Nature inspired composite nanofibers

NATURE INSPIRED COMPOSITE NANOFIBERS TEO WEE EONG B ENG.(HONS.) NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgement Completing this Masters Program is not easy while holding a full time job Work commitment often brings my mind away from thinking about my Masters project I am fortunate to have an understanding boss and supervisor, Prof Seeram Ramakrishna, who I would like to express my thanks for his encouragement and guidance Being an engineer, it is certainly my wish to see my research work being translated into a useful product For this, I have to thank my co-supervisor, Prof Casey Chan for seeing the potential of my project for development into biomedical products Hopefully, in a few years time, the work that begins from this Masters Project is used as a successful clinical product I would like to thank my beloved wife, Karen, for her understanding when I have to spend time studying or reading up research materials during our courtship days To my parents who have shared the ups and downs of my research work, thank you for lending your ears although it must have been really tough for you to understand my work Finally, I have to thank my friends and colleagues in the lab This project will have been much tougher and will probably take longer to complete without you sharing knowledge and experience Table of Contents Acknowledgement Table of Contents i Summary iii List of Tables v List of Figures vi List of Publications viii Chapter 1: Introduction 1.1 Composite Nanofibers 1.2 Nanofiber fabrication techniques 1.3 Biomedical applications 1.4 Motivation 1.5 Objective Chapter 2: Literature Review 2.1 Bone Structure and Organization 2.2 Current Technology 2.3 Electrospinning 11 Chapter 3: Dynamic fluid method to reorganize nanofibers 13 3.1 Introduction 13 3.2 Experimental Procedures 13 3.3 Results and Discussions 16 Remarks 19 3.4 Biomedical applications 23 3.5 Conclusion 25 Chapter 4: Fabrication of 3D Assemblies using fluid properties 26 4.1 Introduction 26 4.2 Experimental Procedures 26 4.3 Results and Discussions 28 i 4.3.1 Physical structure observation 28 4.3.2 Effect of hydrophobicity 31 4.3.3 Effect of cooling rate 35 4.4 Application in Tissue Engineering 37 4.5 Conclusion 38 Chapter 5: Mineralization of three-dimensional matrix 40 5.1 Introduction 40 5.2 Experimental Procedures 40 5.3 Results and Discussions 43 5.4 Conclusion 56 Chapter 6: Research Summary and Future Recommendations 57 Reference 60 ii Summary Natural extracellular matrices are usually constructed from collagen nanofibers and this made nanofibers an attractive candidate for use in biomedical applications in particular regenerative scaffold Since electrospun collagen nanofibers are weak and degrade rapidly in vivo, synthetic polymers are often incorporated to form composite nanofibers Extensive studies carried out on electrospun nanofibrous two-dimensional mesh have shown that the architecture provides a favorable environment for cells to thrive on However, the ability to construct other electrospun architectures has been challenging A modified electrospinning setup using water as a working substrate has been demonstrated here to be capable of fabricating composite nanofibrous yarn and three-dimensional (3D) nanofibrous architectures Nanofiber and yarn diameter has been shown to be affected by the solution feed-rate and concentration of the solution Higher solution feed-rate and concentration gave rise to fiber and yarn of larger diameter The microstructure of the 3D nanofibrous scaffold is affected by the hydrophobicity of the polymer and the drying and freezing condition of the scaffold Using a modified alternate dipping method, calcium phosphate minerals can be deposited throughout the 3D nanofibrous scaffold While conventional static alternate dipping mineralization method yields minerals mainly on the surface of the 3D scaffold and uneven distribution of the minerals on the nanofibers, a flow mineralization method is able to achieve mineralization both at the surface and the