CHAPTER V REGULAR SCAFFOLD FABRICATION TECHNIQUES FOR INVESTIGATIONS IN TISSUE ENGINEERING

C.T. Buckley, K.U. OKelly Centre for Bioengineering, Department of Mechanical and Manufacturing Engineering, Trinity College, Dublin, Ireland Many of the conventional techniques employed for scaffold fabrication in tissue engineering produce scaffolds with random porous architectures which cannot ensure a pertinent and germane environment for new bone formation. Advanced manufacturing technologies such as solid free-form (SFF) fabrication or rapid-prototyping have aided in over coming some of these limitations by producing more regular architectures. The authors will discuss the advantages and limitations of existing techniques and present an alternative method to fabricate regular orthogonal architecture scaffolds for mechanobiology investigations in tissue engineering that will attempt to address the current limitations in cell penetration depth, pore occlusion, cell seeding and nutrient diffusion/flow depth.

1. Introduction Tissue engineering is the use of a scaffolding material to either induce formation of bone from the surrounding tissue or to act as a carrier or template for implanted bone cells or other agents (5). The scaffold or three-dimensional (3D) construct provides the necessary support for cells to proliferate and maintain their differentiated function, and its architecture defines the ultimate shape of the new bone. The engineering of scaffolds has developed over the years from the initial concept of providing an inert biocompatible construct to allow for revascularization
P.J. Prendergast and P.E. McHugh (Eds.), Topics in Bio-Mechanical Engineering, pp. 147-166. 2004 Trinity Centre for Bioengineering & National Centre for Biomedical Engineering Science.

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and cell invasion to a tissue engineered approach, whereby the construct is initially seeded with cells, cultured in vitro, and finally implanted. It is therefore essential to provide a biological environment in which cells can readily attach, proliferate and maintain their differentiated phenotype and to allow deposition of new bone matrix throughout the entire construct. Scaffold characteristics such as interconnectivity, pore size/curvature, microporosity, macroporosity and surface roughness influence cellular responses, but they also collectively control the degree of nutrient delivery, penetration depth of cells and metabolic waste removal. It is also important to allow cell-seeded scaffolds to be subjected to a strain environment, in order to further our understanding of how cells respond to mechanical stimuli. A tissue-engineered scaffold must provide a germane environment for in vitro cell culturing in a bioreactor as well as providing a suitable environment once implanted in vivo. These two environments differ in terms of nutrient concentration gradients, pressure gradients and fluid velocities. In vivo, diffusion is the primary mechanism for transporting nutrients, whereas fluid flow is the principal mechanism for transport of nutrients and provision of mechanical stimuli in vitro. In order for a scaffold to be considered successful, it is essential that it provide a nutrient rich environment within the scaffold core in order for cells to lay down new matrix and minimise cell necrosis. Scaffolds with defined interconnected channels aid in the processes of cell nutrient delivery, waste removal and vascular invasion (2). Many of the conventional techniques used yield scaffolds with random porous architectures which do not necessarily produce a suitable homogeneous environment for bone formation. Non-uniform micro environments produce regions with insufficient nutrient concentrations which can inhibit cellular activity and prevent the formation of new tissue with a homogeneous quality. Advanced manufacturing technologies such as rapid-prototyping have aided in over coming some of these limitations, allowing for greater control over internal scaffold geometry. However even with these technological advances, limitations still remain, and need to be resolved in order to produce the next generation of tissue engineered scaffolds with suitable chemical and mechanical microenvironments. The authors

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will discuss the advantages and limitations of existing techniques and present an alternative method to fabricate regular orthogonal architecture scaffolds for mechanobiology investigations in tissue engineering. 2. Scaffold Properties Ideally a scaffold should possess the following characteristics to bring about the desired biologic response (11): (i) three-dimensional and highly porous with an interconnected pore network for cell/tissue growth and flow transport of nutrients and metabolic waste, (ii) biodegradable or bioresorbable with a controllable degradation and resorption rate to match cell/tissue growth in vitro and/or in vivo, (iii) suitable surface chemistry for cell attachment, proliferation and differentiation, (iv) mechanical properties to match those of tissues at the site of implantation, and (v) be easily processed to form a variety of shapes and sizes. 2.1 Scaffold Materials The three main material types which have been successfully investigated for use in developing scaffolds include (23, 27): (i) Natural polymers, such as collagens, glycosaminoglycan, starch, chitin and chitosan, (ii) Synthetic polymers, based on polylactic acid (PLA), polyglycolic acid (PGA) and their co-polymers (PLGA), and (iii) Ceramics, such as hydroxyapatite (HA) and -tricalcium phosphate (-TCP). While naturally occurring biomaterials offer the greatest potential in terms of biocompatibility, large batch-to-batch variations can exist as well as poor mechanical performance. A concern of material supply limitations has prompted researchers to investigate the use of synthetic polymers. Synthetic polymers have been widely used for over 20 years as surgical sutures, with long established clinical success and many are approved for human use by the FDA. However, synthetic polymers of the poly(-hydroxy acids) family release acidic by-products as they undergo degradation by bulk erosion via hydrolysis when exposed to aqueous environments (15). Although these degradation products are naturally present in the human body and are removed by natural

