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.. .APPLICATION OF RAPID PROTOTYPING TECHNOLOGY TO THE FABRICATION OF 3D CHITOSAN SCAFFOLD FOR TISSUE ENGINEERING GENG LI (B.Eng (Hons)) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING. .. shown that chitosan appears to be a suitable matrix material for tissue engineering applications 3.2.2 The Properties of Chitosan Chitosan is the product of the partial deacetylation of the naturally... use in tissue engineering applications Keywords: Rapid Prototyping, 3D Scaffold, Tissue Engineering, Chitosan, Biocompatibility vi List of Figures List of Figures Figure 3.1 Structure of chitosan
APPLICATION OF RAPID PROTOTYPING TECHNOLOGY TO THE FABRICATION OF 3D CHITOSAN SCAFFOLD FOR TISSUE ENGINEERING GENG LI NATIONAL UNIVERSITY OF SINGAPORE 2004 APPLICATION OF RAPID PROTOTYPING TECHNOLOGY TO THE FABRICATION OF 3D CHITOSAN SCAFFOLD FOR TISSUE ENGINEERING GENG LI (B.Eng. (Hons)) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDEGMENT This project would not have been successfully carried out if not for the support of a number of people. The author would like to express her most sincere gratitude to the following: 1. A/Prof. Wong Yoke San, the project supervisor, for his everlasting patience and for being an inspiring mentor. 2. A/Prof. Loh Han Tong, the project supervisor, for his advice and guidance throughout the course of the project. 3. Dr. Dietmar W. Hutmacher, for his guidance in the area of biomaterials and properties experiments and cell culture work. 4. A/Prof. Fuh Y H, Jerry, for his expertise in the work relating to rapid prototyping. 5. Dr. Feng Wei, for helping to lay the foundation of the system and for sharing his valuable expertise. 6. Miss Diana Tan, for her logistic support and ever helping attitude. 7. All the friends and colleagues from LCEL and BIOMAT, for making a friendly environment in the lab. 8. The author’s family, for their unconditional support, without which the author would not have come this far. The author will also thank the National University of Singapore for awarding the research scholarship and the department of Mechanical Engineering for the use of facilities. i Table of Contents Table of Contents Acknowledgments……………………………………………………………………..….i Table of Contents…………………………………………………………………...……ii Summary……………………………………………………………………………….....v List of Figures…………………………………………………………………………..vii List of Tables……………………………..…………………………………………..….ix Chapter 1 Introduction .................................................................................................... 1 1.1 Tissue Engineering ................................................................................................. 1 1.2 Research Objectives ............................................................................................... 4 1.3 Research Scope ...................................................................................................... 5 1.4 Thesis Outline ........................................................................................................ 5 Chapter 2 Literature Review ........................................................................................... 7 2.1 Scaffolds in Tissue Engineering ............................................................................ 7 2.1.1 Two-dimensional Scaffolds in Tissue Engineering ..................................... 7 2.1.2 Three-dimensional Bioresorbable Scaffolds in Tissue Engineering ........... 8 2.2 Three-dimensional Scaffold Fabrication Techniques ............................................ 9 2.3 Rapid Prototyping ................................................................................................ 12 2.3.1 Popular RP Technologies ......................................................................... 14 2.3.2 RP Materials....... ………………………………………………………...16 2.4 Scaffold Building Using RP Technologies…………………………..…………..17 2.5 Observations……………………………………………………………..………25 Chapter 3 Materials and Methods……………………………………………….….…26 3.1 Materials Used for Tissue Engineering Scaffolds…………………………….....27 3.1.1 Natural Polymers……………………………………………………...…27 ii Table of Contents 3.1.2 3.2 Synthetic Polymers…………………………………………………..…..28 Protocol of Material Preparations………………………………………...……...30 3.2.1 Materials Used in the Research…………………………………………...30 3.2.2 The Properties of Chitosan……………………………………………….30 3.2.3 The Protocol of Chitosan Gel Preparation...……………………………..32 3.2.4 Preparation of Sodium Hydroxide Solution……………………………...33 3.3 Scaffold Fabrication ……...……………………………………………………...33 3.4 Washing Protocol………………………………………………………………...34 3.5 Scaffold Characterization………..……………………………………………….35 3.5.1 Porosity……………………………………………………..…………….35 3.5.2 Morphology…………………………………………………………..…...37 3.5.3 Mechanical Property……………………..……………………………….37 3.5.4 Biocompatibility……………………………..…………………………...39 Chapter 4 The Fabrication Process …………………………………………………..41 4.1 Biomedical RP…………………………………………………………………...41 4.1.1 3D Plotting……………….…………………………………………….…42 4.2 The Rapid Prototyping Robotic Dispensing System………………….…………44 4.2.1 The Control Software…………….………………………………………45 4.3 Cubic Scaffold Fabrication Process……………………………………………...46 4.4 3D Free-form Scaffold Fabrication………………………………………………49 4.4.1 Investigation of Mimics…………….……………………………………50 4.4.2 Data Processing……………………….………………………………….52 4.4.3 Building Free-form Scaffold……….…………………………………….54 iii Table of Contents Chapter 5 Results and Discussion …………………………………………………….57 5.1 Results……………………………………………………………………………57 5.2 Discussion………………………………………………………………………..62 5.2.1 The Requirements for Tissue Engineering Scaffolds…………………...62 5.2.2 Scaffold Fabricated by RPBOD System ………………………………..64 5.2.3 The Requirements for Scaffold fabrication Techniques ...……………...64 5.2.4 The Dual Dispensing Method…………………………………………...66 Chapter 6 Conclusions and Recommendations ..…………………………………….69 6.1 Conclusions...…………………………………………………………………….69 6.2 Recommendations………………………………………………………………..71 REFERENCE…...……………………………………………………………………....73 APPENDICES…………………………………………………………………………..85 iv Summary Summary Bioresorbable three-dimensional scaffolds have special applications in tissue engineering and have been fabricated using different processing techniques. The key to an ideal tissue engineering scaffold might depend on the ability to fabricate scaffolds with suitable shape and inner structure while having the necessary biocompatibility properties for different applications. In this research, rapid prototyping technology is applied to fabricate 3D scaffolds for tissue engineering by using a specially developed desktop RP system. This desktop RP system is a computer-controlled four-axis machine with a multiple-dispenser head. The material used in this study is chitosan dissolved in acetic acid and sodium hydroxide solution. Neutralization of the acetic acid by the sodium hydroxide results in a precipitate to form gel-like chitosan strands. Free-form scaffolds have been built from relevant features extracted from given CT-scan images by this system. The required geometric data for the scaffolds in the form of a solid model can be derived from the CT-scan images through the use of a software to reconstruct images taken from CT/MR into a 3D model and converting the data to the data formats that can be recognized by rapid prototyping systems. The reconstructed computer model is sliced into consecutive two-dimensional layers to generate appropriately formatted data for the desktop RP system to fabricate the scaffolds. The four-axis system enables strands to be laid in a different direction at each layer to form suitable interlacing 3D free-form scaffold structures. v Summary Results from scanning electron microscopy and in-vitro cell seeding showed suitable structure as well as cell compatibility and attachment of the chitosan scaffolds built by the RP system. The study indicated that this RP system has the ability to fabricate 3D free-form scaffolds and the built scaffolds have potential for use in tissue engineering applications. Keywords: Rapid Prototyping, 3D Scaffold, Tissue Engineering, Chitosan, Biocompatibility vi List of Figures List of Figures Figure 3.1 Structure of chitosan.....................................................................................31 Figure 3.2 Dual dispensing method...............................................................................34 Figure 3.3 Instron Microtester.......................................................................................38 Figure 3.4 Typical stress-strain curve of a biological material......................................38 Figure 4.1 A framework of biomedical RP....................................................................42 Figure 4.2 Basic principle of 3D plotting......................................................................43 Figure 4.3 RP dispensing system...................................................................................43 Figure 4.4 The four-axis RPBOD system......................................................................44 Figure 4.5 Mechanical & pneumatic dispenser..............................................................45 Figure 4.6 3D scaffold fabrication controls...................................................................47 Figure 4.7 Tips position.................................................................................................48 Figure 4.8 Scaffold fabrication process by dual dispensing..........................................49 Figure 4.9 Mimics flowchart.........................................................................................50 Figure 4.10 The conversion of CT images to 3D computer mode by Mimics................54 Figure 4.11 Model of skull defect patch shown on the RPBOD monitor.......................55 Figure 4.12 Four consecutive layers with scan lines......................................................56 Figure 4.13 Chitosan scaffold of the patch built by RPBOD (15 layers)........................56 Figure 5.1 Freshly built chitosan scaffold and the air-dried scaffold under optical microscope (15X) shows the uniformity of the pores..................................57 Figure 5.2 ESEM picture of the surface morphology of the freeze-dried chitosan scaffold.........................................................................................................59 Figure 5.3 Stress-Strain curves of scaffolds..................................................................59 Figure 5.4 SEM image shows cell compatibility and attachment..................................61 vii List of Figures Figure A.1 Robokids dimensions.......................................................................................85 Figure B.1 Chitosan information sheet from vendor (Carbomer, Inc, USA) ...................86 viii List of Tables List of Tables Table 2.1 Conventional polymer scaffold processing techniques for tissue engineering…....11 Table 2.2 Comparison of different RP technologies.........................................................24 Table A.1 Machine specifications.....................................................................................85 Table C.1 Sample of macro-porosity calculation..............................................................87 ix Chapter 1. Introduction Chapter 1 Introduction 1.1 Tissue Engineering The need to produce tissues and organs for organ transplant due to the acute shortage of tissues and organs [Mooney and Mikos, 1999], and a possible mismatch of tissue types that can result in organ rejection, astronomical drug therapy costs and the potential development of cancer [Mrunal, 2000], brought about the birth of tissue engineering in the late 1980s [Berthiaume and Yarmush, 1995]. During a National Science Foundation workshop in 1988, tissue engineering was formally defined as [Lewis, 1995]: “The application of principles and methods of engineering and life sciences towards fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue functions.” Still in its infant stage, tissue engineering is intensively researched into to provide for the implantation of an engineered substitute for tissue loss or end-stage organ failure resulting from a disease or an injury. It provides a better alternative to the standard tissues or organ transplant with donated organs. Generally, there are three strategies that are utilized in tissue engineering [Chaignaud et al., 1997]: (1) the replacement of only isolated cells or cell substitutes needed for function; (2) the production and delivery of tissue-inducing substances such as growth factors and signal molecules; (3) the use of a scaffold (matrix) made from synthetic polymers or natural substances to promote cell proliferation. 1 Chapter 1. Introduction Perhaps the most challenging and promising strategy of tissue engineering is the in-vitro generation of autologous tissues by using cells isolated from donor tissues in combination with a scaffold. The success of such an approach offers the possibility of growing functional new tissues and even organs entirely in a laboratory environment. In the study conducted by Vacanti et al. [1988], it was observed that dissociated cells tend to organize themselves to form a tissue structure when they were provided with a guiding template. Therefore, the modern approach in tissue engineering utilizes 2D or porous 3D scaffolds, composed of biodegradable natural or synthetic polymers, to provide a temporary substrate to which transplanted cells could adhere, proliferate and differentiate, in order that a functional tissue can be regenerated. In this scaffold-based tissue engineering strategy, the successful regeneration of tissue and organs relies on the fabrication and application of suitable scaffolds. Different processing techniques have been developed to build TE scaffolds. Conventional scaffold fabrication techniques include fiber bonding [Brauker et al., 1995], phase separation [Ma and Zhang, 1999], solvent casting/particulate leaching [Mikos et al., 1993], membrane lamination [Mikos et al., 1996], melt molding [Thomson et al., 1995], gas foaming/high pressure processing [Baldwin et al., 1995], hydrocarbon templating [Shastri et al., 1997], freeze drying [Healy et al., 1998] and combinations of these techniques (e.g., gas foaming/particulate leaching [Harris et al.,1998], etc.). However, most of them are limited by some forms of flaws that include inconsistent and inflexible processing procedures, use of toxic organic solvents, manual intervention, and shape limitations. Therefore, the scope of their applications is restricted by these drawbacks. 