core of the 3D scaffold with improved mineral distribution on the nanofibers Despite static mineralized scaffold having greater mineral contents, its compressive strength and modulus is much lower than flow mineralized scaffold Therefore, the mechanical strength of the mineralized nanofibrous scaffold is significantly affected by how the iii minerals are deposited on the nanofibers The ability to construct nanofibrous yarn, 3D nanofibrous scaffold and 3D mineralized nanofibrous scaffold has opened up new opportunities for the construction of biomimetic regenerative scaffold The ability to incorporate biological materials into these architectures that mimics the physical characteristic of natural extracellular matrix gave it the potential to replace autologous implants The next step of development will be to test these constructs in in vivo studies iv List of Tables Table 3.1 Polymer solution and average fiber diameter Table 3.2 Fiber diameter with respect to feed rate for PVDF-co-HFP v List of Figures Figure 1.1 Hierarchical organization of bone from the lowest level on the left to the bone macrostructure Figure 2.1 Schematic of collagen fibril and crystal growth The left column shows the arrangement of the collagen triple helix molecules in a fibril in 2-dimension and the right column in 3-dimension [A] Staggered arrangement of the collagen triple helix molecules The 3-dimensional distribution shows the channels resulting from the orderly arrangement of the fibrils [B] Nucleation of the minerals in the gap between the collagen molecules [C] Growth of the minerals between the gaps and along the channel results in a parallel array of coplanar crystals Figure 3.1 Setup used to create a flowing water system for the manipulation of deposited nanofibers Figure 3.2 Electrospun PVDF-co-HFP fibers deposited on a aluminum foil Figure 3.3 SEM images of the collected yarn a) Without going through the drawing process in the air b) After going through the drawing process in the air Figure 3.4 Graph of average PVDF-co-HFP yarn diameter against feed-rate The vertical line for each point depicts the scatter in the yarn diameter for each feed rate Figure 3.5 Nanofibrous yarn used as intra-luminal guidance channel [A] Overview of nerve guidance channel consisting of a nanofibrous conduit and nanofibrous yarn in the lumen [B] SEM image of conduit extracted from rat sciatic nerve after months [Picture courtesy of H S Koh] Figure 4.1 [a] Polycaprolactone (PCL) 3D mesh dried in room condition with no visible pores [b] PCL 3D mesh pre-frozen at -86 oC and freeze-dried in a cylinder with visible pores [c] PCL/col 3D mesh pre-frozen at -86 oC and freeze-dried in a hemispherical container showing visible pores [d] PCL/collagen 3D mesh pre-frozen at -86 oC and freeze-dried in a cylinder with visible pores Figure 4.2 [a] PCL mesh dried under room condition showing yarn stacked closely on top of one another [b] Freeze-dried PCL mesh pre-frozen at -86 oC showing distinct and isolated yarns made out of aligned nanofibres [c] PCL/collagen mesh pre-frozen at -86 o C before freeze-drying showing ridge-like structures [d] Disordered nanofibres that formed the ridges All samples were packed in 15 mm diameter cylinder Figure 4.3 Schematic of the ice crystals pushing the entangled yarn to form disordered fibres and ridges on the PCL/col scaffold [a] Stage Nanofibrous yarns were suspended in the water [b] Stage As the water cools, ice nucleates on the wall of the cylinder and vi on the nanofibres [c] Stage Growing ice front from the cylinder wall pushes against the nanofibres resulting in [d] ridges made out of random nanofibres Figure 4.4 Cross section of the PCL/col scaffold cooled at -86 oC showing inhomogeneous pores and a boundary layer between the displaced nanofibers on the surface and compacted fibers at the inner core Figure 4.5 Freeze-dried PCL/col [a] prefrozen at -86 oC showing “spikes”, [b] mesh prefrozen in liquid nitrogen has a relatively smoother surface [c] Higher magnification showing microstructure of scaffold