Electrospinning of biomimetic and bioactive composite nanofibers

ELECTROSPINNING OF BIOMIMETIC AND BIOACTIVE COMPOSITE NANOFIBERS ZHANG YANZHONG (M.Eng., National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSTIY OF SINGAPORE 2006 Acknowledgements Acknowledgements Most of all, I would like to express my deepest gratitude to my project supervisors, Assoc. Prof. C.T. Lim and Prof. S. Ramakrishna, who had led me into this exciting multidisciplinary research arena of nanobioengineering, and given me great support and continual patience during my years of academic pursuit in the NUS, Singapore. My deepest gratitude is also extended to Prof. Z.M. Huang for his constant encouragement and help in this PhD study. This project could not complete without having help from the friends and colleagues around me. My special thanks go to: Dr. H.W. Ouyang from the Department of Orthopedic Surgery and Dr. J.R. Venugopal in the Nanobioengineering Lab for their expertise in conducting the cell culture work; Dr. J. Li and Dr. X. Wang from the Institute of Materials Research and Engineering (IMRE) for kindly allowing me to use their lab and instruments in my release experiments; Drs. Z.W. Ma, M. Kotaki for their help in XPS analysis, Drs. X.J. Xu, F. Yang, J.X. Zhang, and Mdm X.L. Zhong for their help in electron microscopies; Ms. E. Tan, Mr. G. Lee for phase-imaging with AFM; Dr. T. Song from the Data Storage Institute (DSI) and Ms. M. Wang from the Department of Chemistry for providing magnetic nanoparticles and helped out in some polymer characterization. I would like to thank all the members in the Nanobioengineering Lab, NanoBiomechanics Lab; the staff from the NUSNNI, Division of Bioengineering, and Material Science Division for having given me assistance in different ways. I also own my thanks to my thesis committee members: Assoc/Prof. S.L. Toh, Assoc/Prof. C.H.J. Goh, Asst/Prof. Y. Zhang, and Prof. S.H. Teoh for their kind advices and patience. Last but not least, I would like to thank my family for their constant support and understanding during the past tough years of doctoral study in Singapore. i List of Publications List of Publications Journal papers: 1. Z.M. Huang, Y.Z. Zhang, M. Kotaki and S. Ramakrishna, A review on polymer nanofibers by electrospinning and their applications in nanocomposites, Composites Science and Technology, 2003, 63(15): 2223-2253. 2. Z.M. Huang, Y.Z. Zhang, C.T. Lim and S. Ramakrishna, Electrospinning and mechanical characterization of gelatin nanofibers, Polymer, 2004, 45(15): 5361-5368. 3. Y.Z. Zhang, Z.M. Huang, X.J. Xu, C.T. Lim, S. Ramakrishna, Preparation of core-shell structured PCL-r-Gelatin bi-component nanofibers by coaxial electrospinning, Chemistry of Materials, 2004, 16(18): 3406-3409. 4. Y.Z. Zhang, H.W. Ouyang, C.T. Lim and S. Ramakrishna, Z.M. Huang, Electrospinning of gelatin fibers and Gelatin/PCL composite fibrous scaffolds, Journal of Biomedical Materials Research, 2005, 72B(1): 156-165. 5. Y.Z. Zhang, J. Venugopal, Z.M. Huang, C.T. Lim, S. Ramakrishna, Characterization of the surface biocompatibility of the electrospun PCL-Collagen nanofibers using fibroblasts, Biomacromolecules, 2005, 6(5): 2583-2589. 6. Y.Z. Zhang, C.T. Lim, S. Ramakrishna, and Z.M. Huang, Recent development of polymer nanofibers for biomedical and biotechnological applications, Journal of Materials Science: Materials in Medicine, 2005, 16(10): 933-946. 7. T. Song, Y.Z. Zhang, T.J. Zhu, C.T. Lim, S. Ramakrishna, B. Liu, Encapsulation of self-assembled FePt magnetic nanoparticles in PCL nanofibers by coaxial electrospinning, Chemical Physics Letters, 2005, 415(4-6): 317-322. 8. Z.M. Huang, Y.Z. Zhang, S. Ramakrishna, Double-layered composite nanofibers with better mechanical performance, Journal of Polymer Science Part B: Polymer Physics, 2005, 43(20): 2852-2861. 