2 Chapter 1. Introduction On a separate front, the introduction of rapid prototyping (RP) technologies starts a new revolutionary era for product design and manufacturing industries. The RP technology enables quick and easy transition from concept generation in the form of computer models to the fabrication of physical models. Developed to shorten and simplify the product development cycle, the flexibility and outstanding manufacturing capabilities of RP have already been employed for biomedical applications, especially scaffold fabrication. Its immense potential for producing highly complex macro- and microstructures is widely recognized and studied by many researchers in the manufacturing of TE scaffolds. At present, several RP techniques have been exploited for scaffold fabrication, such as fused deposition modeling (FDM) [Hutmacher et al., 2000], 3D printing (3DP) [Kim et al., 1998] and SLS [Lee and Barlow, 1994]. Landers and co-workers [2002] reported the development of a 3D plotting RP technology to meet the demands for desktop fabrication of hydrogel scaffolds. A key feature of this RP technology is the 3D dispensing of liquids and pastes in liquid media. This RP process prepared scaffolds with a designed external shape and a welldefined porous structure. A fabrication process that resembles the technology reported by Landers [2002] has been adopted to build scaffolds using a specially developed rapid prototyping robotic dispensing (RPBOD) system by researchers at the National University of Singapore [Ang et al., 2002]. This RPBOD system was developed from a computer-guided desktop robot (Robokids, Sony), which is capable of three simultaneous translational movements along the X-, Y- and Z-axis. The scaffolds fabricated by the RPBOD system showed good attachment between layers, which allowed the matrix to form fully interconnected channel architecture, and results of in-vitro cell culture studies revealed the biocompatibility 3 Chapter 1. Introduction of the scaffolds. Ang et al. [2002] demonstrated the potential of the RPBOD system in fabricating 3D TE scaffolds with regular and reproducible macropore architecture. The RPBOD system was subsequently improved by using a new fabrication method, referred to as dual dispensing [Tan, 2002]. A rotary motion about the Z-axis of the base was added. A multiple-dispenser unit was incorporated to the RPBOD system with two kinds of dispensing mechanisms: pneumatic and mechanical. Building of the scaffolds with the desktop RPBOD system has been developed based on the sequential dispensing of chitosan dissolved in acetic acid and sodium hydroxide solution. Neutralization of the acetic acid by the sodium hydroxide results in a precipitate to form a gel-like chitosan strand. The four-axis system enables strands to be laid in a different direction at each layer to form suitable interlacing 3D scaffold structures layer by layer. 1.2 Research Objectives Based on the previous research, the objectives of this research are: I. To optimize the parameters and conditions for fabricating scaffolds by the dual dispensing method with the RPBOD system. II. To design and fabricate 3D free-form scaffolds with relevant features extracted from given medical images (CT/ MRI) using a desktop PC. III. To characterize the built scaffolds and evaluate their potential for application in tissue engineering. 1.3 Research Scope In the first phase of this research, experiments were carried out to determine set of optimized parameters of the fabrication process. At the same time, protocols for 4 Chapter 1. Introduction the preparation of materials for scaffold fabrication were established based on the material properties. In the free-form scaffold fabrication phase, the data conversion process was developed to transfer medical data (CT/MRI) to the appropriate RPcompatible data format. This involves the use of a software to reconstruct images taken from CT/MR into 3D model and convert the data to the format that can be recognized by the developed rapid prototyping systems. Geometric data of the scaffold was generated based on the computer model built from the medical data. During the scaffold characterization and analysis phase, scanning electron microscopy (SEM) was used for scaffold morphology analysis. Porosity and density of the built scaffolds were calculated and compression tests were conducted to evaluate their load capacity. The biocompatibility of the scaffolds was studied by cell seeding. 1.4 Thesis Outline After an overall introduction of this chapter, the rest of this thesis is organized as follows: Chapter 2 provides a literature review on TE scaffold, rapid prototyping and RP-related scaffold fabrication techniques. Chapter 3 investigates the biomaterials used in TE scaffolds, selects the materials and the procedure for material preparation in this research and briefly presents the methods to fabricate scaffolds and the experiments carried out to characterize the scaffolds. Chapter 4 gives a general outline of the RPBOD system and details with the manufacturing process of regular shape and irregular scaffolds using the dual dispensing method. 5 Chapter 1. Introduction Chapter 5 presents the experimental results and discusses the advantages and improvements of the dual dispensing fabrication method and potential of the PRBOD system to desktop manufacture for TE scaffolds. Chapter 6 concludes and recommends for future research. 6 Chapter 2 Literature Review Chapter 2 Literature Review Since scaffolds serve a very important role in TE, there are plenty of existing works about the TE scaffold manufacturing in the literature. Section 2.1 reviews the general applications of TE scaffolds in medical area. Section 2.2 examines the traditional scaffold fabrication technologies and their drawbacks. Section 2.3 reviews the RP technology. Section 2.4 examines some popular RP techniques that are used for TE scaffold manufacturing. Section 2.5 provides general observations based upon the literature reviewed. 2.1 Scaffolds in Tissue Engineering In scaffold-based tissue engineering strategies, the scaffolds, built from synthetic or natural materials, serve as temporary surrogates for the native extra cellular matrix. The challenge in scaffold-based TE is to construct biologic replicas in-vitro such that the engineered composite becomes integrated for transplant in-vivo for the recovery of lost or malfunctioned tissues or organ. Subsequently, the composite should work coordinately with the rest of the body without risk of rejection or complications [Bell, 2000; Martins-Green, 2000]. 2.1.1 Two-dimensional Scaffolds in Tissue Engineering Two-dimensional matrices, in the form of thin films, have special applications in tissue engineering. The earliest and most successful application of 2D matrices in tissue engineering is the regeneration of skin. As a result of the work done in this area for the past two decades, skin regeneration has now become a clinical reality [Cairns et al. 7 Chapter 2 Literature Review 1993; Rastrelli, 1994; Kirsner et al., 1998; Philips, 1998; Teumer et al., 1998]. Bioresorbable polymers in the poly (α-hydroxy esters) family remain the most popular material choice for the fabrication of thin films for tissue engineering applications. For example, poly (lactic-co-glycolic acid) films of thickness 12-133µm have been fabricated using a modified solvent-casting method and shown to support the attachment of human retinal pigment epithelium cells in vitro [Thomson et al., 1996]. Cell proliferation rates on the films were shown to be higher than that on tissue culture polystyrene controls [Lu et al., 1998]. A film of poly (ε-caprolactone) and poly(lactic acid) in a weight ratio of 1:1 and reinforced with woven poly(glycolic acid) has also been developed, made into a tube and used as a matrix for vascular endothelial cells [Burg et al., 1999; Shin'oka et al., 2001]. Recently, 2D films made of synthetic polymers have also been used as potential substrates for developing an artificial salivary gland [Aframian et al., 2000], because native salivary epithelial exists as a single layer. To date, 2D matrices have been applied in the regeneration of such tissues as vascular vessels, retinal epithelium and salivary gland, although the success rate is not as good as in skin. 2.1.2 Three-dimensional Bioresorbable Scaffolds in Tissue Engineering The demand for transplant organs and tissues far outpaces the supply, and this gap will continue to widen [Cohen et al., 1993]. Cell transplantation was proposed as an alternative treatment to whole organ transplantation for malfunctioning organs [Cima et al., 1991]. For the creation of an autologous implant, donor tissue is harvested and dissociated into individual cells. The cells are then attached and cultured onto a proper substrate that is ultimately implanted back at the desired site of 8 Chapter 2 Literature Review the functioning tissue. However, it is believed that isolated cells cannot form new tissues by themselves. Most primary organ cells require specific environments that very often include the presence of a supporting material to act as a template for growth. The currently existing substrates are mainly in the form of 3D tissue engineering scaffold. 2.2 Three-dimensional Scaffold Fabrication Techniques Conventional scaffold fabrication techniques include fiber bonding [Brauker et al.1995, Wang et al., 1993], phase separation [Lo, 1996, Ma and Zhang, 1999], solvent casting/particulate leaching [Mikos et al., 1993, Mooney et al., 1992, Holy et al., 2000, Mikos et al, 1994], membrane lamination [Mikos et al, 1996], melt molding [Thomson et al., 1995], gas foaming/high pressure processing [Baldwin et al., 1995, Mooney et al., 1996], hydrocarbon templating [Shastri et al., 2000], freeze drying [Whang et al., 1995, Healy et al., 1998] and combinations of these techniques (e.g., gas foaming/particulate leaching [Harris et al., 1998], etc.). The principles, procedures and applications or potential applications of these techniques can be found in several research works in literature [Vacanti et al., 1998, Lu and Mikos, 1996, Thomson et al., 2000, Widmer and Mikos, 1998, Yang et al., 2001]. Although conventionally produced scaffolds have been applied to engineer a variety of tissues with varying success, most of the conventional techniques are limited by some flaws, which restrict their scope of applications. Among the main limitations are [Leong et al., 2002]: 1) Manual intervention: All conventional techniques rely on manual processes that are labor-intensive and time-consuming. Most require multi-stage processing of the scaffold materials in order to form the desired scaffolds with the appropriate characteristics. The heavy reliance on user’s skills and experiences often results in 9 Chapter 2 Literature Review inconsistent outcomes and poor repeatability. 2) Inconsistent and inflexible processing procedures: These result in highly inconsistent macro- and micro-structural and material properties that may be adverse to tissue regeneration. Many conventional techniques (e.g., solvent casting, freeze drying, phase separation, etc.) are sensitive to minor variation and as such, may produce results that differ between applications. Hence, the fabricated scaffolds usually possess inconsistent pore sizes, pore morphologies, porosities and internal surface areas over their entire volumes. 3) Use of toxic organic solvents: Most conventional techniques involve extensive use of toxic organic solvents on the scaffold materials in order to convert the raw stock (granules, pellets or powders) into the final scaffold. Incomplete removal of solvents from the fabricated scaffolds, especially in thicker constructs, will result in harmful residues that have adverse effects on adherent cells, incorporated biological active agents or nearby tissues [Healy et al., 1998]. 4) Use of porogens: Salts or waxes are employed as porogens in some conventional techniques (e.g., particulate leaching, hydrocarbon templating, etc.) to create porous scaffolds. The use of porogens limits the scaffolds to thin membranes with thickness of 2mm [Lu and Mikos, 1996] to facilitate complete porogen removal. Porogen particles entrapped by the matrix will remain within the scaffold. Also, it is difficult to prevent the agglomeration of porogen particles and achieve uniform porogen dispersion. These factors will result in uneven pore densities and morphologies that have detrimental impact on the material characteristics of the scaffold. 5) Shape limitations: Molds or containers are used in some techniques to cast 10 Chapter 2 Literature Review scaffolds in thin membrane forms or simple uniform geometries. The melt molding technique, although capable of producing three-dimensional scaffolds, is limited by the complexity in the design and construction of the mold. Although techniques such as membrane lamination can create irregularly shaped scaffolds, the process is tedious and time-consuming due to the lamination of thin membrane layers. It may also result in limited interconnected pore networks. Table 2.1 summarizes the advantages and limitations of these conventional techniques [Leong et al., 2002]. Table2.1 Conventional polymer scaffold processing techniques for tissue engineering Process Advantages Disadvantages Fiber bonding Easy process High porosity High surface area to volume ratio High processing temperature for non-amorphous polymer Limit range of polymers Limit range of polymers Lack of mechanical strength Problems with residual solvent Lack of control over microarchitecture Phase separation Allows incorporation of bioactive agents Highly porous structures Lack of control over microarchitecture Problems with residual solvent Limited range of pore sizes Solvent casting and particulate leaching Highly porous structures Large range of pore sizes Independent control of porosity and pore size Crystallinity can be tailored Limited membrane thickness Lack of mechanical strength Problems with residual solvent Residual porogens Membrane lamination Macro shape control Independent control of porosity and pore size Lack of mechanical strength Problems with residual solvent Tedious and time-consuming Limited interconnected pores Melt molding Independent control of porosity and pore size Macro shape control High processing temperature for nonamorphous polymer Residual porogens 11 Chapter 2 Literature Review Polymer/ceramic fiber composite-foam Good compressive strength Independent control of porosity and pore size Problems with residual solvent Residual porogens High-pressure processing Organic solvent free Allows incorporation of bioactive agents Nonporous external surface Closed pore structure High-pressure processing and particulate leaching Organic solvent free Allows incorporation of bioactive agents Highly porous structures Large range of pore sizes Independent control of porosity and pore size Limited interconnected pores Freeze drying Highly porous structures High pore interconnectivity Limited to small pore sizes Hydrocarbon templating No thickness limitation Independent control of porosity and pore size Problems with residual solvent Residual porogens 2.3 Lack of mechanical strength Residual porogens Rapid Prototyping Rapid Prototyping (RP) is the name given to a family of processes that are used to fabricate objects directly from a 3D computer model. The model is produced either by computer-aided design (CAD), 3D scanning or 3D reconstruction of 2D images. Such technologies are also known as Free-Form Fabrication (FFF), Solid Freeform Fabrication (SFF) or Layered Manufacturing (LM). Rapid prototyping is a relatively new technology, yet tremendous progress has been made in terms of the systems and materials in the last decade. The underlying concept of RP is the generation of a 3D physical model in a layer-by-layer manner through a process that deposits, bonds or fuses material onto the previous layer under computer control [Lamont, 1993]. It is notable that RP uses 12 Chapter 2 Literature Review an "additive" fabrication process, fabricating 3D models by "building-up" rather than "cutting-away" processes, compared with the conventional manufacturing methods such as forming or material removal, etc. In RP process, 3D objects are decomposed into 2D layers, and planning on 2D domain is relatively simple. The planning of the fabrication is largely automatic, demanding little human intervention and robust process planning is easier to implement. RP is especially suitable in areas such as mold production in injection molding industries [Wohlers, 1999], where the high cost is offset by the huge reduction in fabrication time and the flexibility for customized jobs. RP also allows the special capability of fabricating enclosed cavities, something which precision CNC, arguably the closest rival to RP in terms of speed and versatility, cannot achieve. The rapid prototyping technology enables quick and easy transition from concept generation in the form of computer images to the fabrication of physical models. It is an effective technology to expedite the product development. Traditionally, designers required CAD part design, tooling design, tool path programming and tooling machining and molding to test CAD designs, which is a long cycle in the order of months, even years and high cost. RP is of special interest in the non-repetitive fabrication of models with great complexity without high cost. RP technology is now emerging as a major link between part design and manufacturing. In general, the attributes of RP can be summarized as: (1) a material additive process; (2) ability to build complex 3D geometries, including enclosed cavities; (3) process is automatic and based on a CAD model; (4) requires little or no part-specific tooling or fixturing; (5) requires minimal or no human intervention to operate. 