pre-frozen at -86 oC where the “spikes” were ridges formed by disordered nanofibres and [d] mesh pre-frozen in liquid nitrogen with distinct yarns [e] Cross-section of the mesh pre-frozen at -86 oC showing the ridges are only found on the surface while [f] the mesh pre-frozen in liquid nitrogen did not exhibit any apparent differences in the microstructure from the surface to the interior Figure 4.6 Schematic of the ice nucleation and growth on PCL/col mesh cooled at -196 C [a] Stage 1, nanofibrous mesh is suspended in water [b] Numerous and rapid ice nucleation on the surface of the nanofibers while ice nucleation and growth commence from the cylinder wall [c] Complete freezing of mesh before the ice growing from the cylinder wall reaches it o Figure 5.1 Setup for mineralization of the 3D scaffold Figure 5.2 Spectra of PLLA and PLLA/col blended 3D nanofiber scaffold Figure 5.3 SEM images of mineralized [A] PLLA sample and [B] PLLA/col sample Figure 5.4 Distribution of minerals on across the cross-section of static mineralized scaffold Figure 5.5 Distribution of the minerals in the cross-section of the scaffolds using flow mineralization Figure 5.6 XRD showing the presence of amorphous calcium phosphate crystals in the mineralized scaffold Figure 5.7 Ash remains of mineralized scaffold after sintering at 500 oC [A] Static mineralized scaffold showing a hollow core [B] Flow mineralized scaffold showing a solid core Figure 5.8 Mechanical properties of scaffold mineralized under different condition [A] Compressive strength [B] Compressive Modulus Figure 5.9 Comparison of mineral nanoparticles distribution in [A] mineralized nanofibrous scaffold and [B]cancellous bone vii List of Publications W.E Teo, S Liao, C.K Chan, S Ramakrishna (2008) Remodeling of Three-dimensional Hierarchically Organized Nanofibrous Assemblies Current Nanoscience vol pg 361369 Wee-Eong Teo, Renuga Gopal, Ramakrishnan Ramaseshan Kazutoshi Fujihara, Seeram Ramakrishna (2007) A dynamic liquid support system for continuous electrospun yarn fabrication Polymer vol 48 pg 3400-3405 Teo WE, He W, Ramakrishna S (2006) Electrospun scaffold tailored for tissue-specific extracellular matrix Biotechnology Journal vol pg 918-929 (Top download in Biotechnology Journal in September 2006) Wee-Eong Teo, Seeram Ramakrishna (2009) Electrospun nanofibers as a platform for multifunctional, hierarchically organized nanocomposite Compos Sci Technol Vol 69 pg 1804-1817 Wee Eong Teo, Kazutoshi Fujihara, Casey Kwan-Ho Chan, Seeram Ramakrishna Fiber Structures and Process for their preparation WO 2008/036051 27 March 2008 Wee Eong Teo, Kazutoshi Fujihara, Seeram Ramakrishna Method & Apparatus for producing Fiber Yarn WO 2007/013858 A1 01 February 2007 viii Reference Akesson K, Grynpas M, Hancock R, Odselius R, Obrant K Energy-Dispersive Xray 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Teo W, Zhu X, Beuerman R, Ramakrishna S, Yung L, et al Development of a novel collagen-GAG nanofibrous scaffold via electrospinning Mater Sci Eng C 2007;27:262-266 101 Zhong S, Teo W, Zhu X, Beuerman R, Ramakrishna S, Yung L, et al An aligned nanofibrous collagen scaffold by electrospinning and its effect on in vitro fibroblast culture J Biomed Mater Res 2006;79A:456-463 73 102 Zhong S, Teo W, Zhu X, Beuerman R, Ramakrishna S, Yung L, et al Formation of collagen-GAG blended nanofibrous scaffolds and their biological properties Biomacromolecules 2005;6:2998-3004 74 ... 1.1 Composite Nanofibers Nature has been the inspiration for many materials design and architectures and the strongest materials are often made out of composites Closer examination of these composites... constructed from collagen nanofibers and this made nanofibers an attractive candidate for use in biomedical applications in particular regenerative scaffold Since electrospun collagen nanofibers are weak... has been inspired by the structure of nature? ??s design and this has been used in macro-level designs It is only in recent years where advances in nanotechnology have allowed us to mimic nature? ??s