Bioinspired scaffold for regenerative medicine: production engineering and scaffold characterization

UNIVERSITÀ CAMPUS BIO-MEDICO DI ROMA FACOLTÀ DI INGEGNERIA DOTTORATO DI RICERCA IN INGEGNERIA BIOMEDICA XXIV CICLO Bioinspired scaffold for regenerative medicine: production engineering and scaffold characterization Supervisor Prof. Marcella Trombetta Candidate Sara Maria Giannitelli, M Eng. Co-supervisor Alberto Rainer, PhD

Index Index... 2 Abstract... 5 Introduction... 6 Chapter 1: Scaffolds in Tissue Engineering... 8 1.1 Scaffold requirements... 8 1.2 Scaffold materials... 9 1.2.1 Natural versus Synthetic polymers... 10 1.2.2 Poly(ε-caprolactone) (PCL)... 11 1.3 Biomimetic scaffolds... 13 1.4 Fabrication techniques... 15 1.4.1 Conventional fabrication methods... 16 1.4.2 Electrospinning... 17 1.4.3 Solid freeform fabrication... 18 1.5 Scaffold characterization... 20 1.5.1 Morphological... 21 1.5.2 Chemical and thermal... 22 1.5.3 Physical... 24 1.5.4 Biological: in vitro-in vivo... 25 1.5.5 Finite element analysis in scaffolds characterization... 27 Objective and thesis roadmap... 27 Chapter 2: Development of an extrusion based deposition system... 29 Overview... 29 2.1 Extrusion-based techniques... 29 2.2 Home made RP system... 31 2.2.1 Development of a dispensing head... 33 2.3 Printing parameters... 37 2

2.4Development of dedicated software modules... 39 Conclusions... 44 Chapter 3: Rapid prototyping of biopolymeric functionalized scaffolds for bone regeneration.. 45 Overview... 45 3.1 Rapid prototyped functionally graded scaffolds... 45 3.1.1 Biomaterials choice... 46 3.1.2 Implemented design... 47 3.1.3 Functionalization protocol... 48 3.2 Home-made RP system performance... 50 3.3 PCL scaffold fabrication... 53 3.3.1 Morphological study... 54 3.3.2 Porosity... 55 3.3.3 Mechanical characterization... 56 3.4 PCL-HA scaffold fabrication... 57 3.4.1 Morphological study... 58 3.5 Scaffold functionalization... 59 3.6 In-vitro experiments on tissue-engineered constructs... 60 3.6.1 Scaffolds biocompatibility... 60 3.6.2 Cell differentiation... 61 Conclusions... 64 Chapter 4: A bioinspired approach to the design of porous additively manufactured scaffolds with optimized mechanical properties... 65 Overview... 65 4.1 Design of scaffold micro-architecture in extrusion based techniques... 66 4.2 Load Adaptive Scaffold Architecturing (LASA) algorithm... 69 4.2.1 Theory and calculation... 70 4.2.2 Design and Finite Element Simulations... 72 4.2.3 Samples fabrication... 76 3

4.2.4 Mechanical testing... 77 4.3 Application of LASA... 78 Conclusions... 81 Chapter 5: Electrospun fibers with nanostructured surface morphology... 82 Overview... 82 5.1 Electrospinning in drug delivery... 83 5.2 Scaffold fabrication and characterization... 85 5.2.1 Scanning electron microscopy (SEM)... 86 5.2.2 Differential scanning calorimetry (DSC)... 88 5.2.3 Attenuated Total Reflectance (ATR)-Fourier transform infrared spectroscopy (FTIR)... 92 5.3 Release study... 97 5.3.1 Short term study... 97 5.3.2 Long term study... 99 5.3.3 Mechanical characterization... 102 5.3.4 Three-stages release model... 103 5.4 Biocompatibility assay... 104 Conclusions... 106 Chapter 6: Conclusions and future work... 107 6.1 Ongoing research... 108 6.1.1 Biomimetic micro-fibrous PLLA scaffold for tendon regeneration: a preliminary in vitro study... 108 6.1.2 Preparation of a polycaprolactone/bioglass hybrid scaffold through a direct sol-gel method... 111 References... 114 Appendix... 122 4