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metabolic pathways (21), the local pH of the surrounding microenvironment can be reduced below that of the natural physiological pH and thus elicit an immunological response. The effect of this acidic environment can cause cell necrosis as well as acting as an autocatalyst, further accelerating the degradation of the polymer. Ceramics have also been widely used, due to their high biocompatibility and resemblance to the natural inorganic component of bone and teeth (6, 7). Ceramics are inherently brittle and limit their applicability in tissue engineering/mechanobiology investigations to load bearing applications, since ceramics are stronger in compression than in tension. As synthetic polymers are deemed to be ductile with insufficient rigidity, some researchers have developed composite materials (e.g. polymers with ceramic particles embedded within the polymer matrix) to improve mechanical performance and render the material more suitable for load bearing applications. The added advantage of this is that the embedded ceramic particles act as a buffer to the degradation of byproducts produced (13). The development of materials for tissue engineering scaffolds presents many challenges in obtaining specific mechanical and bioresorbable properties, as well as developing materials suitable for various fabrication processes. 2.2 Pore Size and Curvature Many investigators have defined scaffold pores based on size as either micro (diameter < 100m) or macro (diameter > 100m). For colonisation of macropores to occur, the minimum pore size in which bone will form is claimed to be approximately 100m (6). Other researchers have created scaffolds with pore sizes of between 150-300m and 500-710m to promote bone formation (12, 18). However many of these pore sizes were determined using random pore geometries, and hence do not define optimum pore sizes accurately; rather they define the range of pore sizes in which bone formation was observed. The pore size employed may also be dependent on the tissue-type desired. For example scaffolds with pore sizes less than 150m have been successfully used for regeneration of skin in burn patients (20).

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Osteoblasts appear to exhibit greater cellular response when pore sizes of between 200 and 400m are employed (6). This may be due to the curvature of the pore which may provide optimum compression and tension on the cells mechanoreceptors and allows them to migrate into openings of such a size (4). 2.3 Interconnectivity, Macroporosity and Microporosity A scaffold should provide an open porous networked structure allowing for easier vascularisation, which is important for the maintenance of penetrating cells from surrounding tissues and the development of new bone in vivo. The higher the macroporosity the easier it is for vascularisation to occur. Failure to develop an adequate vascular network will mean that only peripheral cells may survive or differentiate, supported by diffusion. Chang et al. (6) proposed that the degree of interconnectivity rather than the actual pore size has a greater influence on osteoconduction. Interconnectivity is a physical characteristic that aids in the delivery of nutrients and removal of metabolic waste products. Studies have shown that bone normally forms in the outer 300m periphery of scaffolds and that this may be explained by the lack of nutrient delivery and waste removal (12). When the pore size used is too small, pore occlusion can occur by cells preventing further cell penetration and bone formation (14). It is pertinent to note that much higher rates of mass transfer exist at the periphery of a scaffold, and that these higher rates promote mineralisation, further limiting the mass transfer of nutrients to the core of a scaffold (17). It is essential that a scaffold possess a high degree of interconnectivity in conjunction with a suitable pore size, in order to minimize diffusion limitations and pore occlusion. The incorporation of microporosity within the scaffold material may have added advantages with regard to nutrient delivery and cellular response. Taboas et al. (22), successfully incorporated microporosity (Fig. 1) within a scaffold material (PLA) consisting of interconnected plate structures, yielding 5-11m void openings, through an emulsionsolvent diffusion technique. The microporosity of a scaffold gives it the potential to be preconditioned with bone morphogenetic proteins (BMPs)

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(1), anti-inflammatory drugs (Dexamethasone) (29) and oxygen release agents (ORAs) such as perfluorocarbons. Tancred et al. (24) assessed the fluid-retention characteristics (Fig. 2) of ceramic based scaffolds produced by identically replicating the architecture of bovine cancellous bone and observed that these scaffolds were capable of retaining water at the level of at least 50 wt% of the mass of the mineral, at less than 65% porosity, to about 150 wt% of the mass of the mineral at 80-85% porosity, indicating that these -TCP replicated structures could also be useful as a carrier for osteogenic agents such as BMP.

Fig. 1. Interconnected plate structures (7x5m average) yielding 5-11m void openings within PLA material (22).

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Fig. 2. Water retention characteristics of porous -TCP replicas of bovine cancellous bone showing the increase in water retention (wt% of matrix mineral) with increasing construct porosity (24).

