in tissue engineering is significant since this nano-dimensional scaffold made of natural ECM closely mimics native ECM found in human body and may eventually support more active tissue regeneration. The incorporated GAG component was found to enhance cell growth as GAG is an important ECM component. This novel nanofibrous scaffold may facilitate cell-matrix interactions and speed up cell growth or tissue regeneration by introducing cellspecific ligands or extracellular signaling molecules, such as peptides and oligosaccharides. The aligned collagen scaffold was fabricated by one controllable electrospinning apparatus with a rotating wheel collector. This scaffold exhibited a distinct fiber VII Summary alignment compared with the random fibrous scaffold (as control) using a static plate collector. The elongated proliferation pattern of the cells growing on the aligned scaffold coincided with the cell morphology found in many native tissues, indicating that the controllable electrospinning technique to produce nanofibrous scaffolds with well-defined architecture can be very useful for engineering different specific tissues or organs. This study also suggests that the topography of the extracellular matrix (ECM) may affect cellular behavior, and controlling this environment is essential in the design of scaffolds for tissue engineering. Nanofibrous collagen scaffolds with aligned pattern architecture which assemble the structure of native cornea also have significant potentials for many other specific tissue engineering and organs regeneration applications. The research of my project may lead to the development of novel platforms for generating functional soft tissues and improved cellular scaffolds through precisely controlling tissue assembly at the nanometer level. 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Res., 1996. 30(1): p. 53-65. 174 [...]... in vitro culturing of corneal cells scaffolds Their in vivo and in vitro tissue regeneration needs to be further developed in future 4 Chapter 1 Background and progress on tissue engineering, especially corneal tissue engineering and the status use of the electrospinning will be extensively reviewed in chapter 2 5 Chapter 2 Chapter 2 Background and literature Review 2.1 Overview of tissue engineering. .. topic in the 21st century The principles of tissue engineering are as old as interventional surgery, but new tools and techniques have been emerging [24, 35, 36] Tissue engineering is an interdisciplinary field that blends classical engineering and the life sciences It has been considered as one of the most influential new technologies for the future of biomedicine with great insights into mimicking tissue. .. usability of the scaffolds by assaying cell attachment and proliferation rate 4 And a functional dendrimer was incorporated for modifying GTA and EDC crosslinking of collagen in order to increase the biostability and biological properties of collagen gel/sponge This research focuses on nanofabrication and architecture controlling of collagen scaffolds using electrospinning Nano-dimension and high surface... engineering is the choice of suitable materials for scaffolding The materials for tissue engineering could be gradually degraded and eventually absorbed in the body [46] Potential materials with these characteristics include natural, synthetic polymers, ceramics, metals and combination of these materials [48-52] Most of these materials have been used in the medical field before the advent of tissue engineering. .. delivery systems and tissue engineering may lead to a better understanding of pathological diseases [76, 77] The concepts of high binding affinity and specificity play a critical role in targeting delivery Collagen- based biomaterials are expected to become a useful matrix substance for various biomedical applications in the future Currently, the collagen used in tissue engineering applications is derived... cells and stimulates new growth in the shape dictated by the scaffold so as to replace the damaged tissue The requirements of scaffolds for tissue engineering are complex and specific to the structure and function of the targeted tissue, and thus the resultant living tissue constructs should mimic the replaced tissues functionally, structurally, and mechanically An ideal scaffold generally should have the. .. suitable interconnected architecture and a defined 3-dimensional structure for the target tissue From an engineering and 9 Chapter 2 biology standpoint, both scaffolds materials and fabrication techniques are crucial to produce the scaffolds with the above chemical and physical characteristics for specific goals in tissue engineering fields 2.2 Scaffold Materials The first issue with regard to tissue engineering. .. network to retain cells But the gels have disadvantage of low strength and high degradation Reinforcement with solid components and alignment during gelation and culture mature can improve performance [87, 88] The main function of collagen is mechanical reinforcement and physical support of the connective tissues of vertebrates [89] However, the application of collagen in tissue engineering has been... below 2.2.1.1 Collagen and their applications Collagen itself is the most abundant and ubiquitous structural protein, constituting approximately 30% of all vertebrate body protein For example, more than 90% of the extracellular protein in the tendon and bone, and more than 50% in the skin consist of collagen Collagen is the most frequently used natural polymer for various biomedical applications [53,... mechanical strength and durability The electrospinning technique has gained attention and popularity in the last 10 years due to an increased interest in nano-scale properties and technologies One major attractive feature of electrospinning is the simplicity and inexpensive nature of its setup During the electrospinning process, numerous tiny fibers with diameters on the order of several nanometers . NATIONAL UNIVERSITY OF SINGAPORE 2007 NANOFABRICATION, ARCHITECTURE CONTROL, AND CROSSLINKING OF COLLAGEN SCAFFOLDS AND THE POTENTIAL IN CORNEAL TISSUE ENGINEERING APPLICATION . NANOFABRICATION, ARCHITECTURE CONTROL, AND CROSSLINKING OF COLLAGEN SCAFFOLDS AND THE POTENTIAL IN CORNEAL TISSUE ENGINEERING APPLICATION ZHONG SHAOPING. needs to be further developed in future. Chapter 1 5 Background and progress on tissue engineering, especially corneal tissue engineering and the status use of the electrospinning will be