Chapter One: Introduction Chapter One Introduction Chapter One: Introduction 1.1 Background At present, many strategies for tissue engineering (TE) require scaffolds that, by acting as a surrogate extracellular matrix, provide an initial site for cells to anchor, grow and proliferate. Furthermore, a biodegradable scaffold that could be implanted into a defect site, promote regeneration of tissue, and then degrade over time as new tissue reclaims the site (Han et al, 1998). Such scaffolds can be cultured with cells prior to implantation (cellular strategy) or alternatively implanted without cells (guided tissue regeneration strategy) (Patrick et al, 1998). These strategies have been studied for the regeneration of many tissues, including bone and cartilage (Perka et al, 2000; Whang et al, 1998). A number of materials as well as scaffold design have been experimentally and/or clinically studied. For the treatment of tissues/organs in malfunction, one of the challenges in the field of tissue engineering is the design of various types of scaffolds that can mimic the structure and biological functions of the natural extracellular matrices (ECM) of different tissues. The minimum requirements for TE scaffolds are as follows (Hutmacher, 2001a): a) Three-dimensional and highly porous with an interconnected pore network to favour flow transport of nutrients and metabolic waste, and tissue integration and vascularization b) Biodegradable or bioresorbable with a controllable degradation and resorption rate to match cell/tissue growth in vitro and/or in vivo c) Suitable surface chemistry for cell attachment, proliferation, and differentiation Chapter One: Introduction d) Favourable mechanical properties to match those of the tissues at the site of implantation e) Be easily fabricated to form a variety of shapes and sizes In the early days of tissue engineering, FDA approved devices and implants made of polymers of synthetic origin, such as sutures, meshes etc. were used (Chaignaud et al, 1997). Later, techniques were developed to fabricate polymeric scaffolds based on either heating macromolecules or dissolving them in a suitable organic solvent. In these techniques, the viscous behaviour of the polymers above their melting temperatures, and their solubility in various organic solvents are the important characteristics, which determine the type of process that will be used. Based on the use of organic solvents, a number of techniques have been developed to design and fabricate porous 3D bioresorbable scaffolds for tissue engineering applications (Lu et al, 1996). These scaffold-processing techniques, called conventional/traditional techniques include solvent casting and particulate leaching, gas foaming, fibre meshes/fibre bonding, temperature-induced phase separation (TIPS), melt moulding, emulsion freeze drying, solution casting, membrane lamination and freeze drying (Sachlos and Czernuszka, 2003). A wide range of scaffold characteristics such as pore morphology, pore size and porosity, has been reported using such conventional fabrication techniques (Widmer and Mikos, 1998). However, these techniques often remain impractical because they cannot provide control over scaffold design (e.g. complex & reproducible architectures and compositional variation across the entire structure) to achieve specified properties within required limits on morphological Chapter One: Introduction characteristics (Lin et al, 2004). To overcome this hurdle, rapid prototyping (RP), called solid free form (SFF) fabrication techniques have been introduced with high encouragement for scaffold fabrication. RP is a fast growing popular technology that enables quick and easy fabrication of customised forms directly from computer aided design (CAD) model to solid model. The needs for quick product development, decreased time to market, and highly customised and low quantity parts are driving the demand for RP technology (Kochan, 1997). The flexible manufacturing capabilities of RP techniques have been applied to biomedical engineering applications ranging from the production of scale replicas of human bones (D’Urso et al, 2000) and body organs (Sanghera et al, 2001) to advanced customized drug delivery devices (Leong et al, 2001) and other areas of medical sciences including anthropology (Recheis et al, 1999), palaeontology (Zollikofer and De Leon, 1995) and medical forensics (Vanezi et al, 2000). Today, RP technique is regarded as an efficient tool to reproducibly generate scaffolds with tailored properties on a large scale (Sacholos and Czernuszka, 2003; Hollister et al, 2000). The widely studied RP techniques include stereolithography apparatus (SLA), selective laser sintering (SLS), threedimensional printing (3DP), fused deposition modeling (FDM), 3D plotting, multiphase jet solidification (MJS), 3D fiber deposition and precise extrusion manufacturing (PEM) (Wu et al, 1996; Koch et al, 1998; Zein et al, 2002, Woodfield et al, 2004; Wang et al, 2004; Williams et al, 2005). These techniques will be elaborated in the next chapter (literature review). Chapter One: Introduction 1.2 Hypothesis The variation in macro/micro architecture and material would modulate the morphological and biomechanical characteristics of the scaffold. Therefore, the rapid prototyping (RP) technology in combination with novel synthetic polymer(s) would allow developing a scaffold family that could cater broad tissue engineering applications. 1.3 Significance of Hypothesis Tissue engineering leads to new medical therapies for injury and organ disease through use of living cells together with polymeric biomaterials. Cells live in a nano- and/or micro-featured environment with natural extracellular matrix comprised of a complex mixture of pores, ridges, fibres and networks. Fundamental understanding of interactions between cells and their living environments, such as extracellular matrix, is critical for design and development of efficient tissue engineering devices. In this regard, the biochemical and topographic cues of the scaffolds play significant roles in the cell behaviours, such as cell adhesion, proliferation, migration and differentiation. On top of that, the mechanical strength of the bioresorbable scaffold should match that of the host tissue at the time of implantation as closely as possible, and should retain until the tissue engineered transplant is fully constructed that can assume its structural role (Hutmacher, 2000). Therefore, in the development of a tissue engineering scaffold, there remains a great challenge to mimic the structural and biological functions of natural ECM. The novel polymer aims to modulate the hydrophilicity, biodegradability and mechanical properties as required for targeted tissue. Besides, the RP Chapter One: Introduction technique would allow generation of 3D scaffolds with fully interconnected and highly reproducible pore networks similar to honeycomb-like pattern that will accomplish morphological and biomechanical requirements for tailored tissue engineered applications. 