13 Chapter 2 Literature Review 2.3.1 Popular RP Technologies There are six well-known RP technologies available in the market and these are stereolithography (SL), fused deposition modeling (FDM), solid ground curing (SGC), laminated object manufacturing (LOM), selective laser sintering (SLS), and 3D printing (3DP). 1) Stereolithography (SL) [Lu et al., 2001]: Stereolithography, which is a combination of computer graphics, laser technology and photochemistry, creates 3D parts by selectively solidifying polymeric materials layer-by-layer upon exposure to ultra-violet radiation or laser beams. It is currently the most accurate RP process in terms of dimensional accuracy and capability in creating small fine features. However, prototype parts created by currently available SL systems exhibit weak mechanical properties and significant amount of shrinkage. 2) Fused Deposition Modeling (FDM) [Kochan, 1997; Hutmacher, 2000]: An FDM machine consists of a movable head which deposits a thread of molten material onto a substrate. After a layer is completed, the platform on which the material is extruded is lowered by one layer thickness, and the extrusion process repeats. FDM employs the concept of melt extrusion to deposit a parallel series of material roads that forms a material layer. In FDM, filament material stock (generally thermoplastics) is fed and melted inside a heated head before being extruded through a nozzle with a small orifice. The material is deposited in very thin layers and bonds onto the previous when the material solidifies. After a layer is completed, the table is lowered by one layer thickness, and the extrusion process begins again. 3) Solid Ground Curing (SGC) [Kochan, 1993]: This system utilizes photo- polymer resins and ultra-violet (UV) light. Data from the CAD model is used to 14 Chapter 2 Literature Review produce a mask which is placed above the resin surface. When the layer has been cured, the excess resin is wiped away and spaces are filled with wax. The wax is cooled and the wax chips removed. A new layer of resin is applied and the process is repeated. The advantages of SGC are that the entire layer is solidified at once; reducing the part creation time, especially for multi-part builds. Also, no post-curing is required. The disadvantages of this system are that it is noisy, large and needs to be constantly manned. It wastes a large amount of wax which cannot be recycled and is also prone to breakdowns. 4) Laminated Object Manufacturing (LOM) [Cooper, 2001]: The build material is applied to the part from a roll, and then bonded to the previous layers using a hot roller which activates a heat sensitive adhesive. The contour of each layer is cut with a laser that is carefully modulated to penetrate to the exact depth of one layer. After the layer has been completed and the build platform lowered, the process repeats itself. However, there is a need to separate the finished parts from the build platform, which affects their surface finish, creating a large amount of scrap. There is also a need to hand polish the finished parts. 5) Selective Laser Sintering (SLS) [Lu et al., 2001]: In the SLS process, a layer of powder is deposited on a support and leveled by a rolling device. A laser beam then scans and sinters a 2D pattern on the deposited powder layer. After sintering a layer, a new layer of powder is deposited in the same manner. By successive powder deposition and laser scanning, a 3D part is built. 6) 3D Printing (3DP) [Stephen et al., 1998]: Developed at the Massachusetts Institute of Technology (MIT), this technology is based on the bubble jet printing of a binder, much like a conventional desktop printer. In fact, some commercial models of 3DP machines utilize the same print cartridge packages as commercial printers. 15 Chapter 2 Literature Review Instead of printing on paper, a print head prints onto a bed of powdered material following the object’s profile as generated by the system computer. The binder is delivered to the powder bed to produce the first layer and the bed is then lowered by a fixed distance. Powder is then deposited and spread evenly across the bed with a roller mechanism, and a second layer is built. This is repeated until the entire model is fabricated. The completed object is embedded inside unprocessed powders and is extracted by brushing away the loose powders. The process can produce porous parts but lack strength without post fabrication processing 2.3.2 RP Materials There is a wide choice of materials for RP processing, which can be generally classified into these few categories: 1) Reactive liquids that change into solids with application of radiation (photopolymers), e.g. stereolithography (SL); 2) Powder bonding, directly or with a binder, e.g. selective laser sintering (SLS), 3D printing (3DP); 3) Plastic or wax extruded under heat or in pre-cured form, e.g. fused deposition modelling (FDM), 3D plotting; 4) Laminates cut to shape and stacked together, e.g. laminated object manufacturing (LOM). A wide range of materials can fit into these categories; hence increase the flexibility and applicability of the various RP processes in different areas. Among these, the powder bonding and polymer extrusion processes have the widest applications, especially in the biomedical field [Rüdiger Landers & Rolf Mülhaupt, 2000]. 16 Chapter 2 Literature Review 2.4 Scaffold Building Using RP Technologies With the advent of cell and tissue culture technologies and the long-term biocompatibility advantages of such implants over non-biological materials, the drive is towards culturing matching cell types within biodegradable scaffolds. The speed and customizability of RP enable construction of individual, patient-specific scaffolds, and the capability of internal cavities allows specially designed internal structures. The inherent porosity of many RP processes, such as 3DP and FDM, is critical here for the successful establishment of the cells into the structure. Some significant advantages derivable with scaffold fabrication using RP technology include: 1) Customized design: Direct utilization of CAD models as inputs for scaffold fabrication allows complex scaffold designs to be realized. Patient-specific data and scaffold structural properties required for regenerating specific tissues can be incorporated into the scaffold design via CAD. 2) Computer-controlled fabrication: The use of automated computerized fabrication will result in high throughput production with minimal manpower requirements. The high build resolution of RP technologies coupled with the ability to define and control individual process parameters will enable the creation of highly accurate and consistent pore morphologies. Using CAD and RP technologies, scaffolds with porosities exceeding 90% and complete pore interconnectivity can be realized. The ability to optimize scaffold designs will facilitate cell attachment, colonization and proper ECM (extra cell matrix) formation. 3) Anisotrophic scaffold microstructures: The use of CAD and RP will allow user to control the localized pore morphologies and porosities to suit the requirements 17 Chapter 2 Literature Review of different cell types within the same scaffold volume. This is achieved by incorporating different controllable macroscopic and microscopic design features on different regions of the same scaffold. Having an anisotrophic scaffold microstructure is advantageous in TE applications where multiple cell types arranged in hierarchical structures are necessary [Park et al., 1998, Hutmacher et al., 2001]. 4) Processing conditions: RP techniques employ a diverse range of processing conditions that include solvent and porogen-free processes and room temperature processing. Some RP techniques allow pharmaceutical and biological agents to be incorporated into the scaffolds during fabrication [Leong et al., 2001, Low et al., 2001, Wu et al., 1996]. At present, several RP techniques have been exploited for scaffold fabrication, which include: 1) Three-dimensional printing (3DP) The versatility and simplicity of 3DP allow the processing of a wide variety of powder materials including polymers, metals and ceramics. So far, 3DP is perhaps the most widely investigated RP technique for scaffold fabrication. Kim et al. [1998] employed 3DP with particulate leaching technique for creating porous scaffolds using polylactide-coglycolide (PLGA) powder mixed with salt particles and a suitable organic solvent. Cylindrical scaffolds measuring 8mm (diameter) by 7mm (height) were fabricated. The salt particles were leached using distilled water after 3DP fabrication to result in scaffolds with pore sizes of 45–150 mm and 60% porosity. To improve pore interconnectivity and porosity, the scaffolds were constructed by printing and horizontally stacking 800 mm diameter longitudinal channels that were arranged in parallel throughout the scaffolds’ height. 18 Chapter 2 Literature Review Zeltinger et al. [2001] employed 3DP fabricated porous poly (l-lactic acid) (lPLA) disc shaped scaffolds measuring 10mm (diameter) by 2mm (height) to investigate the influence of pore size and porosity on cell adhesion, proliferation and matrix deposition. The scaffolds were constructed with two different porosities (75% and 90%) and four different pore size distributions (>38, 38–63, 63–106 and 106–150 mm) that were formed using salt and leaching methods. Lam et al. [2002] developed a blend of starch-based powder containing cornstarch (50%), dextran (30%) and gelatin (20%) that can be bound by printing distilled water. Cylindrical scaffolds measuring 12.5mm (diameter) by 12.5mm (height) were printed and characterized. Both solid cylindrical scaffolds as well as structures constructed by stacking cylindrical (2.5mm in diameter) or rectangular (2.5mm_2.5 mm) cross sectional channels were fabricated. Other research works that have exploited the capabilities of 3DP include the work of Park et al. [1998]. In their work, 3DP was employed with surface modification methods for creating scaffolds with controllable anisotrophic microstructures and surface chemistry. Stephen et al. [1998] have been able to illustrate that micro-porous 3D scaffold can be created using 3DP on copolymers of polylactide-coglycolide. The advantage of this process is the relatively fast speed and the ability to fabricate complex geometries with overhang due to the support of the powder bed. However, one disadvantage is that small pore size cannot be achieved with this technology. 2) Fused deposition modeling (FDM) 19 Chapter 2 Literature Review Developed by Stratasys Inc. FDM process is one of the most successful RP systems in the market. It is gaining more and more market because of its ability to build parts with thermoplastic material, which is widely used and relatively cheap. Zein et al. [2002] employed FDM for producing poly (e-caprolactone) (PCL) scaffolds with different geometrically consistent honeycomb-like patterns and fully interconnected porous channels. The scaffolds were constructed with pore/channel sizes ranging from 160 to 700 mm and 48% to 77% porosities. The different honeycomb designs were obtained by employing different laydown patterns for each consecutively deposited layer. Different channel sizes were obtained by varying the spacing between the extruded roads of polymeric material. Hutmacher et al. [2001] investigated the in-vitro cell cultural response of primary human fibroblast and osteoblast cells on FDM-fabricated PCL scaffolds. Rectangular scaffolds measuring 32mm (length) by 25.5mm (width) by 13.5mm (height) and comprising of two different microstructures formed using two different laydown patterns (i.e., (1) 0_/60_/120_ and (2) 0_/72_/144_/36_/108_) were fabricated. The scaffold porosities were measured to be 61%. Both microstructures exhibited complete pore interconnectivity. Bose et al. [1998] and Hattiangadi et al. [1999] applied an indirect fabrication method involving FDM for producing porous bioceramic implants. In their research, FDM was employed to fabricate wax molds containing the negative profiles of the desired scaffold microstructure. Ceramic scaffolds were then cast from the molds via a mold technique. Ceramic scaffolds with 31–55% porosities, pore sizes of 150–750 mm and complete pore interconnectivity were produced. 20 Chapter 2 Literature Review Other FDM-based researches conducted include the work carried out by Leong et al., [2002]. In their work, FDM filaments made from different grades of high density polyethylene (HDPE) were processed on a FDM1650 system (Stratasys Inc.). By changing the direction of material deposition for consecutively deposited layers and the spacing between the material roads, scaffolds with highly uniform internal honeycomb-like structures, controllable pore morphology and complete pore interconnectivity are obtained. In order to fabricate scaffold designs with overhanging features, removable supporting structures are deposited alongside the scaffold to support such features. However, the main disadvantage of this process is the restriction on thermoplastic materials. The high temperature required to melt the material rule out the possibility of using any heat sensitive materials as base material or as additives. 