Abstract Scaffolds play a pivotal role in the tissue engineering paradigm, as they provide an extra-cellular matrices onto which cells can attach, grow, and form new tissues. Cellseeded scaffolds can either be cultured in vitro, or directly implanted in vivo, using the body s own system as a bioreactor. The efficacy of the first approach is often questioned due to requirement for at least two surgical procedures and the delay in treatment while the construct is being cultured in vitro before implantation. Thus, the desired biological scaffold performance has consequently shifted from a passive role, where scaffolds were merely accepted by the body, to an active role in which they instruct their biological surroundings in a predictable and controlled fashion. Due to the primary importance of scaffold design, one of the challenges in tissue engineering is to reproduce an analog of the in vivo scenario, mimicking the microenvironment that promote cell-cell and cell-matrix interactions. Between the main issues that need to be considered in the engineering of functional constructs, material selection, tissue specific micro-architecture design and developing methods for scaffolds fabrication, are often included. Aim of this thesis is to explore alternative strategies for the development of bioactive scaffolds, off-the-shelf available and intended to be used to guide tissue regeneration, exploiting the endogenous regenerative abilities of the body. This challenge has been addressed by combining the synthesis of novel biologically inspired design with innovative production techniques, in order to optimize scaffold architecture, composition and mechanical properties, leading to what would more conventionally be termed a smart scaffold. 5

Introduction Disease, injury and trauma can lead to damage and degeneration of tissues in the human body, which necessitates treatments to facilitate their repair, replacement or regeneration. Treatment typically focuses on transplanting tissue from one site to another in the same patient (autograft) or from one individual to another (transplant or allograft). While these treatments have been revolutionary and lifesaving, major problems exist with both techniques. Harvesting autografts is expensive, painful, constrained by anatomical limitations and associated with donor-site morbidity due to infection and hematoma. Similarly, allografts and transplants also have serious constraints due to problems with accessing enough tissue for all of the patients who require them and the fact that there are risks of rejection by the patient s immune system as well as the possibility of introducing infection or disease from the donor to the patient. Tissue engineering (TE) has emerged in the last decade of the 20th century as an alternative approach to circumvent the existent limitations in the current therapies for organ failure or replacement. It has been defined as the application of scientific principles to the design, construction, modification and growth of living tissues using biomaterials, cells and growth factors, alone or in combination. There are three strategies in tissue engineering; (1) the use of isolated cells or cell substitutes to replace those cells that supply the needed function, including genetic or other manipulations before infusion. (2) The delivery of tissue-inducing substances, such as growth and differentiation factors, to targeted locations. (3) The introduction of three-dimensional (3D) matrices (scaffolds), where cells can be either recruited from the host tissues in vivo or seeded in vitro. Scaffold guided tissue engineering approach applies methods from materials engineering and cell/molecular biology to create artificial constructs for tissue regeneration. Cell-seeded scaffolds are either cultured in vitro, to synthesize tissues which can then be implanted into an injured site, or are directly implanted, using the body s own systems as bioreactor. Many clinicians question the efficacy of in vitro tissue engineering due to the requirement for at least two procedures and the delay in treatment, while the construct is 6

being cultured in vitro. From a commercial perspective, this approach also poses problems due to the prolonged regulatory process required before such construct can be approved for clinical use. However, in tissues such as cartilage, which do not have the ability to regenerate themselves when damaged, long term in vitro tissue engineering is currently the only solution to prevent the requirement of an eventual joint arthroplasty. Other tissues, such as bone for example, have an intrinsic ability to repair, remodel and regenerate. TE in this case aims to harness this innate regenerative capacity. One way to do so might be to engineer the scaffold in such a way that itself provides regenerative signals to the cells which might avoid the requirement for prolonged in vitro culture, prior to implantation. Therefore, much ongoing research is devoted to developing more sophisticated scaffolds that optimally recreate extra cellular matrix (ECM) environment in a temporally coordinated and spatially organized structure. In the following Chapter, a general overview of various aspects of scaffold design will be provided, highlighting the specific topics handled in this thesis. Functional requirements involved in scaffold design, types of materials, fabrication and characterization techniques used in developing state of the art scaffolds, challenges and future research direction, will be presented. 7