9. Y.Z. Zhang, Y. Feng, Z.M. Huang, S. Ramakrishna, C.T. Lim, Fabrication of porous electrospun nanofibers, Nanotechnology, 2006, 17(3): 901-908. 10. Y.Z. Zhang, J. Venugopal, Z.M. Huang, C.T. Lim, S. Ramakrishna, Crosslinking of the electrospun gelatin nanofibers, Polymer, 2006, 47(8):2911-2917. 11. Y.Z. Zhang, X. Wang, Y. Feng, J. Li, C.T. Lim, S. Ramakrishna, Coaxial electrospinning of fitcBSA encapsulated PCL nanofibers for sustained release, Biomacromolecules, 2006, 7(4): 1049-1057. ii List of Publications Conference paper: 1. Y.Z. Zhang, X. Wang, J. Li, C.T. Lim, S. Ramakrishna, Proteinencapsulated PCL composite nanofibers via coaxial electrospinning for controlled releases in tissue engineering applications, 3rd International Conference on Materials for Advanced Technologies (ICMAT), 3-8 July, 2005, Singapore. Patent: 1. S.S Chia, C.T. Lim, Y.Z. Zhang, S. Ramakrishna, Highly productive electrospinning apparatus with multiple spinneret system, US provisional application no. 60/475921, filed on 05 June 2003. Book Chapter: 1. S. Ramakrishna, K. Jayaraman, Z.M. Huang, Y.Z. Zhang and X.M. Mo, "Polymeric Nanofibers and Structures: A Review of Processing Methods, Characterization Techniques, Modeling and Applications", Advances in Nanoscience and Nanotechnology, National Institute of Science Communication & Information Resources, Editors Ashutosh. Sharma, J. Bellare and Archana Sharma (July 2004):pp113-140. 2. Tan EPS, Zhang YZ, Ramakrishna S and Lim CT, Polymer nanofibers: Fabrication, applications and characterization, Specialty Polymers, IK International, New Delhi, India, 2006. (in press) iii Table of Contents Table of Contents Acknowledgements i List of publications ii Table of contents iv List of abbreviations ix List of figures x List of tables xv Summary xvi Chapter Introduction 1.1 Background 1.2 Objectives 1.3 Scope of the thesis Chapter 2.1 Literature review Electrospinning 2.1.1 Principle and mechanism 2.1.2 Controlling the electrospinning process 2.1.2.1 Processing variables 2.1.2.2 Arrayed nanofiber assembly 10 2.1.3 Applications of nanofibers 2.2 Scaffolds technology 2.2.1 Extracellular matrix 13 13 15 2.2.1.1 Constituents of the ECM 15 2.2.1.2 The structure of fibrous collagen 17 2.2.2 Scaffold materials 20 iv Table of Contents 2.2.2.1 Synthetic aliphatic polyesters 21 2.2.2.2 Natural biopolymer of collagen-derived gelatin 24 2.2.3 Scaffolds fabrication 26 2.2.4 General standards in scaffold design 28 2.3 The prior art of materials hybridization for scaffolding applications 29 2.4 Electrospun nanofibrous scaffolds for tissue engineering 34 2.5 Summary 37 Chapter Electrospinning of gelatin nanofibers 39 3.1 Introduction 39 3.2 Experimental details 40 3.2.1 Electrospinning of gelatin nanofibers 40 3.2.1.1 Materials 41 3.2.1.2 Electrospinning 42 3.2.1.3 Fiber morphology 42 3.2.2 Crosslinking of the electrospun gelatin nanofibers 44 3.2.2.1 Sample preparation 44 3.2.2.2 GTA vapor crosslinking 44 3.2.2.3 Solubility test 44 3.2.2.4 Physical characterization 45 3.2.3 Evaluation of cytotoxicity of the crosslinked gelatin nanofibers 46 3.3 Results & discussion 3.3.1 Electrospinnability of gelatin 47 47 3.3.1.1 Solvent effect 47 3.3.1.2 Concentration effect 48 3.3.2 Crosslinking 49 3.3.2.1 Fiber morphologies before and after crosslinking 49 3.3.2.2 Thermal and mechanical properties 52 3.3.3 Cytotoxicity 56 v Table of Contents 3.4 Conclusions Chapter Randomly blended composite nanofibers 57 59 4.1 Introduction 59 4.2 Electrospinning of Gt/PCL composite nanofibrous scaffolds 60 4.2.1 Objectives 60 4.2.2 Experimental details 60 4.2.2.1 Materials 60 4.2.2.2 Electrospinning 61 4.2.2.3 Characterization 61 4.2.2.4 Cell culture 62 4.2.3 Results & discussion 64 4.2.3.1 Characterization of the Gt/PCL composite fibers 64 4.2.3.2 Bioactivity of Gt/PCL composite fibers 68 4.2.4 Conclusions 4.3 Formation of porous fibers 75 4.3.1 Introduction 75 4.3.2 Experimental details 