3) Selective laser sintering (SLS) SLS employs a CO2 laser beam to selectively sinter polymer or composite (polymer/ceramic, multiphase metal) powders to form material layers. The laser beam is directed onto the powder bed by a high-precision laser scanning system. The fusion of material layers that are stacked on top of one another replicates the object’s height. During fabrication, the object is supported and embedded by the surrounding unprocessed powders and has to be extracted from the powder bed after fabrication. Since the powders are subjected to low compaction forces during their deposition to form new layers, SLS-fabricated objects are usually porous. The porosity of SLSfabricated objects can be controlled by adjusting the SLS process parameters [Leong et al., 2001, Low et al., 2001]. 21 Chapter 2 Literature Review Lee and Barlow [1994, 1993] prepared various forms of calcium phosphate powders with Ca/P ratios ranging from 0.5 to 1 by reacting hydroxyapatite (Ca 5 (OH)(PO4)3 ) with phosphoric acid. The pore sizes of the SLS-fabricated ceramic parts were reported to measure around 50 mm and were well interconnected. Problems encountered were mainly due to shrinkage of the parts during sintering. 4) Three-dimensional plotting (3D-Plotting) In these types of systems, a dispenser head is controlled by a 3-axis platform, typically a CNC machine or robot. The process generates an object by building micro strands or dots in a layered fashion. Depending on the type of dispenser head, a variety of materials can be used to build scaffolds. The advantage of such a system is its versatility with a wide range of fluids and the absence of hot processes, often adverse for biological materials. This method is generally low cost with minimal specialized equipment required. A simple adaptation of dispenser heads will allow a wide variety of thermoplastic polymers as well as practically any pastes and solutions. Landers et al. [2000] presented a new technique that can make use of a wide variety of polymer hot melts as well as pastes, solutions and dispersions of polymers and reactive oligomers. However, resolution is the primary limiting factor, determined by the size of dispensing tip. Vozzi et al. [2001] achieved resolution as low as 10µm through the use of microsyringes and electronically regulated air pressure valves. A microsyringe-based 3D scaffolds fabrication technique was also described by Vozzi et al [2002]. It employs a highly accurate three-dimensional micropositioning system with a pressure-controlled syringe to deposit biopolymer structures with a lateral resolution of 5 mm. The pressure-activated microsyringe is 22 Chapter 2 Literature Review equipped with a fine-bore exit needle, through which tiny amounts of polymer ooze out when pressure is applied to the syringe. A wide variety of two- and threedimensional patterns can be fabricated. Poly-L-lactic acid (PLLA), polycaprolactone (PCL) and blend of PLLA and PCL were used in their research. Experiments indicated the simplicity and possibility of scaffold fabrication with various biopolymers. 5) Laminated object manufacturing (LOM) LOM is a process where individual layers are cut from a sheet by a computer- controlled laser, after which the individual layers are bonded together to form a 3D object. LOM has been used for fabrication of bioactive bone implants, using HA and calcium phosphate laminates [Steidle et al., 1999]. A HA/glass tape is laid down on the working platform. The outside profile of the layer to be built is cut using a laser directed by an XY plotter. The laser only cuts to the depth of a single layer. A second layer of the HA/glass tape is laid down on top of the first and a heated roller passes over the two squeezing and bonding them together. The entire object is formed in this way. The downside in this process is the burnt edges due to the laser cut, not an issue with most applications, but creates unwanted and possibly harmful debris in biomedical applications. Material degradation in the heated zone may also occur. 6) Multiphase Jet Solidification (MJS) The basic principle of MJS is to extrude melted material through a jet, similar to FDM. In contrast to FDM, the MJS process is mainly designed to produce highdensity metallic and ceramic parts. 23 Chapter 2 Literature Review In Koch and coworker’s research [1998], the MJS technology was used to create bio-compatible implants. The material used in this research is a biocompatible and bioresorbable poly-lactide material instead of the usual powder-binder-mixture of stainless steel. Table 2.2 summaries the advantages and disadvantages of different RP technologies. Table 2.2 Comparison of different RP technologies RP Technique 3D printing Materials Advantage Disadvantage Ink+powder of bulk polymers, ceramics No inherent toxic components Fast processing Low costs Weak bonding between powder particles Bad accuracy-rough surface FDM/FDC Some thermoplastic polymers/ceramics Low costs Elevated temperatures Small range of bulk materials Medium accuracy 3D plotting Swollen polymers (hydrogels) thermoplastic polymers reactive resins, ceramics Broad range of materials Broad range of conditions Incorporation of cells proteins and fillers Slow processing Low accuracy No standard conditiontime consuming adjustment to new materials SLS Metals, ceramics, bulk polymers compounds High accuracy, Good mechanical strength, Broad range of bulk materials Materials trapped in small inner holes is difficult to be removed, biodegradable materials maybe degrade in the chamber SLA Photopolymer resins Relative easy to remove support materials, Relative easy to achieve small feature Limited by the development of photopolymerizable and biocompatible, biodegradable liquid polymer material 24 Chapter 2 Literature Review SGC Photo-polymer resins LOM Paper, nylon, polyester MJS Stainless steel, alumina and bronze powder 2.5 Relative less part creation time, especially for multi-part builds. No post-curing required. Low cost No chemical reaction involved, parts can be made quite large. Accurate to approximately 0.1 mm. Low cost Fast Noisy, large and needs to be constantly manned. Wasting a large amount of wax which cannot be recycled. Prone to breakdowns. Need to separate and hand polish the finished parts from the build platform Material degradation in the heated zone may also occur. Shrinkage occurs during sintering Observations The literature has shown that much research effort has been put to the TE scaffold fabrication and application. In general, Rapid prototyping (RP) is suitable for tailoring individual patient-specific scaffold parts because of its flexibility to build complex structures. Though studies have shown these RP techniques have the potential to produce biomedical scaffolds, each has its shortcomings. 3DP requires post processing to improve the mechanical properties of the scaffold. FDM, on the other hand, allows the application of only thermoplastic polymers. This prevents the implementation and application of biological agents and natural polymers as temperature induces protein inactivation. Additionally, the major proportion of the scaffold fabrication supported by RP technology was based upon melt and powder processing. Future research in customized scaffold fabrication concerns greater flexibility and low cost for clinical reality. 25 Chapter 3 Materials and Methods Chapter 3 Materials and Methods The selection of materials plays a key role in the design and development of scaffolds suitable for tissue engineering [Vacanti et al., 1994]. Ideally, the material used for TE scaffolds should meet the following design criteria [Freed et al., 1994]: 1) Surface property: During the implantation of the cells in-vitro, the scaffold is soaked in a suspension of cells. The surface should promote proper attachment and growth of the cells onto the structure. Often, additives such as hydroxyapatite (HA) are added to the basic scaffold material to promote cell attachment on biomedical scaffolds. 2) Material degradation: A scaffold should fully degrade once it has served its purpose of providing a template for the regenerating tissue. The material needs to be reabsorbed by the tissue after the cells have established themselves; hence its degradation products should not provoke acute inflammation or toxicity when implanted in-vivo and a controllable degradation rate is critical to ensure that proper cell growth is achieved before absorption. 3) Material processable ability: Material should be reproducibly processable into a variety of shapes and sizes. 4) Mechanical properties: Lastly, the area and type of implant will require scaffold material of suitable mechanical properties, e.g. more rigid PCL scaffolds favor applications in bone cell cultures while soft chitosan scaffolds are more suitable for soft tissues. 26 Chapter 3 Materials and Methods 3.1 Materials Used for Tissue Engineering Scaffolds In literature there are basically two classifications of biomaterials used in the fabrication of scaffolds for tissue engineering. They are natural or biologically derived polymer and synthetic polymer. 3.1.1 Natural Polymers The rationale behind the use of natural polymers as matrix materials is to mimic, as closely as possible, the natural environment of the extra cellular matrix (ECM) that forms the framework of all tissues in the body. Natural polymers, being very similar to macromolecular substances which the biological environment is capable of interacting with, may thereby minimize the problems of poor biocompatibility and stimulation of a chronic inflammatory reaction. Such positive cell-matrix interactions also introduce the possibility of designing matrices which function biologically at the molecular level. This means that besides providing a structural role, matrices could also play a part in enhancing tissue regeneration. The natural polymers used as matrix materials thus include those that exist in natural ECM, such as collagen and various glycosaminoglycans and those that exist elsewhere in nature, such as chitosan. Collagen Collagen is a major constituent of all ECM in the human body, and is thus a natural choice as a matrix material. There are many types of collagen [Mayne and Burgeson, 1987], which are tissue-specific, and their primary role is to provide structural support. The shape and properties of each type of collage is dependent on the structure of the triple-helix of the molecule [Linsenmayer, 1991]. However, it has become clear now that collagens have also numerous developmental and 27 Chapter 3 Materials and Methods physiological functions. The pioneers of skin tissue engineering focused on natural bioresorbable collagen lattices as cell matrices. Bell et al. [1981a, 1981b, 1983] used collagen lattices to develop bilayered skin equivalents. Collagen has also been used in cartilage [Roche et al., 2001], bone [Du et al., 1999], muscle [van Wachem et al., 1996] and liver [Ranucci et al., 2000] regeneration. Glycosaminoglycans (GAGs) The next group of natural polymers used is glycosaminoglycans (GAGs). GAGs are negatively charged and heavily hydrated linear polymers of repeating disaccharides that exist in the ECM, usually as part of proteoglycans. There are four classes of GAGs: hyaluronic acid (HA), chondroitin sulfate (CS), keratan sulfate (KS) and heparan sulfate (HS). To date, HA and CS are the most commonly used GAGs as matrix materials for tissue engineering. The attractiveness of using HA lies in its ability to promote cell proliferation, migration and define the space in which cells differentiate and form new matrices [Toole, 1991; Wight et al., 1991]. Chitosan Another natural polymer, which has been commonly studied as a matrix material, but exists outside the human body in nature, is chitosan. It has been extensively studied both as a drug delivery medium [Chellat et al., 2000] and as a cell carrier [Mori et al., 1997; Chuang et al., 1999]. In brief, results from the use of natural biodegradable polymers as cell matrices have been encouraging. 3.1.2 Synthetic Polymers The advantages of synthetic bioresorbable polymers over natural ones include better mechanical properties, more readily processable into a variety of shapes and 28 Chapter 3 Materials and Methods forms using various fabrication techniques [Thomson et al., 1997] and more easily modified degradation and resorption profiles. Poly (α-hydroxy esters) Aliphatic polyesters in the family of poly (α-hydroxy esters), including poly (glycolic acid) (PGA), poly (lactic acid) (PLA), and their copolymers, have been most widely studied as matrix materials. These polymers were approved for in-vivo use by the Food and Drug Administration (FDA) and have been developed for use as various sutures, drug delivery systems and matrices for tissue regeneration [Park and Park, 1994; Chu, 1995; Dunn, 1995; Athanasiou, 1996; Hutmacher et al., 1996]. Poly (ε-caprolactone) Poly (ε-caprolactone) (PCL) is a semi-crystalline aliphatic polyester which belongs to the family of poly (ω-hydroxy esters) [Kimura, 1993]. It is soluble in solvents such as chloroform and methyl chloride but only partially soluble in acetone and ethyl acetate. A novel method of fabricating 3D porous scaffolds with PCL, via fused deposition modeling, was developed and studied as potential matrices for tissue engineering by Hutmacher[2000] and his coworkers [Hutmacher et al, 2001]. The scaffolds have been used in the studies of bone [Hutmacher et al., 2000a] and cartilage [Hutmacher et al., 2000b] regeneration. Promising results have been shown in terms of the scaffolds’ ability to support the attachment and proliferation of different cell types, and the formation of preliminary cartilage and bone-like tissue both in-vitro and in-vivo. 29 Chapter 3 Materials and Methods 3.2 Protocol of Material Preparations 3.2.1 Materials Used in the Research Among the commonly used biomaterials in TE, chitosan is a promising materials considering of its biocompatibility and abundant source. Besides the use as a drug delivery medium [Chellat et al., 2000] and as a cell carrier [Mori et al., 1997; Chuang et al., 1999], It has also been shown that chitosan has the potential to be fabricated into various porous structures, including membranes, blocks, tubes and beads [Madihally and Matthew, 1999]. Furthermore, chitosan contains hydroxyl and amine groups, and therefore has the potential to cross-link with other useful biomaterials, such as poly (vinyl alcohol) [Chuang et al., 1999] and rayon [Yunlin et al., 1998]. Chitosan cross-linked with collagen and polyvinyl pyrrolidone has been used as matrices in skin regeneration [Shahabeddin et al., 1990; Risbud et al., 2000]. Park et al. [2000] also showed that platelet-derived growth factor incorporated chitosan sponge has the ability to induce better attachment and higher proliferation rate of osteoblastic cells in-vitro. Lahiji et al. [2000] seeded human osteoblasts and chondrocytes on chitosan films cast onto plastic cover slips and showed that the films support the expression of collagen type I in the osteoblast cultures and collagen type II in the chondrocyte cultures. Various groups have shown that chitosan appears to be a suitable matrix material for tissue engineering applications. 