Chapter 1: Scaffolds in Tissue Engineering 1.1 Scaffold requirements Within TE, one of the major research themes is scaffold design, which ultimately determines the functionality of the construct and of the grown tissue. It comprehends the material and fabrication method used, as well as other features like scaffold shape, size, architecture, and surface topography. Although the final requirements depend on the specific TE application, several general guidelines can be considered for all the designs. The scaffold should be/have: -biocompatible; it should provoke an appropriate biological response of the host tissue and prevent any adverse reaction. -biodegradable; the scaffold should degrade into non- toxic and easily metabolized products without interfering with the function of the surrounding tissue. Rate of scaffold s degradation must be tuned appropriately so that it retains sufficient structural integrity until the neo-tissue (cells and extracellular matrix without vascularisation) is developed. This property strictly depends on the intrinsic characteristics of biomaterial used for scaffold fabrication, including the chemical structure, the presence of hydrolytically unstable bonds, the level of hydrophilicity/hydrophobicity, crystalline/amorphous morphology, the copolymer ratio, and the molecular weight. Other physical and chemical factors such as the overall porosity, the pore size distribution, the scaffold morphology (fiber, foams, 3D printed structure) and the ph at the implantation site, can also contribute to regulate degradation kinetic. -suitable mechanical properties; engineered tissues must possess the appropriate mechanical properties to fulfill their structural role. The required properties depend on the specific application, in e.g. cardiovascular versus bone prostheses. Particularly, in the reconstruction of hard, load-bearing tissues such as bone and cartilages, the mechanical strength to retain the scaffold s structure after implantation is essential. The degradable scaffold should maintain sufficient mechanical strength to manage any in vivo stresses and physiological loadings imposed on the engineered construct. For 8

tissue-engineered vascular grafts, hemodynamic competence and suturing characteristics are also critical. -appropriate porosity; in term of magnitude of the porosity, pore size, distribution and interconnectivity. A highly porous scaffold with an open, fully interconnected porosity and a large surface-to-area volume ratio is desirable to allow cell in-growth and uniform distribution within the material. Compared to a closed pore structure, an interconnected network enhances the diffusion rates to and from the center of the scaffold and facilitates vascularization, thus improving oxygen and nutrient supply and waste removal. Furthermore, a micro-porosity is also required in order to promote capillary ingrowth. The effects of pore size on tissue regeneration have been testified by literature data demonstrating optimum pore size for neovascularization (5µm), fibroblast ingrowth (5-15µm), hepatocytes ingrowth (20µm), for regeneration of adult mammalian skin (20-125µm), and for bone regeneration (100-700µm). Besides size and porosity, shape and tortuosity of pores can also affect the rate and extent of tissue in-growth. However the mechanical strength of a scaffold tends to decrease as the porosity increases. Thus, there may be a conflict between optimizing the porosity and maximizing mechanical properties. - tunable shape; scaffold must have appropriate geometry and size in order to exactly fits the site of replacement. For this purpose it should be manufactured in a reproducible, controlled and cost effective fashion in a variety of shapes and sizes. The interdisciplinary character of these features, combined with the extensiveness of the application field, yields the high level of complexity involved with TE scaffold design. All pieces of the puzzle need to fall in place before the engineered construct can be translated into clinical application. 1.2 Scaffold materials One of the aspect to which all of the criteria listed above are dependent upon, is the choice of biomaterial. Various materials, including biopolymers, bioceramics and metals, have been used to produce scaffolds for several TE purposes. Due to the variation in properties required in soft versus hard applications, especially in term of mechanical behavior, the constructs for these two sub- categories generally use different 9