77 4.3.2.1 Sample preparation 77 4.3.2.2 Phase separation characteristics of Gt/PCL fibers 77 4.3.2.3 Release of (leaching) gelatin 79 4.3.2.4 BET surface area measurement 79 4.3.3 Results and discussion 80 4.3.3.1 Phase separation 80 4.3.3.2 Formation of 3-D porous fibers 83 4.3.4 Conclusions Chapter 5.1 74 Core-sheath structured composite nanofibers Introduction 90 91 91 vi Table of Contents 5.2 Development of the coaxial electrospinning process 93 5.2.1 Objective 93 5.2.2 Core-shell nanofibers from two electrospinnable solutions 94 5.2.2.1 Materials 94 5.2.2.2 A simple setup for coaxial electrospinning 94 5.2.2.3 Characterization of the core-shell structure 96 5.2.2.4 Concentration effect 99 5.2.3 Core-shell nanofibers with non-electrospinnable inner solution 102 5.2.3.1 Materials 102 5.2.3.2 Design and fabrication of coaxial electrospinneret 102 5.2.3.3 Encapsulation of FePt magnetic nanoparticles in PCL nanofibers 5.2.3.4 Characterization of microstruture 104 5.2.3.5 Flow rate effect 107 5.2.3.6 Patterned magnetic nanofibers 108 5.2.4 Conclusions 5.3 104 Core-shell composite nanofibers as cellular scaffolds 109 110 5.3.1 Objective 110 5.3.2 Experimental details 110 5.3.2.1 Materials 110 5.3.2.2 Sample preparation 111 5.3.2.3 Electron microscopy 112 5.3.2.4 In vitro cell culture 112 5.3.2.5 Statistical analysis 113 5.3.3 Results 113 5.3.3.1 Morphology of the nanofibers 113 5.3.3.2 Cell proliferation 115 5.3.3.3 Cell morphology 116 5.3.4 Discussion 119 vii Table of Contents 5.3.4.1 Core-shell structured composite nanofibers 119 5.3.4.2 Cell-scaffold interaction 120 5.3.5 Conclusions 5.4 Core-sheath composite nanofibers for sustained release 122 122 5.4.1 Introduction 122 5.4.2 Experimental details 124 5.4.2.1 Materials 124 5.4.2.2 Coaxial electrospinning 125 5.4.2.3 Characterization 125 5.4.2.4 In vitro release 126 5.4.3 Results & discussion 127 5.4.3.1 Fiber morphology 127 5.4.3.2 Characterization of the encapsulation 131 5.4.3.3 In vitro release 135 5.4.4 Conclusions Chapter Conclusions & recommendations 142 144 6.1 Conclusions 144 6.2 Recommendations 148 Bibliography 150 viii List of Abbreviations List of Abbreviations 3-D AFM AGM BET BMSC BSA CFDA DMEM DSC ECM EHD FBS FESEM FITC Gt GTA HAp or HA: HDF HFIP LCSM MTS assay PBS PCL PCLEEP: PEG PEUU: PGA PHBV: PLA PLLA PLGA PVA PVP SEM TCPS TEM TFE TGA XPS three-dimensional atomic force microscopy alternating gradient magnetometer Brunauer-Emmett-Teller bone marrow stromal cell bovine serum albumin carboxy fluorescein diacetate dulbecco’s modified eagle’s medium differential scanning calorimetry extracellular matrix electro-hydrodynamic fetal bovine saline field emission scanning electron microscopy fluorescein isothiocyanate gelatin glutaraldehyde hydroxyapatite human dermal fibroblast hexaluoroisopropanol laser confocal scanning microscopy CellTiter96TM Aqueous assay phosphate buffered saline poly(ε-caprolacton) poly(ε-caprolactone-co-ethyl ethylene phosphate) poly(ethylene glycol) poly(ester urethane)urea poly(glycotic acid) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) poly(lactide acid) poly(L-lactic acid) poly(D,L-lactic acid-co-glycolic acid) poly(vinyl alcohol) poly(vinylpyrrolidone) scanning electron microscopy tissue culture polystyrene transmission electron microscopy trifluoroethanol thermo gravimetric analysis x-ray photoelectron spectroscopy ix Chapter LCSM and XPS. It was found that varying inner flow rate of fitcBSA/PEG/TFE solution from 0.2 mL/h, 0.4 mL/h to 0.6 mL/h led to increase in overall fiber diameters of PCL-r-fitcBSA/PEG nanofibers from about 270 nm, 330 nm to 380 nm, respectively. In vitro release study indicated the core-shell structured nanofibers are capable of releasing the BSA protein continuously over a period of more than months. Compared to the nanofibers of fitcBSA/PEG/PCL blend, core-shell structured PCL-r-fitcBSA/PEG nanofibers