1.4 Research Aims The overall objective of this PhD research is to develop and characterize a 3D scaffold family with a range of morphological and biomechanical properties by the combination of synthetic polymer(s) and in house built rapid prototyping (RP) technique. To achieve the above-mentioned objective, this research aims at the following: ¾ In house design and development of a melt extrusion based system, called desktop robot based rapid prototyping (DRBRP) technique to process synthetic biopolymers into 3D scaffolds ¾ Design and fabrication of 3D scaffold family with various architectures utilizing a range of biopolymers ¾ Physical (morphological and mechanical) characterization of the scaffolds ¾ Evaluation of degradation behavior and kinetics of the scaffolds ¾ Cell culture study that would investigate the cell compatibility and neotissue formation onto the scaffolds Chapter One: Introduction 1.5 Structure of Thesis The overall research program can be carried out in five phases. Phase I focuses on development of the DRBRP technique to process synthetic biopolymers into 3D scaffolds of honeycomb-like architecture with various pore shape, size and porosity while maintaining complete interconnectivity and reproducibility throughout the structure. The DRBRP technique allows direct extrusion of the material from its granulated or any other form into 3D scaffold in contrast to traditional FDM system that requires preparation of filament or any other pre-cursor. This work involves design and manufacturing of the necessary hardware namely, liquefier or melting chamber, insulating jacket for the chamber, extrusion nozzle etc. and further incorporation with the commercial “Sony Robot” operated by RPBOD software. Virtually, DRBRP system allows to process any thermoplastic material into 3D scaffold with any lay-down pattern. Phase II initially investigates the efficacy of the DRBRP technique to process a wide range of biopolymers namely, PCL and PCL-based copolymers (PCLPEG, PCL-PEG-PCL and PEG-PCL-PLA). In the beginning, four polymers prepared by ring-opening polymerization were experimented to investigate the feasibility of these polymers to be processed into 3D scaffold by the DRBRP technique. These materials were taken into consideration because they had the potential to modulate hydrophilicity and/or degradability and consequently, the biomechanical properties of the matrices for tailored tissue engineered Chapter One: Introduction applications by varying the ratio of the polymer components. Afterward, this work focuses on using two polymers (PCL and PCL-PEG) throughout the project assuming that it would produce ample data to justify the objective of this research and ultimately, to avoid overwhelming of data. The fabrication process was optimized by varying the process parameters (liquefier temperature, extrusion pressure and deposition speed). Finally, a family of scaffolds was fabricated with various lay-down patterns and filament distances applying the optimized process parameters. Phase III performs the physical characterization of the developed scaffolds that evaluates morphological the morphological characterization and investigates mechanical the properties. influence of The process parameters, lay-down patterns and filament distances on the pore size, shape and porosity of the scaffolds. Likewise, the mechanical characterization studies the influence of process parameters, lay-down patterns and filament distances on the mechanical properties of the scaffolds. The mechanical characterization also reveals the effect of strain rate, simulated physiological environment and loading direction on the scaffolds’ mechanical properties. Phase IV evaluates the degradation behavior and kinetics of the scaffolds. This study investigates mainly, the effect of the variation in material and laydown pattern on the degradation kinetics of the scaffolds in terms of mass loss, and change in morphological and mechanical properties. Chapter One: Introduction Phase V focuses on the in vitro cell culture study that investigates the cell compatibility and neotissue formation onto the scaffolds. Scaffold surface and morphology to which the cells adhere, affect bio- and electrochemical signals that control cell migration, proliferation and differentiation. The qualitative and quantitative analyses of cell adhesion and proliferation to the scaffold surface and architecture are the basic fundamentals of studying biocompatibility of new scaffold in tissue engineering research. In the preliminary study, human fibroblasts are used to assess the general biocompatibility of PEG-PCL-PLA scaffolds. Further, rabbit smooth muscle cells are cultured to evaluate biocompatibility and cellular functions onto the PCL and PCL-PEG scaffolds. This study investigates the variation in cell adhesion and function due to the variation of material and scaffold architecture. Light, scanning electron, and confocal laser microscopy are used to assess qualitatively cell adhesion, proliferation and extracellular matrix production while, the PicoGreen DNA allows to study cell proliferation from a quantitative point of view. . match those of the tissues at the site of implantation e) Be easily fabricated to form a variety of shapes and sizes In the early days of tissue engineering, FDA approved devices and implants. field of tissue engineering is the design of various types of scaffolds that can mimic the structure and biological functions of the natural extracellular matrices (ECM) of different tissues. . number of materials as well as scaffold design have been experimentally and/ or clinically studied. For the treatment of tissues/organs in malfunction, one of the challenges in the field of tissue