classes of biomaterials and different processing techniques. For soft TE scaffold, e.g. skeletal muscle or cardiovascular field, a wide variety of polymers are generally applied. On the other hand, hard tissue replacements, e.g. bone substitutes, are generally based on more rigid polymers, ceramics and metals. It s clear that each class of biomaterial has unique advantages and limitations. Clinical studies and laboratory experiments have a main role in the choice of the right material for each application or in the identification of a successful combination of biomaterials. In the past decades, a tendency toward the synthesis of new composites, or hybrid materials was observed. The rationale for designing composites is to provide many attractive properties that each individual material cannot have. For example, in the case of organic/inorganic composites, like polymers with ceramic particles, inorganic component provides bioactivity and higher biocompatibility whereas polymer matrix will ensure mechanical stability. Furthermore, the addition of ceramic particles will act as a buffer to the degradation of byproducts produced by the synthetic polymer. 1.2.1 Natural versus Synthetic polymers Polymeric biomaterials originate from a wide range of natural as well as synthetic sources. Naturally-derived polymer, such as collagen, fibrin, glycosaminoglycans (GAGs), chitosan, alginates and starch, can be extracted from plants, animals or human tissues. They show excellent biocompatibility, low toxicity and low chronic inflammatory response due to the high similarity with native cellular environments, especially when the compounds are naturally present in ECM. Furthermore they are easily degradable by an enzymatic or hydrolytic mechanism. However, they show poor mechanical performance, difficult processing and they can undergo to batch-to-batch variations. Other concerns are the potential pathogen transmission, antigenecity and lack of constant material supply. Synthetic polymers do not show variation in chemic/physical properties and offer high versatility and good workability. The greatest advantages are the ability to tailor mechanical properties and degradation kinetics on the basis of specific application. However, biocompatibility is generally lower than natural polymers. For acidic degradation products, high local concentrations can affect cell growth on the scaffolds, in vitro and cause inflammatory responses, in vivo. 10

One of the most widely used biomaterials is a group of synthetic polymers known as polyesters. These include aliphatic polysters such as polylactic acid (PLA), polyglycolic acid (PGA), poly-ε-caprolactone (PCL), as well as their copolymers or blends. In addition, other synthetic polymers also have significance especially in bone tissue engineering, including poly(methyl methacrylate), polyethylene, polypropylene, polyurethane, poly(-ethylene terephthalate), polyetherketone, and polysulfone. It is beyond the scope of this chapter to discuss all polymers in detail; particular attention will be paid on PCL because of its relevance in the present work. 1.2.2 Poly(ε-caprolactone) (PCL) PCL is an aliphatic linear polyester, obtained by a ring-opening polymerization of ε - caprolactone using a catalyst such as stannous octanoate (Figure 1). It has a glass transition temperature of -62 C and a melting point of about 60 C, depending on the degree of crystallinity, which in turn is dictated by the molecular weight (normally 3000 100 000 g/mol) and, to some extent, by the scaffold fabrication process. It is biocompatible, bioresorbable and a low-cost synthetic polymer. Due to its semicrystalline and hydrophobic nature, it exhibits a very slow degradation rate (2 4 years depending on the starting molecular weight and fabrication process) and has mechanical properties suitable for a variety of applications. It is a Food and Drug Administration (FDA) approved material and has been clinically used as a slow release drug delivery device and suture material since the 1980s (i.e. Capronor, SynBiosys, Monocryl suture). Recent clinical trials, ranging from plugging cranial burr holes, orbital floor support, craniosynostosis, craniofacial reconstructions, to dental applications, have demonstrated favorable results and drawn positive responses. Schantz and Lim et al.[1] reported on clinical use of PCL scaffolds over a 12-month period, in cranial reconstruction of burr holes. They concluded that the burr plugs have excellent biocompatibility and are well tolerated by patients with no detectable signs of acute local or systemic immune reactions. The implant s strength and fracture-resistant properties enable it to be firmly anchored in the surrounding calvarium, leading to stable reconstruction achieving the functional and aesthetic objectives of cranioplasty. However, these developments thus far have been limited to low load-bearing maxillofacial treatments only. 11