could obviously suppress the burst release. The suppression efficiency was loading dependent. SEM examinations of post-release samples suggested that the release kinetics is diffusion controlled but are obviously related to the dispersion status of the proteins in the nanofibers. Present results provide basis for further design and optimization of processing conditions to control the nanostructure of core-sheath composite nanofibers in order to achieve highly sustainable and controllable protein-release kinetics in practical tissue engineering applications. In this regard, coaxial electrospinning should be a viable technique of coupling the structural integrity and functionalization in one nanofiber to ultimately fulfill the success of nanofibers as tissue engineering scaffolds. Overall, two different biomimetic composite nanofibers in the form of blending and core-sheath structure had been successfully fabricated via the electrospinning technology. Physical, mechanical, chemical and biological characterizations performed suggested the applicability of these biomimetic composite nanofibers as scaffolding elements for tissue engineering applications. Moreover, the electrospinning both natural with synthetic polymers together provides a feasible and effective approach to make biomimicking and bioactive nanofibrous scaffolds. The 147 Chapter concept of combining synthetic materials with cell-recognition sites of naturally derived biomaterials at nanoscale is very attractive in the construction of biomimetic scaffolds. 6.2 Recommendations In Chapter 3, electrospun gelatin nanofibers had been successfully crosslinked with a GTA vapor method. In the future, it will be desirable to use other crosslinking techniques to replace current GTA method because: 1) GTA vapor crosslinking in a moist environment would result in bonding between fiber junctions. Although this has led to water-insoluble and improved mechanical and thermal properties, it would potentially limit cellular ingrowth with the bonded fiber structure; and 2) as demonstrated with a cellular proliferation study, GTA would possess potential toxic effect to the cells. In Chapter 4, electrospinnability and cellular responses had been evaluated based on the composite nanofibers Gt/PCL at a blending ratio of 1:1. For optimization purpose in terms of pore morphology, porosity, relevant mechanical properties and cellular activities, different formulation ratios between gelatin and PCL can be further investigated. This is also the case for the formation of 3-D porous fibers. Furthermore, from our preliminary results from an animal wound healing test, Gt/PCL matrix seeded with animal cells in a larger biomimetic nanofibrous matrices can be prepared for further in vivo animal experiments. 148 Chapter In Chapter 5, coaxial electrospinning is able to create interesting compound nanofiber structures, but the complex electrohydrodynamics involved in the coaxial electrospinning are yet to be investigated. 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Biomacromolecules 2004;5:326-333. 162 [...]... segment of the nanofibers with skewed inner component 97 Figure 5.5 XPS spectra of the PCL-r-Gelatin composite nanofibers (a), and 99 xii List of Figures pure Gelatin nanofibers (b) Figure 5.6 Effects of inner dope concentrations on the diameter of core component and total dimension of composite nanofibers (a), and on the content of the wrapped component (b) 100 Figure 5.7 Typical TEM photograph of PCL... core) composite nanofibers, and (b) actual setup and the coaxial spinneret consisting of one syringe needle and a fitting attached to a syringe tip 95 Figure 5.4 TEM images of core-shell structured PCL-r-Gelatin composite nanofibers electrospun from 10w/v % Gelatin/TFE and 10w/v % PCL/TFE: (a) overview of nanofibers on a copper grid; (b) and (c) segments of the nanofibers with a