3.2.2 The Properties of Chitosan Chitosan is the product of the partial deacetylation of the naturally occurring polysaccharide chitin, which is found widely in nature as the skeletal material of crustaceans. It has a chemical structure similar to chitin, but it is less produced in nature compared to chitin. Chitosan has been proposed as a biomaterial because it has 30 Chapter 3 Materials and Methods been shown to be non-toxic [Chandy and Sharma, 1990; Rao and Sharma, 1997], biodegradable [Hirano et al., 1989] and possesses good biocompatibility properties [Lee et al., 1995; Shigemasa and Minami, 1996]. Figure 3.1 Structure of chitosan Because of the stable, crystalline structure, chitosan is normally insoluble in aqueous solutions above pH 7. However, in dilute acids, the free amino groups are protonated and the molecule becomes fully soluble below pH 6.5. The pH-dependent solubility of chitosan provides a convenient mechanism for processing under mild conditions. Viscous solutions can be extruded and gelled in high pH solutions or baths of non-solvents such as methanol. Such gel fibers can be subsequently drawn and dried to form high-strength fibers [Hirano, 1996]. Structurally, chitosan is a linear polysaccharide. In general, these natural polymers have been found to evoke a minimal foreign body reaction. Formation of normal granulation tissue appears to be the typical course of healing and chronic inflammatory response does not develop. The effects of chitosan and chitosan fragments on immune cells may play a role in inducing local cell proliferation and ultimately integration of the implanted material with the host tissue. In vivo, chitosan is degraded by enzymatic hydrolysis. The degradation products are chitosan oligosaccharides of variable length. The degradation kinetics appears to be inversely related to the degree of crystallinity, which is controlled mainly by the degree of deacetylation [Shigemasa and Minami, 1996]. 31 Chapter 3 Materials and Methods One of chitosan’s most promising features is its excellent ability to be processed into porous structures for use in cell transplantation and tissue regeneration. Freezing and lyophilizing chitosan acetic acid solutions can form porous chitosan structures. A number of researchers have studied chitosan-based scaffolds in various tissue engineering applications [Onishi and Machida, 1999; Eser et al., 1998; Elcin et al., 1999; Madihally and Matthew, 1999; Sechriest et al., 2000; Janeen and Angela, 2000]. For future applications it is important that the chitosan-scaffold meet mechanical properties to withstand in vitro and in vivo forces in order to provide seeded cells with a proper biomechanical environment. Hence, the scaffold should be able to withstand the contraction forces of blood clot, tissue reaction and finally tissue formation as well as remodeling. For example, development of chitosan-based matrix that can support chondrogenesis may be significant not only in terms of the quality of tissue produced, but also in terms of the ability of that tissue to integrate with the host matrix. 3.2.3 The Protocol of Chitosan Gel Preparation In this research, chitosan was used as the scaffold material. High-purity and high-viscosity chitosan with a deacetylation degree of more than 80% was obtained from CarboMer, Inc, Washington, USA. Sodium Hydroxide Pellets were obtained from J.T. Baker, Phillipsburg, NJ, USA. Chitosan is insoluble in water due to its stable crystalline structure and has to be prepared by dissolving the chitosan powder in acetic acid. All chitosan powder is soluble at pH lower than 6.5 to form viscous solutions [Tachibana et al., 1988]. The percentage of acetic acid used for dissolution ranged from 1.2% (v/v) to 5% (v/v) in 32 Chapter 3 Materials and Methods water. In this research, tests done showed that 2%v/v acetic acid was high enough to dissolve the chitosan powder completely to form transparent chitosan gel, so 2% (v/v) was used in this case. The chitosan gel was prepared by dissolving 3%w/v chitosan in 2%v/v acetic acid. The mixture was magnetically stirred for 2 hours at room temperature in a beaker, the mouth of which was covered with a film during stirring. The reason for covering the beaker was to prevent evaporation during stirring, which would change the material concentration. The chitosan gel was then filtered and placed into a vacuum oven for half an hour to remove air bubbles introduced into the gel during stirring. This step is of utmost importance as the viscous gel must be completely bubble-free for consistent extrusion. 3.2.4 Preparation of Sodium Hydroxide Solution The NaOH solution was prepared by 8%v/v NaOH and 100% high-grade ethanol mixed in a ratio of 7:3 in this study. These solutions were prepared fresh just before fabricating the scaffolds. 3.3 Scaffold Fabrication A micro-syringing robotic system (RPBOD) with multiple-dispensr head built in-house was used for this project. The prepared chitosan gel was contained in one of the plastic syringe barrel and dispensed by pressurized purified air. Purified air was used for this purpose to ensure a clean environment. NaOH solution was used as coagulation and dispensed via another syringe using a motorized plunger. To fabricate a scaffold, the chitosan and NaOH are sequentially dispensed in each strand pass. The chitosan fluid is extruded and allowed to contact the base. The coagulation medium (NaOH) followed closely after the chitosan spread out. The two 33 Chapter 3 Materials and Methods materials react to precipitate into a strand. As shown in Figure 3.2, the tip on the right dispenses the chitosan and that on the left dispenses the NaOH solution, positioned approximately 5 mm apart. The dispense sequence is from left to right, and the nozzles are individually timed to dispense only when over the same section. In the dispensing process, the chitosan gel is dispensed as the dispenser moves (from left to right), leaving the chitosan gel on the base. Immediately following, the mechanical dispenser drops the NaOH solution to precipitate the chitosan gel. Motion NaOH NaOH tip clears the strand by 0.1~0.2mm Chitosan Chitosan tip clears the surface by 0.1~0.2mm Figure 3.2 Dual dispensing method After the first layer, the base is rotated by 90 degrees and the dispensers are lifted to a higher level that allows for the chitosan gel for the next layer to lie on the previous layer. The robot then generates the second layer and the scaffold is progressively built as layers are sequentially generated in this manner. 3.4 Washing Protocol Once the scaffold was made, it was rinsed three times with distilled water and left in 100%, 70% ethanol sequentially for 30mins each and subsequently washed three times with distilled water. Finally, the scaffold was left in deionized water in the refrigerator overnight. This was a precautionary step to leach out any residual NaOH solution. The excess water was drained and the scaffold was left in the oven at 400C for 2 hours. This step was taken because previous experiments indicated that the 34 Chapter 3 Materials and Methods material properties of the scaffolds were improved by heating before freeze-drying [Sultana, 2002]. The washed scaffold was then transferred to a freezer and frozen for at least 8 hours. After that, the frozen scaffold was transferred to a freeze-drier and dried for 2 days. 3.5 Scaffold Characterization Besides material issues, the macro- and micro-structural properties of the scaffold are also very important [Cima et al., 1991; Wake et al., 1994]. In general, the scaffolds require individual external shape and well defined internal structure with interconnected porosity. Ideally, a scaffold should have the following characteristics: [Thomson et al, 1995; Hutmacher, 2001] (1) be highly porous with an interconnected pore network for cell growth and flow transport of nutrients and metabolic waste; (2) have suitable surface chemistry for cell attachment, proliferation, and differentiation; (3) possess mechanical properties to match those of the tissues at the site of implantation; (d) be easily fabricated into a variety of shapes and sizes and (e) possess interconnecting porosity so as to favor tissue integration and vascularity. 3.5.1 Porosity The pore structure is a very important parameter of the scaffold structure because it determines permeability, mechanical properties and cell growth. The ratio of scaffold apparent volume (V), which means the volume of the whole scaffold 35 Chapter 3 Materials and Methods including the inside pores, to scaffold true volume(V’), which means the volume of the scaffold materials was used to calculate the porosity (ε). The porosity was calculated by the following method [Hutmacher et al., 2000]: (1) Measuring the weight (m) and apparent volume (V) of each sample. (2) Calculating from these measurements, the apparent density of the scaffolds ρ* = m / V (3.1) (3) Using the formula ε= 1 - ρ*/ρ × 100%. (3.2) The protocol used for calculating the apparent and true density of scaffold was proposed by Zhang et al. [2001]. To obtain the volume of the scaffold, a sample of weight W was immersed in a graduated cylinder containing a known volume, V1, of ethanol. In this method ethanol was used as the displacement liquid because it penetrated easily into the pores and did not induce swelling or shrinkage [Xiong et al., 2001]. The scaffold sample was kept in the ethanol for 5 minutes. Then the graduated cylinder was put into vacuum oven for half an hour and then taken out. These evacuation and re-pressurization cycles were continued until no air bubbles emerged from the scaffolds and this volume was taken as V2. The volume difference (V2-V1) was taken as the true volume of the scaffold (V’), which is the volume of the scaffold material. The soaked scaffold was then removed and the residual ethanol volume was recorded as V3. Because the ethanol had entered into the pores of scaffold and was removed with the scaffold, the volume difference (V1-V3) can be considered as the volume of the pores of scaffold. Then the scaffold apparent volume (V), which means the volume of the whole scaffold including the inside pores, can be calculated by (V2V1) plus (V1-V3), as shown by equation (3.3). The true density (ρ) and apparent 36 Chapter 3 Materials and Methods density (ρ*) were calculated using equations (3.4) and (3.5), respectively. Porosity of the scaffolds was calculated using equation (3.6). Apparent Volume, V= (V2 - V1) + (V1 – V3) = V2 – V3 (3.3) True Density, ρ = W / V’ = W/ (V2 –V1) (3.4) Apparent Density, ρ* = W/ V =W / (V2-V3) (3.5) Porosity, 3.5.2 ε =1 - ρ*/ρ× 100% = (V1 –V3) / (V2 – V3) × 100% (3.6) Morphology SEM (scanning electron microscope) is a popular technique used in the analysis of surface topography of materials and the interaction between the substrate and cells. Specimens to be viewed with SEM need to be dried and fixed. The freeze-dried chitosan scaffolds were mounted on an aluminium specimen holder with double sided carbon tape and observed under a Jeol JSM-5800LV (JEOL USA, Peabody, MA) environmental scanning electron microscope (ESEM) at 15V to assess the gross morphology and microstructures. 3.5.3 Mechanical property Compression tests were conducted with an Instron Microtester material testing system (Instron, MA, USA) using a 10N load cell to find out the load bearing capacity of the scaffolds. The compressive modulus and failure point were evaluated from the stress–strain graph. A sample set of three scaffolds, which were cut into 8x8x3 mm cuboids, was used for the compression test. The test was conducted four times using different sample sets and the average value was calculated. 37 Chapter 3 Materials and Methods Scaffold Figure 3.3 Instron Microtester A typical stress-strain curve for a biological material is shown in Figure 3.4 [Mow. et al., 1997]. Ei stands for the Initial Compressive Modulus, while Ef stands for the Final Compressive Modulus. Ei and Ef can be obtained from the stress-strain curve by calculating the slops of the two tangents of the curve at the initial and final point, ε-f is the intersection point of the tangents. Since the scaffolds have the structure of foam, they do not have a particular fracture point like that of a crystalline Stress material. ε-f is taken to be the failure point in such a case. ε-f Strain Figure 3.4 Typical stress-strain curve of a biological material 38 Chapter 3 Materials and Methods 3.5.4 Biocompatibility Preliminary evaluation of scaffold biocompatibility was conducted by in-vitro culture studies. The cell seeding protocol is described below. The lyophilized scaffold was left in 100% ethanol for 1 hour to rehydrate the scaffolds. Then it was cut into 8x8x3mm cuboids and immersed in 70% ethanol followed by 50% ethanol each for 1 hour of continued rehydration of the scaffold. The scaffolds were finally washed three times with phosphate buffer solution (PBS) to equilibrate the pH of the scaffolds. The scaffolds were then left in the sterile hood for 3 hours to dry. This was done to ensure that all the ethanol had evaporated off as ethanol retention could kill the cells. The scaffolds were left overnight in sterile PBS. The following day, the scaffolds were left overnight in M199 culture medium for osteoblasts/bone cells at 370C in a self-sterilizable incubator. Finally, the scaffolds were left to dry for two days. This was done to help the cells attach better onto the scaffolds. Just before seeding, the scaffolds were dampened with one drop of medium. The scaffolds were transferred into the bottom of 12-well plate. Porcine osteoblasts (Passage 3) were lifted from the bottom of a culture flask by 0.25% trypsin, and the cell density was adjusted to 1x105/ml by medium. Into each well 2ml of cell suspension was added, and the plate was transferred into the cell incubator. 24 hours after cell seeding, medium was removed from the wells and the specimens were rinsed with PBS (pH7.4) for three times. Then the specimens were fixed with 3.7% formaldehyde and dehydrated in a graded ethanol series of 70%, 80%, 90% and 100% for 10 minutes each time and left to dry at room temperature. Once dry, the specimens 39 Chapter 3 Materials and Methods were left in an oven at 400C to remove the excess moisture. The specimens were then coated with gold and observed under scanning electron microscope at 10 kv. 40 Chapter 4 The Fabrication Process Chapter 4 The Fabrication Process 4.1 Biomedical RP A general framework for the application of rapid prototyping in the area of tissue engineering is shown in Figure 4.1 [Landers et al, 2002]. A specific area of the patient is scanned by computer tomography (CT) or magnetic resonance (MR) and the data are imported into a CAD software. The scaffold is designed according to the individual requirements using the CAD software, and the post-processed data for the fabrication of the scaffold is then transferred to a RP system to produce the scaffold with a biocompatible and biodegradable material. Living cells are seeded onto the surface of the scaffold during or after the RP process. When the cell number increases following cell culture treatment, the scaffold is implanted into the human body and eventually replaced by natural tissue. 