PCL also has some rheological and viscoelastic properties that allow it to be formed from a wide range of scaffold fabrication technologies. The ease unto which a polymeric material is accommodated into different scaffold fabrication technologies is a property that should not be underestimated. PCL and its copolymers have demonstrated this utility by being successfully used in electrospinning, gravity spinning [2], phase separation, solid freeform fabrication [3] and microparticles [4, 5], due to the low melting temperature, very good blend-compatibility, FDA approval and low cost. The wide use of PCL and the increasing interest of TE community toward this polymer is testified by the trend of publications regarding only the electrospun PCL meshes, during the last 10 years (Figure 1). Figure 1: PCL molecular structure and publications regarding PCL electrospun meshes during the last 10 years, until March 2011. Sourced from ISI Web of Knowledge [6] One disadvantage of PCL, however, is its high hydrophobic nature, resulting in poor wettability and uncontrolled biological interactions with the material [7]. Surfaces with moderate hydrophilicity are able to absorb adequate amount of proteins, while preserving their natural conformation, unlike hydrophobic or very hydrophilic surfaces. In order to rectify this issue, surface modification techniques can be adopted to alter 12

chemical and/or physical surface properties. The optimal degree of hydrophilicity, however, depends on the cell type and on the specific surface treatment applied. Four main approaches have been tested on PCL surface [6]: - Plasma treatment, which improves the hydrophilicity by forming oxygen-containing groups on the surface. -Chemical treatment with reagents such as sodium hydroxide (NaOH). Scaffold immersion in NaOH aqueous solution introduces hydroxyl groups and side chain modification on PCL surface by the formation of carboxylate ( COOH) groups. -Coating or adsorbing natural ECM proteins, which introduce cell recognition sites for improved cell biomaterial interaction. -Blending biologically active materials with PCL to provide signals to increase cell affinity. Some of these approaches can be adopted also to encourage common used biomaterials to have, for example, bioinstructive and stimuli-responsive properties. 1.3 Biomimetic scaffolds In order to achieve tissue formation the scaffold does not only have to provide a support for cell adhesion and proliferation but also stimulate the desired differentiation of cells and thus promote the secretion of new ECM constituents. Both physical and chemical surface properties are of major importance for this purpose. Physical factors include the wettability, surface energy and micro-nanotopographic properties, such as roughness. From a chemical point of view, the presence of biological binding sites and the released products are also critical. Recently, several strategies have been developed to enhance tissue regeneration tuning this chemical and physical factors in order to mimic the natural environment in which cells grow. ECM has an instructive role providing a dynamic and spatially heterogeneous constellation of microstructural, compositional and mechanical cues that influence cell behavior. Harnessing the mechanosensitive capacity of cells, for example, provides immense opportunities for tissue regeneration. Scaffold mechanical properties have profound biological consequences in terms of implant bioactivity versus failure, transmission of mechanical stimuli, and for a wide range of processes at the tissue, cell and sub-cellular levels. Key roles in molecular signaling pathways are played by cell adhesion 13

complexes and cellular cytoskeleton, whose contractile forces are transmitted through transcellular structures. Therefore, the mechanical properties of the substrate to which cells are attached are critical to the regulation of cellular mechanotransduction and subsequent cellular behavior (attachment, proliferation and differentiation). It is now clear, for example, that substrate stiffness can regulate both the behavior of mature cells and the differentiation pathway of stem cells. In fact, when MSCs were grown on firm gels that mimic the elasticity of muscle, differentiation down a myogenic lineage was observed, whereas when MSCs were grown on rigid gels that mimic pre-calcified bone, they differentiated down an osteogenic pathway. Therefore, increasing research is now being directed at utilizing the mechanosensitive capacity of cells to develop scaffolds and biomaterials with specific mechanical properties which can be used to direct the behavior of the cells with which they interact. [8, 9] In addition to biomechanical signals, cellular behavior is strongly influenced by biological and biochemical cues. Appreciation of the complexity of the cell response to ECM signaling has helped in the development of 3D scaffolds that imitate its properties. Generally, cells bind to ECM trough the receptors on the cell walls. One class of receptors are integrins, which bind selectively to specific binding sites such as arginineglycine-aspartic acid (RGD) tripeptide found in cell adhesive proteins such as vibronectin, laminin and fibronectin. Beside attachment these connections mediate several intracellular signals that define mobility, cellular shape and regulate cell cycle. Therefore, several attempts have been made to develop biomimetic materials that mimic integrin-binding in various biological systems. In addition, ECM proteins such as collagen I, laminin, fibronectin, vitronectin, and fibrinogen as well as peptides designed from these proteins have been applied onto scaffolds to enhance cell adhesion and proliferation. The use of scaffolds as delivery systems for growth factors, adhesion peptides and cytokines is receiving considerable attention in TE field. Incorporation of angiogenic growth factors in scaffolds in order to improve their vascular potential is one of the most investigated topic. [10, 11] Another area of critical importance is controlling, and understanding, the host immune response and preventing infection following implantation. To this end, the incorporation of drugs (i.e. inflammatory inhibitors and/or antibiotics) into scaffolds has been proposed as a method to reduce the possibility of infection after surgery. Finally, the use 14