sharp boundary; and (d)... hydrophilicity and cellular affinity than that of the synthetic counterpart, and suggested the applicability of these nanofibers as scaffolding elements for engineering tissues The co -electrospinning of natural and synthetic polymers demonstrated here will provide a facile and effective approach for making other bioactive and functional nanofibrous structures, and ultimately fulfill the success of using nanofibers. .. engineering, a survey of the prior arts of materials hybridization for scaffold fabrications, and the state of the art of electrospun nanofibers as scaffolds for engineering tissues Chapter 3 is on electrospinning of gelatin nanofibers Chapter 4 is on the fabrication of composite nanofibers using a random polymers-blending or hybridization approach Chapter 5 is on the coaxial electrospinning to develop... 2 aligned nanofibrous membranes over a large area, instead of a strand of nanofibers from the previous technique It also provides a convenient means to collect single nanofibers for nanofiber characterization [38, 44] Further, larger area of nanofibers can be collected by rotating a multi-frame cylindrical structure [3, 45, 46] 2.1.3 Applications of nanofibers Electrospun polymeric nanofibers possess... following scope of work: 1) To develop a means to electrospin biopolymer of gelatin3 into nanofibers, and have the resultant nanofibers crosslinked This is to make the generated gelatin nanofibers a practical nanofiber material as useful as its counterpart forms such as films, large-diameter fibers and microspheres, and to provide feasibility for subsequent fabrication of Gt/PCL composite nanofibers 2)... encapsulated inside the PCL nanofibers and the feasibility of using core-sheath composite nanofibers for sustained release of proteins was investigated To conclude, two different composite nanofibers in the form of random blending (e.g., Gt/PCL) and core-sheath structure (e.g., collagen-r-PCL) had been successfully fabricated via the electrospinning process The physical, mechanical, chemical and biological characterization...List of Figures List of Figures Figure 2.1 Schematic of a basic laboratory setup for electrospinning, and a representative SEM image showing randomly arrayed nanofibers produced 8 Figure 2.2 (a) A setup used to collect uni-axial nanofiber strands, (b) aligned PCL nanofibers thus obtained 11 Figure 2.3 Paralleled array of carbon nanofibers (A) and stacked alignment structure... 1 way for modifying and tailoring the material properties Depending on the application, our biomimetic composite nanofibers can be designed in the form of either randomly blended or in an ordered structure e.g., core-sheath from the available synthetic and natural polymers The conceivable merits of such composite nanofibers will be as follows: 1) Physically, the new composite nanofibers can provide... successful electrospinning of gelatin, the first type of composite Gt/PCL nanofibers was fabricated from electrospinning blends of Gelatin and PCL Compared to the synthetic PCL nanofibers, it was found that such randomly blended Gt/PCL nanofibers became hydrophilic and had improved mechanical properties Biologically, the Gt/PCL scaffolds supported the cellular growth very favorably and encouraged cellular . overview of nanofibers on a copper grid; (b) and (c) segments of the nanofibers with a sharp boundary; and (d) segment of the nanofibers with skewed inner component 97 Figure 5.5 XPS spectra of. of gelatin, the first type of composite Gt/PCL nanofibers was fabricated from electrospinning blends of Gelatin and PCL. Compared to the synthetic PCL nanofibers, it was found that such randomly. functionality of the core-sheath nanofibers, bovine serum albumin was encapsulated inside the PCL nanofibers and the feasibility of using core-sheath composite nanofibers for sustained release of proteins