4.1.1 3D Plotting A new rapid prototyping (RP) technology was reported as 3D plotting [Landers et al., 2002] to meet the demands for desktop fabrication of hydrogel scaffolds. The prototype of the 3D plotting was built on a CNC-milling machine tool for 3D positioning of a dispenser. It placed the dispensing process into a bath of a liquid with matched density. The resulting gravity force compensation allowed the dispensing of low-viscosity plotting materials and widened the plotting material spectra. Therefore a very large variety of materials could be processed, including melts, pastes, reactive resins or hydrogels. 41 Chapter 4 The Fabrication Process Figure 4.1 A framework of biomedical RP [Landers et al, 2002] The basic principle of the 3D plotting is illustrated in Figure 4.2. In this project, a desktop rapid prototyping (RP) system was used to fabricate scaffolds using a principle very similar to 3D plotting (Figure 4.3). Previous project demonstrated the potential of the RPBOD system in fabricating 3D scaffolds with regular and reproducible macropore architecture [Ang et al., 2002]. 42 Chapter 4 The Fabrication Process z y Moving directions of the dispenser Plotting medium x Plotting material Figure 4.2 Basic principle of 3D plotting Figure 4.3 RP dispensing system Although this 3D-plotting process of dispensing plotting material into a fluid medium has the advantages such as buoyancy support for the scaffold as it forms, a main disadvantage of the method is the high sensitivity to material concentration. In the process, the coagulation started at the gel/coagulation medium interface immediately upon exposure. When the gel-like precipitate was formed too quickly due to the high concentration of the fluid medium, there was a tendency for a coagulated lump to form at the nozzle. The plotting material became coagulated before it contacted the base layer, resulting in poor adhesion and failure to hold the strands down as they were dispensed, eventually leading to further clumping and dragging. Conversely, when the coagulation was too slow as concentration of the fluid medium decreased, the dispensed strands did not precipitate fast enough to hold its shape in the fluid medium as it tended to spread out, resulting in the merging of adjacent strands. Slow coagulation would also cause the possible dragging of material because the dispensed material spread into the path of the subsequent parallel strands that were to be built. 43 Chapter 4 The Fabrication Process To eliminate the high sensitivity to medium material concentration and improve the scaffold fabrication process, the RPBOD system was improved for a new manufacture method, referred to as dual dispensing. A multiple-dispenser and a rotary base were added to the RP dispensing system (Figure 4.3) to develop a four-axis multiple-dispenser robotic system. 4.2 The Rapid Prototyping Robotic Dispensing System The rapid prototyping system shown in Figure 4.4 for the fabrication of scaffolds is a four-axis multiple-dispenser robotic system (RPBOD) based on the Sony Robokits. It is capable of three simultaneous translational movements along the X-, Yand Z-axis with an added rotary motion about the Z-axis. The three translational movements have positioning accuracy of up to 0.05mm and a minimum step resolution of 0.014mm. X-axis Z-axis Y-axis Rotation about Z-axis Figure 4.4 The four-axis RPBOD system 44 Chapter 4 The Fabrication Process There are two kinds of dispensing mechanisms: pneumatic and mechanical dispensing (Figure 4.5). The pneumatically driven syringe dispenser is controlled by a solenoid-operated pneumatic valve. The mechanical dispenser is controlled by a plunger driven by a stepper motor. By controlling the displacement of the plunger, the dispensing rate can be precisely regulated, particularly at very low flow rate (such as 0.5 µl/sec). Compressed air Stepper motor Threaded rod Plunger Syringe Nozzle Tubing Clamp Adapter Assembly Syringe Nozzle Figure 4.5 Mechanical & pneumatic dispenser 4.2.1 The Control Software The SONY RoboKids is a low-cost microprocess-based desktop robot for low payload. As a platform for RP dispensing system, it has been developed to be controlled by a PC through a serial port connection, with a developed user interface software to ease the operation. The Robokids has three levels of codes responsible for controlling it, from the machine codes -- LUNA Programming Language and RoboDLL, which is the 45 Chapter 4 The Fabrication Process communication interface, to the user interface -- RPBOD software. Robokids is programmable via the Sony’s Robotic LUNA Language. LUNA commands are used to run the various functions of the robot. There is a special library of standard command functions in the SONY Robokids Programming Manual which allow the user to control the robot. LUNA also allows user-specific functions to be created externally. RoboDLL is the second level of codes, which is a communication interface to allow the use of VC++ written program to call the LUNA functions. The RPBOD software is the graphic user interface software written in Visual C++ to control the fabrication system. The fabrication technique offered by the RPBOD allows the generation of geometric data of 3D scaffolds through the control software. The geometric data are generated by using its slicing function to segment the 3D computer model to generate sliced layers to fabricate free-form customized scaffolds layer-by-layer. Additionally, to build simple shape scaffolds, like cubic ones, the geometrical data of the scaffold can be generated by simply specifying the required control parameters directly at a user-friendly dialog box provided by the RPBOD software. 4.3 Cubic Scaffold Fabrication Process Figure 4.6 shows a part of the fabrication controls to build regular-shape scaffolds. To fabricate simple cubic scaffold, the length and width of the scaffold is set from the scaffold dimensions control box (Figure 4.6). Other control parameters such as number of layers, dispensing pressure, dispensing speed and height increment of each layer etc. are also set in this dialog box. 46 Chapter 4 The Fabrication Process Length and width of the cubic scaffold Number of layers of the scaffold 3D Scaffold Dispense Control Dialog _________________________________________________________________________________ Other parameters as dispensing speed and height increment of each layer etc Figure 4.6 3D scaffold fabrication controls The rotation centre of the base should be calibrated accurately to allow the edges of a layer to fall neatly over the previous layer, such that the cubic scaffold is able to maintain its shape. This is possible because the orientation is 90 degree to each other. Once the start point is set properly according to the rotation centre, the ‘edge wrapping’ of the scaffold is obtained, which is possible only if both the start and end 47 Chapter 4 The Fabrication Process of the dispensed strand extend very slightly over the previous layer. Improper calibration of the centre will result in ragged edges of the cube. The position of the two dispenser tips must also be properly set. The tip of the NaOH dispenser must be set so that it just clears the chitosan strand in order for the NaOH droplet point to continuously contact the strand as it dispenses. Figure 4.7 shows the position of the two tips. Pneumatic dispenser for Chitosan gel Mechanical dispenser for NaOH solution Figure 4.7 Tips position There are other process control parameters to be set for the fabrication. Among these parameters, dispensing pressure, dispensing speed and height increment of each layer have significant effect on the quality of the built part. An optimized set of parameters was picked out by single-layer experiments carried out initially. In this research, a pressure of 2 bars, a dispensing speed of 6mm/s and a height increment of 3.5mm were selected. Figure 4.8 shows the process of scaffold fabrication, referred to as the dual dispensing method. During the dispensing process, the pneumatic dispenser moves form left to right, dispensing the chitosan gel. The following mechanical dispenser delivers the NaOH solution immediately to precipitate the chitosan gel. The strands are left on the base as the dispenser moves. 48 Chapter 4 The Fabrication Process (a) (b) (a) (c) (b) (c) Figure 4. 8 Scaffold fabrication process by dual dispensing (a) Fabrication of first layer; (b) Start of dispensing for second layer; (c) Scaffold building layer by layer. After the first layer, the base is rotated by 90 degrees and the dispensers are lifted to a higher level that allows for the chitosan gel for the next layer to be laid on the previous layer. The robot then generates the second layer similarly (Figure 4.8b) and the scaffold is progressively built as layers are sequentially generated in this manner (Figure 4.8c). 4.4 3D Free-form Scaffold Fabrication A key advantage of the RP technology is its ability to produce complex 3D shape from a given computer model. To fabricate 3D free-form customized scaffold by the RP system, a CAD model is needed. One important step in the generation of CAD model is reconstructing images taken from CT/MR into 3D model of significant complexity and converting highly detailed medical imaging data to the data format that can be recognized by the rapid prototyping system. The accuracy and facility of this data conversion critically affect the overall utility of the scaffolds. Now several kinds of software in the market can act as the visualization medium to capture morphological data on a biological structure and to process such 49 Chapter 4 The Fabrication Process data by a computer to generate the code required to manufacture the structure by a rapid prototyping equipment. In this work, the Materialise’s Interactive Medical Image Control System (Mimics) software was investigated for its ability of processing medical data. 4.4.1 Investigation of Mimics Mimics is an interactive tool for the visualization and segmentation of CT/MRI images as well as 3D rendering of objects. A very flexible interface to rapid prototyping or to CAD systems is included in the software for building distinctive segmentation objects. Figure 4.9 indicates the flowchart of Mimics. MedCAD and CTM are two modules of Mimics software. CT/MR Scanner MedCAD Interface to CAD CAD Systems CTM Model Generation Rapid Prototyping Mimics Figure 4.9. Mimics flowchart MedCAD interfaces from the medical scan data to surface files, which are directly usable for the design of custom made prosthesis in any CAD system. To verify the CAD design, the CAD 3D model can be imported into MedCAD (as an STL file). The implants can be visualized in 2D cross sections on the images as well as in 3D model. MedCAD is designed to make a bridge between medical imaging (CT/MRI) and CAD design. This means that it can export data from the imaging system to the CAD system and vice versa. 50 Chapter 4 The Fabrication Process Interpretations are made on the images in MedCAD, taking into account all the gray value information (soft tissue, different types of bone, tendons, etc.). On the images, basic features can be recognized, and converted to geometric entities (basic axes, reference curves, anatomical landmarks). Only the surfaces, which are needed to make a fit, are actually converted to B-spline surfaces. The CAD design can also be imported in the MedCAD system via the STL interface. The designs are visualized in 2D sections together with the actual images, or in 3D shaded representations, with the anatomical data in a transparent mode. With this method, it is possible to bridge the images and the CAD system in a very short time. CTM interpolates the medical slice data into very thin layers, and interfaces directly with most rapid prototyping systems. After visualization of the image data that are imported into Mimics, the data can be interfaced directly with most rapid prototyping systems by CTM. Because of this direct interface and because of the higher-order interpolation mathematical algorithms, such as Bilinear and C-Spline functions, it produces the most accurate models in a very short time. As the pixel size of the input images can vary from only 0.2mm to more than 1mm, a resolution enhancement technique is necessary when creating the RP models so as to minimize the effect of stair-stepping, and to retain the natural curvature of the surface. Two techniques are used to increase the resolution of the contour: a bilinear interpolation increases the in-plane resolution of the contours with a factor of up to 100; and an inter-plane linear interpolation of cubic alpine convolution is performed to decrease the slice thickness of the data set. A slice thickness of 1 to 3mm is very common for the input images, whereas the slice thickness used on the RP machines is normally less than 0.25mm. 51 Chapter 4 The Fabrication Process Before performing segmentation of the anatomy, the data is loaded into Mimics and converted into the proprietary data format of Mimics. The image is processed by applying suitable threshold value to differentiate regions of interest. The interested tissue can be separated from other tissues by setting a fixed threshold value. Once visualized, the file is interfaced with CTM. CTM uses a higher-order interpolation algorithm to interpolate the slice data of the scan images to generate vertices and triangulated surfaces from the 3D models for rapid prototyping machine. The format exported is specific and dependent on the RP system, which includes the Standard Triangulation Language (STL), Initial Graphic Exchange Specification (IGES), Standard for the Exchange of Product Model Data (STEP), Common Layer Interface (CLI) and Virtual Reality Modeling Language (VRML), etc. 4.4.2 Data processing Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) systems are the two most commonly used medical scanning systems. Through CT or MRI scan, a series of digitized gray-scale slice images of the scanned body is obtained. The purpose of the data processing is to produce 3D reconstructions of objects directly from the medical data and to convert the medical data to the data that can be processed by rapid prototyping systems. This involves separating the data of the tissue of interest from the scan data sets, or generating a certain part of the tissue from the available data. In some cases, the missing part of the tissue is extracted to create the implant for the scaffold building. Once certain tissue part is separated or created, it can be converted into data formats that are compatible with RP systems. The MIMICS software can also generate high-resolution 3D renderings in different colors directly from the slice information. Contrast enhancement can be carried out interactively to improve the model. Culling of the “unnecessary” parts or 52 Chapter 4 The Fabrication Process unwanted noise is achieved with sophisticated dimensional selection and editing tools. Mimics also provides several segmentation techniques for processing different kinds of images or structures to extract interested objects. Among these techniques, segmentation by thresholding can be used for segmentation in CT images; segmentation by region growing permits the user to split an object of interest into several distinctive parts; recovering an ROI (Region Of Interest) makes it possible to recover the original (thresholded) segmentation area within specified area; segmentation into masks can create up to 16 different segmentation objects in one session; cavity fill can perform 2D or 3D fill functions. And the Boolean operations allow making all different kinds of combinations based on two masks. It is a very useful tool to reduce the work that needs to be done when separating two joints. In this research, a 3D computer model is derived from the CT scanned images using the Mimics software. Figure 4.10 shows the model of a skull generated from its CT scan images by Mimics. Nearly a hundred of the CT grey value images were imported into Mimics and converted into Mimics’ proprietary data format. The bone was separated from other soft tissues by setting a suitable threshold value. Based on the reconstructed 3D computer model of the skull, a model of a patch has also been interactively created that can fill the hole on the skull by using several editing and segmentation tools, such as drawing, erasing and region growing, provided by Mimics software. 