of scaffolds as delivery systems for therapeutic genes is undergoing considerable investigation. Gene therapy approaches (viral and non-viral) which utilize DNA encoding for therapeutic genes potentially provide a stable and effective approach to allow sustained and controlled release of therapeutic factors. [8] 1.4 Fabrication techniques Considerable research effort has been devoted to develop and optimize fabrication techniques apt to the production of scaffolds with architectures mimicking the structure of the tissue to be repaired. Conventional methods include: solvent casting and particulate leaching, gas foaming, fiber meshes and fiber bonding, phase separation, melt molding, emulsion freeze drying, solution casting and freeze drying. More innovative and promising approaches concerns the use of electrospinning and rapid prototyping. Yet, also these fabrication methods have some disadvantages if considered alone. On the basis of the selected technique, scaffold design can be divided into two broad categories. The first design incorporates a precise geometrical layout and scaffolds that fall into this category include construct with regular pore (Figure 2a) and woven textile meshes (Figure 2b). Controlled pore structures can be achieved by rapid prototyping, while woven textile meshes via precise weaving techniques or particular electropsinning set-up. The second category involves the formation and deposition of scaffold struts or walls in a non-precise manner. Structures which belong to this category include foams (Figure 2c), obtained via porogen decomposition, gas forming or salt leaching, and random micro-nanofiber meshes (Figure 2d), fabricated by electrospinning or phase separation methods. [12] 15

Figure 2: SEM pictures o different type of matrices. (a) PCL scaffold with a laydown pattern 0 /90 created by RP technique. (b) PLGA woven textile mesh. (c) Hybrid foams obtained by gas foaming process. (d) Electrospun PCL random fibres 1.4.1 Conventional fabrication methods Conventional fabrication techniques have been broadly used to make 3D scaffolds for tissue-engineering and drug-release applications. However, a number of drawbacks can be outlined in term of control of tissue formation and drug release profile. Although the pore size and shape of these matrices are controllable to some extent, not completely interconnected and tortuous pathways are created. Furthermore, the incorporation of drugs, growth factors, or other biological agents is hampered by the processing conditions. In thermoplastic technologies, the high temperatures involved in the manufacture can compromise the stability of the compound to be integrated, resulting in its denaturation and loss of activity. In solution-based techniques the solvents used can also hinder the stability of the desired biological factor due to a ph change, which will promote aggregation and loss of activity.[13] 16

Tesi di dottorato in Ingegneria Biomedica, di Sara Maria Giannitelli, 1.4.2 Electrospinning Electrospinning has attracted great interest due to its ability to process polymeric solutions into fibrous structures at the micro/nanoscale by simply controlling few process parameters. Such matrices are characterized by a high surface area-to-volume ratio and resemble the physical structure of protein fibrils in native extracellular matrix. Furthermore, its low cost in constructing different set-up, its versatility a wide range of possible polymer solutions can be employed for the obtainment of scaffolds with different features and reproducibility are to be included between major advantages. The electrospinning process is based on the application of an electric field between a polymeric solution and a metal ground collector. When the electric field reaches a critical value, the electrostatic force overcomes the surface tension of the polymeric solution and a charged polymer jet is ejected from a capillary tip or needle. As the jet travels towards the grounded collector, it undergoes a stretching process together with a fast solvent evaporation. This process results in the formation of a random non-woven mesh composed of solid and continuous fibers. Varying the process parameters, e.g. strength of the electric field, distance needle collector, polymer concentration, allows tuning of the fiber diameter. Figure 3: electrospun nanofibers with various morphologies and assembled patterns. [14] 17