53 Chapter 4 The Fabrication Process Mimics 3D computer models of the skull and the patch CT images of a skull with a hole Figure 4.10 The conversion of CT images to 3D computer mode by Mimics. 4.4.3 Building Free-form Scaffold The model reconstructed by Mimics (Figure 4.10) was transferred in STL format to the RPBOD system. Figure 4.11 shows the model displayed on the monitor of the RPBOD. The RPBOD software’s slicing program provides users with interfaces to do the following: 1) Open and read files in STL format; 2) Display both the STL model as well as the sliced results with some of its 3D operations (e.g. layer display, fit display, rotate and translate); 3) Generate sliced layers in the +Z-direction based on user-defined scan and slice parameters (i.e. scan pitch (x, y) and layer thickness respectively); and 4) Generate raster scan pattern data into RP slicing files (CLI format). 54 Chapter 4 The Fabrication Process Figure 4.11 Model of skull defect patch shown on the RPBOD monitor The model was appropriately rotated before slicing in the Z-direction. The information of these layers was saved as CLI file, which is a simple, efficient and unambiguous format for data input to fabricate the model layer-by-layer. Figure 4.12 show four consecutive scanned layers. The direction of the scan lines is set to intersect that of the preceding layer at 90 degrees. Hence, the built strands crossed at each layer to form the scaffold. The quality of built scaffold depends on the characteristics of the materials and experimental conditions, including the concentration, dispenser speed, and dispensing rate. 55 Chapter 4 The Fabrication Process Figure 4.12 Four consecutive layers with scan lines The built part shown in Figure 4.13 is based on the model shown in Figure 4.11 and indicates the potential of the system to build customized free-form scaffold from computer model. Parameters, such as strand distance and layer height, also have significant effect on the quality of the built part. Figure 4.13 Chitosan scaffold of the patch built by RPBOD (15 layers) 56 Chapter 5 Results and Discussion Chapter 5 Results and Discussion 5.1 Results The chitosan scaffolds built by this dual dispensing method exhibited excellent uniformity and strength. This method had practically eliminated the occurrence of edge curling, the primary cause of strand dragging in chitosan scaffolding fabrication process. Edges of scaffolds are also well defined and good surface uniformity of the top layer is maintained (Figure 5.1). The process has good reproducibility, once properly calibrated [Tan, 2002]. Figure 5. 1 Freshly built chitosan scaffold and the air-dried scaffold under optical microscope (15X) shows the uniformity of the pores Greater flexibility and advantage can be achieved with the method of dual dispensing with different dispensers to suit the nature of the fluid to be dispensed. The pneumatic dispenser extrudes the viscous gel through a small diameter (0.1 ~ 0.2 mm) 57 Chapter 5 Results and Discussion needle at a pressure from 2 to 4 bars, depending on the dispensing rate and the size of the needle. However, when the solution is of low viscosity, it flows in an uncontrollable way. Therefore the pneumatic dispenser is not suitable for the dispensing of low-viscosity solution, such as NaOH solution. On the other hand, the mechanical dispenser can achieve dispensing of low-viscosity fluids at low flow rate of 0.5µl/sec. Thus, the mechanical dispenser was used to dispense NaOH solution in the study. Moreover, in the case of single dispensing of one solution into a container of another solution, there are the problems of gradual lowering of concentration and agitation of the solution in the container. These problems are eliminated in the dual and sequential dispensing of the solutions. Additionally, improved adhesion is achieved and the operating speed is also improved since agitation of the solution that is dispensed into is not a problem. To characterize the built scaffolds, the macro-porosity and density of the scaffolds were measured following the method described earlier in chapter 3. The overall porosity of the freeze-dried scaffolds was about 90%, which is desirable to provide high surface area for cell polymer interactions [Freed et al., 1997]. The calculated apparent density of the freeze-dried chitosan scaffold was about 0.06g/ml. Figure 5.2 shows the morphology of the scaffold under ESEM at 15v. Macropore diameters of 300-500 µm were observed which was desirably for cell growth into scaffolds [Lu and Mikos, 1996]. 58 Chapter 5 Results and Discussion Figure 5. 2 ESEM picture of the surface morphology of the freeze-dried chitosan scaffold The stress-strain curves shown on Figure 5.3 were obtained from the compression testing results using the Instron Microtester, which was described in chapter 3. The values of initial compression modulus (referred to as Ei), and final compression modulus (referred to as Ef) were calculated from the test to evaluate the mechanical properties of the scaffolds. Dry Scaffolds Curve tangent Wet Scaffolds ε-f Curve tangent Strain Figure 5.3 Stress-Strain curves of scaffolds 59 Chapter 5 Results and Discussion The stress-strain curve of the wet scaffold is quite similar in shape to the typical stress-strain curve for a biological material (refer to Figure 3.4). The curve was obtained by compression test using the Instron Microtester with results of over a few hundred points. The initial compression modulus (Ei) and final compression modulus (Ef) can be obtained from the stress-strain curve by calculating the slops of the two tangents of the curve at the initial and final point, as shown in the Figure 5.3. The Ei and Ef for wet scaffold are about 0.013 + 0.002 MPa and 0.051+ 0.012MPa, while for dry scaffolds they are about 0.11+ 0.06 MPa and 0.28+ 0.06MPa. The failure point for the wet scaffolds is at about 0.5. The fracture point of the dry scaffolds is very low at about 0.25. This is due to the highly porous nature of the scaffolds. The strands began to break very soon after being subjected to load. It should be noted that the values of Ei and Ef of the chitosan scaffolds are generally very low and that these scaffolds should not be considered for load bearing applications, such as being part of knee or hip prosthesis that undergoes heavy compressive stresses. Fillers and additives for the chitosan gel may improve the material properties during fabrication. The incorporation of bioceramics such as hydroxyapatite (HA) into chitosan scaffolds to improve mechanical properties has also been reported [Ito et al., 1999; Yamaguchi et al., 2000]. Preliminary evaluation was made by in-intro studies on the cell compatibility and attachment of the fabricated scaffolds. The scaffold was seeded with porcine osteoblasts (Passage 3). Adhesion and distribution of cells was studied via the SEM. 60 Chapter 5 Results and Discussion (a) (b) Figure 5.4 SEM image shows cell compatibility and attachment. (a) shows cells (circled) attached on the scaffold. (b) osteoblasts adhered and spread well on the surface of the scaffold and kept the initial shape of polygonal Figure 5.4 is the SEM picture taken 24h after seeding. The cells can be seen to be attached on the scaffold surface in Figure 5.4(a) (indicated by the cycle), which is 120 times enlarged. Though the amount of the cells is not large, it is acceptable considering the short time after seeding. Figure 5.4(b) is a closer view that is enlarged by 900 times. On this picture, osteoblasts can be observed adhering and spreading well on the surface of the scaffold and the initial shape of polygonal is kept, which indicated the good biocompatibility of the scaffold. 61 Chapter 5 Results and Discussion 5.2 Discussion 5.2.1 The Requirements for Tissue Engineering Scaffolds In order to provide a temporary substrate to which transplanted cells can adhere, proliferate and differentiate, both scaffold chemistry and architecture are important. The scaffolds for tissue engineering are supposed to be implanted in vivo which confines the viable scaffold materials to those that are non-mutagenic, nonantigenic, non-carcinogenic, non-toxic, non-teratogenic and possess high cell/tissue biocompatibility as described in chapter 3. Besides the requirements for materials, 3D scaffolds require certain macroand micro-structural properties in order to provide a suitable base for cell growth, apart from providing mechanical structure. The following have been identified as essential scaffold macro and micro-structural properties critical for rapid cell growth [Freed et al., 1994, Grande et al., 1993]: 1) Macrostructure: A temporary 3D scaffold that mimics the physiological functions of the native ECM (extra cell matrix) is vital to maintaining the cells’ ability to express their native differentiated phenotypes. An optimal scaffold design will promote cell proliferation and cell-specific matrix production that would eventually take over the supporting role of the degrading scaffold. 2) Porosity and pore interconnectivity: Scaffolds must possess an open-pore geometry with a highly porous surface and microstructure that allows cell in-growth and reorganization in vitro and provides the necessary space for neovascularization from surrounding tissues in vivo. The highly porous microstructure with interconnected porous networks is critical in ensuring spatially uniform cell 62 Chapter 5 Results and Discussion distribution, cell survival, proliferation and migration in vitro [LeGeros et al., 1995]. The scaffold’s porosity (exceeding 90%) and degree of pore interconnectivity directly affect the diffusion of physiological nutrients and gases to and the removal of metabolic waste and by-products from cells that have penetrated the scaffold [Vacanti et al., 1988, Mikos et al., 1993]. In larger scaffolds, high porosity and pore interconnectivity will enable the use of bioreactors for creating hydrodynamic microenvironments with minimal diffusion constraints that closely resemble natural interstitial fluid conditions in-vivo for achieving large and well-organized cell communities [Hutmacher, 2000]. 3) Pore size: The diverse nature of tissue architectures requires different microenvironment for their regeneration that includes the employment of scaffolds with optimal pore sizes. In regenerating bone tissues in vitro, some researchers [Robinson et al., 1995, Boyan et al., 1996] indicated the need for pore sizes ranging from 200 to 400 µm, while Yoshikawa et al. [1996] successfully employed scaffolds with 500 µm nominal pore size. Scaffolds with pore sizes between 20 and 125 µm have been used for regenerating adult mammalian skin [Yannas et al., 1989] and 45– 150 µm for regenerating liver tissues [Kim et al., 1998]. When the pores employed are too small, pore occlusion [Rout et al., 1988] by cells will prevent cellular penetration and matrix elaboration within the scaffolds. 4) Mechanical properties: As templates to guide tissue regeneration, scaffolds should have sufficient mechanical strength during in vitro culturing to maintain the spaces required for cell in-growth and matrix elaboration [Brekke, 1996]. In regenerating load-bearing tissues (e.g., cartilage, bone), additional issues relating to the scaffolds’ mechanical properties would have to be resolved [Mikos et al., 1993]. To allow early mobilization of the treated site, the degradable scaffold should retain 63 Chapter 5 Results and Discussion sufficient mechanical strength to manage any in vivo stresses and physiological loadings imposed on the engineered construct. The scaffolds’ degradation must be tuned appropriately such that it retains sufficient structural integrity until the newly grown tissue had replaced the scaffolds’ supporting function [Mikos et al., 1993]. 5.2.2 Scaffold Fabricated by RPBOD System Since the scaffolds fabricated with the RPBOD system are to be applied in TE, they should satisfy the requirements for TE scaffolds. First of all, the biocompatibility of chitosan fulfils the requirement for the scaffold materials. Moreover, the 3D scaffolds show the potential for TE application by possessing the following macroand micro-structural properties: 1) Macrostructure: The 3D scaffold was fabricated with designed shape and interconnected pore structure, which mimics the physiological functions of the native ECM (extra cell matrix). 2) Porosity: The overall porosity of the freeze-dried scaffolds was about 90%, which is desirable to provide high surface area for cell polymer interactions. 3) Pore size: The scaffold has macro-pore diameters of 300-500 µm, which was desirable for cell growth into scaffolds. 4) Mechanical properties: Due to the highly porous nature of the scaffolds, the value of failure point for the chitosan scaffolds is generally very low. It is suitable in low load bearing applications. The mechanical properties may be improved by the incorporation of bioceramics into chitosan scaffolds. 5.2.3 The Requirements for Scaffold Fabrication Techniques Based on the structural pre-requisites listed previously, one major goal in scaffold production is to maintain high levels of accurate control over their macro64 Chapter 5 Results and Discussion (e.g., spatial form, mechanical strength, density, porosity) and micro-structural (e.g., pore size, pore distribution, pore interconnectivity) properties. Although a variety of conventional manual-based fabrication techniques are available for scaffold production [Widmer and Mikos, 1998], most of them were tailored to create scaffolds that will meet the key requirements of specific TE applications and are not usually generic. Besides the fabrication techniques that largely define the scaffolds’ macroand microstructure, there are a large variety of natural or synthetic scaffolding biomaterials to consider. Each scaffolding material or combinations of materials possesses different processing requirements and varying degrees of process ability to form scaffolds. Among the key requirements necessary to assess a fabrication technique for scaffold production are: [Leong et al., 2002] 1) Processing conditions: The material processing procedures and conditions should not adversely affect the material properties and subsequent clinical utility of the scaffolds. As such, a key requirement is that the technique should not change the chemical properties and biocompatibility of the scaffold nor cause any degradation in its mechanical properties. 