Additionally, changing the type of collector or the set-up configuration, different fibers organization can be achieved (Figure 3). For example, while depositing fibers on a static collector plate produces a randomly oriented nonwoven fiber matrix, deposition on a high speed rotating drum or mandrel produces aligned fiber matrices. 1.4.3 Solid freeform fabrication Solid freeform fabrication (SFF) techniques have been explored as an alternative method to improve scaffolds manufacturing. The main advantages are both the capacity to rapidly produce complex 3D models and the ability to process various raw materials. In TE field, SFF have been used to produce scaffolds with customised external shape and predefined internal morphology, allowing the control of pore size and pore distribution [15]. Although there are several commercial variants, the general process involves the use of a computer model derived from CAD or CT/MRI data, converted into.stl file format and sliced into thin cross-sections (Figure 4). Figure 4: Tissue engineering of patient-specific bone grafts. CT scan data of the patient defect (a) are used to generate a computer-based 3D model (b). This model is then imported into RP system software to be sliced into thin horizontal layers, with the tool path specified for each layer (c). The sliced data are used to instruct the RP machine (d) to build a scaffold (e) layer by layer, based on the actual shape of the computer model (c). 18

Utilization of computer-aided technologies in tissue engineering has evolved in the development of a new field of Computer-Aided Tissue Engineering (CATE). It can be defined as the application of enabling computer-aided technologies, including computer-aided design (CAD), image processing, computer-aided manufacturing (CAM), and rapid prototyping (RP) and/or solid freeform fabrication (SFF) for modeling, designing, simulation, and manufacturing of biological tissue and organ substitutes. According to broad diffused classification [16], CATE embraces three major applications: 1) computer-aided tissue modeling, including anatomic, biophysics and CAD-based modeling; 2) computer-aided tissue informatics, including tissue classification applied to tumor detection, morphometric and cytometric study; 3) computer-aided tissue scaffold design and manufacturing. In this latter case, the final architecture can be produced either directly using an additive manufactured process, or indirectly, by producing a sacrificial mould into which a biomaterial is cast. In this alternative route, pioneered by Chu et al. [17], the negative mold, which encompasses both the external shape and the internal porous architecture of the bone scaffold, is designed using CAD software and produced using RP techniques. The mold is, finally, removed chemically by using solvents or thermally by melting or burning the mold. Indirect RP scaffolds manufacturing methods offer the possibility of using biomaterials and design that cannot be processed directly via additive manufacturing but are inappropriate for developing hydrogel scaffolds, because it cannot be removed without damaging both internal and external architecture. Other disadvantages are the longer and more complex production process, and increased toxicity due to solvents presence. Commercially available additive manufacturing techniques may be categorized into three major groups based on the way materials are deposited [18]. The first group includes laser-based machines that either photopolymerize liquid monomer (Figure 5d) or sinter powdered materials (Figure 5c). The second major group actually prints material, including printing a chemical binder onto powdered material (Figure 5a) or directly printing wax. The third major group is of nozzle-based systems, which process material either thermally or chemically as it passes through a nozzle (Figure 5b). According to the type of feedstock (filament, powder, granulate) and to the extrusion 19

mechanism (mechanical or pneumatic), extrusion-based techniques have been further classified as Fused Deposition Modeling (FDM), Precision Extrusion Deposition (PED), Bioplotting and other variants. Figure 5: (a) 3D printing process, (b) fused deposition modelling process, (c) selective laser sintering process, (d) stereolithography system [15] 1.5 Scaffold characterization In vitro and in vivo biological experiments are of major importance but beforehand scaffold mechanical, structural and chemical analysis is required. First of all, scaffolds characterization enables to evaluate advantages and drawbacks of the fabrication methods applied. Secondly, the architecture of a scaffold will influence the mechanical properties and the biological performance. It is thus crucial to understand how these characteristics are correlated. A brief overview of some of the most important scaffold parameters with the corresponding characterization techniques has been reported in Table 1 and discussed in following paragraphs. Particular attention has been devoted to such properties considered in this thesis work. 20