2) Process accuracy: The technique should be capable of producing spatially and anatomically accurate 3D scaffolds that fit the intended spaces at the implant site. Accuracy of the created pore sizes and morphologies as determined by the user is an advantage of RP technology that is difficult or sometimes impossible to achieve with conventional fabrication. The capability to vary and maintain accurate pore morphologies will enable a wide variety of scaffolds to be created to suit different TE applications. Having accurately constructed scaffolds will enable the application of computer-aided engineering (CAE) methods to perform strength and degradation analyses to accurately predict the scaffolds’ performance. As such, optimized scaffold 65 Chapter 5 Results and Discussion designs can be realized with reduced experimentation. 3) Consistency: The technique should produce scaffolds with highly consistent pore sizes with a narrow size distribution range over their entire volume. In addition, consistency in pore characteristics, morphologies, pore distribution, pore density and interconnectivity in all three dimensions is required in order to produce highly regular 3D structures. 4) Repeatability: Different scaffold batches should exhibit minimal variations in physical forms and properties when produced from the same set of processing parameters and conditions. The technique should allow highly consistent and reproducible results to be achieved with ease. 5.2.4 The Dual Dispensing Method The dual dispensing method provided by the four-axis RPBOD system presented in this research fulfills the requirements for scaffold fabrication techniques as flowing: 1) Processing conditions: The RPBOD system fabricates scaffolds by extruding chitosan gel, which was prepared by dissolving chitosan acetic acid, forced by purified air during process. The material processing procedures do not change the chemical properties and biocompatibility of the scaffold nor cause any degradation in its mechanical properties. Additionally, it does not require heating and can apply reactive components as well as thermally sensitive bio-components into the fabrication process. 2) Process accuracy: The RPBOD system is capable of three simultaneous translational movements along the X-, Y- and Z-axis, which have positioning accuracy of up to 0.05mm and a minimum step resolution of 0.014mm. As a RP 66 Chapter 5 Results and Discussion technology, the RPBOD system is capable of spatially and anatomically accurate 3D scaffolds according to the custom design. The system has the capability to vary and maintain accurate pore morphologies of scaffolds to suit different TE applications. 3) Consistency: The scaffolds fabricated with the RPBOD system showed well- defined external and internal structure. As shown in Figure 5.3, the scaffold exhibited the uniformity of the pores. 4) Repeatability: The process has good reproducibility, once properly calibrated. It ensures that different scaffold batches exhibit minimal variations in physical forms and properties when produced from the same set of processing parameters and conditions. Furthermore, the dual dispensing method embodies the following additional advantages: 1. It can apply a wide variety of polymer hotmelts as well as hydrogel and pastes. Hence, it becomes possible to control the solidification process and the properties of the resulting scaffold with the use of multiple materials. 2. It does not require heating and can apply reactive components as well as thermally sensitive bio-components into the fabrication process. For tissue engineering, this means that the living cells and may be integrated into the scaffold material, enabling precise control and optimal conditions for tissue growth right from the start of fabrication. However, the disadvantages of the technology are the relatively slow processing, and time-consuming adjustment required when a new material is used to build scaffolds. 67 Chapter 5 Results and Discussion The scaffolds fabricated with the RPBOD system showed well-defined external and internal structure, high porosity and cell compatibility. Considering the requirements for TE scaffolds described above, the scaffolds fabricated are potential for TE application. In general, the advantages of the manufacture process and the properties of scaffold demonstrated the potential of the RPBOD system in fabricating 3D TE scaffolds with regular and reproducible macro-pore architecture. 68 Chapter 6 Conclusions and Recommendations Chapter 6 Conclusions and Recommendations 6.1 Conclusions A rapid prototyping robotic dispensing (RPBOD) system was used in this project to fabricate 3D scaffolds for tissue engineering applications. The entire system includes a multiple dispensing head with dual-dispensing tips for chitosan gel and NaOH, a rotary table and a desktop robot and is interfaced to and controlled by a personal computer. Experiments were carried out to build chitosan scaffolds using the dual dispensing methods provided by RPBOD system. The factors and mechanisms that affect the scaffold fabrication process have been investigated. Through a series of experiments, the dispensing parameters and concentrations that yield the best result were identified as a pressure of 2 bars, a dispensing speed of 6mm/s and a height increment of each layer of 3.5mm. A standardized protocol for preparation of the manufacturing materials was also drawn up: to form the chitosan gel, 3%w/v chitosan was dissolved in 2%v/v acetic acid; then the mixture was magnetically stirred for 2 hours at room temperature in a beaker, the mouth of which was covered with a film during stirring to prevent evaporation during stirring; after that the chitosan gel was filtered and placed into a vacuum oven for half an hour to remove air bubbles introduced into the gel during stirring to make the viscous gel be completely bubblefree for consistent extrusion. This protocol was adhered to during the experiments to eliminate problems due to variation of materials. 3D free-from chitosan scaffold fabrication was based on computer model created from CT images, converted and extracted using the Mimics software. This 69 Chapter 6 Conclusions and Recommendations involves obtaining the required geometric data for the scaffold in the form of a solid model from CT-scan images. The extracted scaffold model was then sliced into consecutive two-dimensional (2D) layers by the RPBOD software to generate appropriately formatted data for this rapid prototyping system to fabricate the scaffolds. This fabrication process shows the advantage of RP technologies in its ability to produce complex 3D shape from a given computer model. Testing and analysis of the properties of scaffolds built by RPBOD system were carried out. Macro-pore diameters of 300-500 µm were observed which are suitable for cell growth into scaffolds. The calculated overall porosity of the freezedried scaffolds was about 90%, which is desirable to provide high surface area for cell polymer interactions. The stress-strain curves of chitosan scaffolds indicated that these scaffolds should be considered for use in low load bearing applications. Preliminary in-vitro culture studies were conducted by seeding with porcine osteoblasts. Adhesion and distribution of cells was studied via the scanning electron micrograph (SEM). Osteoblasts can be observed adhering and spreading well on the surface of the scaffold and the initial shape of polygonal is kept. The tests indicated the cell compatibility and attachment of the scaffold. In general, the RP robotic dispensing system (RPBOD), combining RP technology with tissue engineering, provides much potential for the design and desktop manufacturing of biomedical scaffolds. Rapid prototyping of scaffolds by the RPBOD is presented using a biocompatible chitosan gel. During the scaffold fabrication, high temperature is not required and with the multiple-dispenser feature, it allows fabrication with materials or material additives, which otherwise decompose under heat, as well as the incorporation of proteins and living cells. The high porosity of the resulting scaffolds can be obtained to facilitate good ventilation and cell growth. 70 Chapter 6 Conclusions and Recommendations 6.2 Recommendations As a continuation to this work, the following recommendations are made for future research to further develop the RPBOD system for tissue engineering applications. 1. In-vivo experiments can be carried out to study the tissue compatibility in the body. This is because materials do not behave in the same manner inside and outside the body. 2. Post-processing for the chitosan scaffolds, such as leaching [Lu et al., 2000], may be conducted to change the smooth surface of the hydrogel strands and generate a rougher surface in order to improve the cell attachment. 3. Modify the algorithm for dual dispensing to generate more patterns of scaffold strands, (e.g. the strands in orientations of 60 degrees) to investigate the effect of different orientations of adjacent layers on the strength and structure of the scaffolds, as well as the cell cultivation. 4. Fabricate other regularly-shaped scaffolds, like cylindrical, other than the current cubic scaffolds to increase the variety of scaffolds. 5. With multiple dispensing capabilities, different multiple-feeding 3Ddispensing techniques using various materials can now be investigated. A particularly interesting area is the use of fibrinogen and thrombin, which require precise low flow rates and simultaneous-dispensing capabilities. 6. Instigate the incorporation of cell seeding into the 3D dispensing plotting materials. 7. Improvements regarding the mechanical properties are necessary. 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Zhang Y. and Zhang M., Synthesis and Characterization of Macroporous Chitosan/Calcium Phosphate Composite Scaffolds for Tissue Engineering, Journal of Biomedical Materials Research, Vol. 55, pp. 304-312. 2001. 84 Appendix A. Machine Specifications APPENDICES Appendix A. Specifications of Sony Robokits. Figure A.1 RoboKids dimensions Table A.1 Machine specifications Item Work envelope Maximum speeds Pose-repeatability (Positioning accuracy) Maximum payload Resolution X-axis 250mm 300mm/s Y-axis 210mm 300mm/s Z-axis 70mm 100mm/s ±0.05mm ±0.05mm ±0.05mm 0.014mm 5kg 0.014mm 2kg 0.014mm 85 Appendix B. Materials Information Appendix B. Materials Information Physical/Chemical Characteristics of Chitosan: Appearance and odor – Chitosan is fine, odorless and tasteless powder manufacturing from crab shells. Solubility – Insoluble in water and alcohols; soluble in dilute organic acids. Chitosan, 50g Catalog# UOM Product Size 4-00022 EA 50g Details Appearance: High Purity Powder Ash: [...]... transfer medical data (CT/MRI) to the appropriate RPcompatible data format This involves the use of a software to reconstruct images taken from CT/MR into 3D model and convert the data to the format that can be recognized by the developed rapid prototyping systems Geometric data of the scaffold was generated based on the computer model built from the medical data During the scaffold characterization and... porogens Rapid Prototyping Rapid Prototyping (RP) is the name given to a family of processes that are used to fabricate objects directly from a 3D computer model The model is produced either by computer-aided design (CAD), 3D scanning or 3D reconstruction of 2D images Such technologies are also known as Free-Form Fabrication (FFF), Solid Freeform Fabrication (SFF) or Layered Manufacturing (LM) Rapid prototyping. .. Research Scope In the first phase of this research, experiments were carried out to determine set of optimized parameters of the fabrication process At the same time, protocols for 4 Chapter 1 Introduction the preparation of materials for scaffold fabrication were established based on the material properties In the free-form scaffold fabrication phase, the data conversion process was developed to transfer... which the individual layers are bonded together to form a 3D object LOM has been used for fabrication of bioactive bone implants, using HA and calcium phosphate laminates [Steidle et al., 1999] A HA/glass tape is laid down on the working platform The outside profile of the layer to be built is cut using a laser directed by an XY plotter The laser only cuts to the depth of a single layer A second layer of. .. the objectives of this research are: I To optimize the parameters and conditions for fabricating scaffolds by the dual dispensing method with the RPBOD system II To design and fabricate 3D free-form scaffolds with relevant features extracted from given medical images (CT/ MRI) using a desktop PC III To characterize the built scaffolds and evaluate their potential for application in tissue engineering. .. where the high cost is offset by the huge reduction in fabrication time and the flexibility for customized jobs RP also allows the special capability of fabricating enclosed cavities, something which precision CNC, arguably the closest rival to RP in terms of speed and versatility, cannot achieve The rapid prototyping technology enables quick and easy transition from concept generation in the form of. .. back at the desired site of 8 Chapter 2 Literature Review the functioning tissue However, it is believed that isolated cells cannot form new tissues by themselves Most primary organ cells require specific environments that very often include the presence of a supporting material to act as a template for growth The currently existing substrates are mainly in the form of 3D tissue engineering scaffold. .. 3DP requires post processing to improve the mechanical properties of the scaffold FDM, on the other hand, allows the application of only thermoplastic polymers This prevents the implementation and application of biological agents and natural polymers as temperature induces protein inactivation Additionally, the major proportion of the scaffold fabrication supported by RP technology was based upon melt... rapid prototyping and RP-related scaffold fabrication techniques Chapter 3 investigates the biomaterials used in TE scaffolds, selects the materials and the procedure for material preparation in this research and briefly presents the methods to fabricate scaffolds and the experiments carried out to characterize the scaffolds Chapter 4 gives a general outline of the RPBOD system and details with the manufacturing... bonded to the previous layers using a hot roller which activates a heat sensitive adhesive The contour of each layer is cut with a laser that is carefully modulated to penetrate to the exact depth of one layer After the layer has been completed and the build platform lowered, the process repeats itself However, there is a need to separate the finished parts from the build platform, which affects their