3. Traditional Scaffold Fabrication Techniques Several techniques have been developed to fabricate scaffolds. These include solvent-casting and particulate-leaching, gas foaming, fibre meshes/fibre bonding, phase separation, melt moulding, emulsion freeze drying, solution casting and freeze drying (Table 1). Traditional methods of fabricating scaffolds, through material processing and casting, have largely been unsuccessful in controlling the internal architecture to a high degree of accuracy or homogeneity (Fig. 3), since the resulting interior architectures are determined by the processing technique. For example, particulate leaching is a process whereby the internal architecture is determined by embedding a high density of salt crystals into a dissolved polymer or ceramic matrix. The dissolved mixture is then poured into a mould and treated under heat and pressure to form the external shape. The salt particles are subsequently leached out to leave interconnecting

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Fig. 3. Random porous architecture of PLGA created via liquid-liquid phase separation (16).

interior channels. Running the salt crystals through a sieve to obtain a specific range of pore size can control the pore diameters; although the agglomeration of salt particles can alter the eventual pore size and pore distribution during leaching (14). Particulate leaching techniques are limited to producing thin membranes (2-3mm), due to the difficulty in ensuring complete removal of the embedded particles. Also, there is little control over the orientation and the degree of interconnectivity. However the degree of interconnectivity can be improved by having a high density of salt particles (8, 12) and also by fusion of the salt crystals prior to infiltration (19). Creation of scaffolds with identical internal architectures for mass transfer and mechanobiology investigations is essential. Previous researchers have demonstrated that control over the interior architecture is crucial to ensure scaffold vascularisation and bone deposition (7, 18).

Regular Scaffold Fabrication Techniques Table 1. Conventional scaffold processing techniques for tissue engineering (14, 27). Process Solvent casting and particulate leaching Advantages Large range of pore sizes Independent control of porosity and pore size Crystallinity can be tailored Highly porous structures Disadvantages Limited membrane thickness (3mm) Limited interconnectivity Residual porogens Poor control over internal architecture

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Fibre bonding

High porosity

Limited range of polymers Residual solvents Lack of mechanical strength

Phase separation

Highly porous structures Permits incorporation of bioactive agents

Poor control over internal architecture Limited range of pore sizes High temperature required for nonamorphous polymer Residual porogens Lack of mechanical strength Limited interconnectivity Problems with residual solvent Residual porogens Nonporous external surface Closed-pore structure

Melt moulding

Independent control of porosity and pore size Macro shape control

Membrane Lamination

Macro shape control Independent control of porosity and pore size Independent control of porosity and pore size Superior compressive strength No organic solvents

Polymer/ceramic fibre composite foam High-pressure processing 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

Residual solvents Residual porogens

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3.1 Solid Free Form (SFF) Fabrication Technologies Rapid prototyping (RP) or solid free form (SFF) fabrication technologies are currently being used by investigators to manufacture scaffolds for use in tissue engineering (3, 7, 9, 10, 25, 26). SFF methods are based on the premise that a material in either powdered or liquid form is solidified one layer at a time. It is thus an additive process unlike traditional methods of manufacturing which are subtractive based. Each layer is created as defined by a computer-generated file. Once the layer is complete, the build platform is indexed downward by one layer thickness and the process is repeated. The main systems that fall under this category are: (1) stereolithography (SLA), (2) selective laser sintering (SLS), (3) fused deposition modelling (FDM) and (4) three-dimensional printing (3-DP). Each SFF fabrication process has its own advantages and disadvantages in fabricating scaffolds as summarised in Table 2.
Table 2. Advantages and limitations of SFF fabrication techniques. Technique SLA Advantages Relatively easy to remove support materials Accurate small features SLS Good compressive strengths Greater material choice Solvent free FDM No material trapping within small features Solvent free Good compressive strengths 3D-P Greater material choice Low heat effect on raw material Limitations Limited by the development of photopolymerizeable and biocompatible, biodegradable liquid polymer material High processing temperatures Materials trapped in small inner features is difficult to remove Requires support material for irregular structures Anisotropy between XY and Z direction

Materials trapped in small inner features is difficult to remove Use of toxic organic solvents Lack of mechanical strength

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The main limitations include the use of toxic binders, poor feature symmetry (Fig. 4c) and materials. Due to these material limitations, researchers have also used SFF techniques to indirectly cast scaffolds with controlled internal and external architecture by means of a lost mould process (7, 22). Manufacturing of these scaffolds consists of three different types of development and optimisation work. They include: (a) mould design (b) sacrificial mould fabrication (c) material casting and (d) thermal or chemical removal of mould.

Fig. 4. (A) & (B) Solid free form (SFF) fabricated scaffold produced using selective laser sintering (SLS) technique, material is Duraform polyamide. (C) Threshold image of a single pore showing poor symmetry formed through selective laser sintering.

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Lost mould processes are mainly suited to ceramic infiltrates as ceramics are typically sintered to temperatures in excess of 1000C, which ensures complete removal of a polymer mould. When attempting to fabricate polymer scaffolds though indirect fabrication methods, an extra step of creating a ceramic-type mould is required. This ceramic mould is infiltrated through melt or solvent casting depending on the desired polymer. Once cured, the ceramic mould can be removed through solvent dissolution. The choice of solvent for mould dissolution is dependent on the cast and mould material. This iteration further reduces the quality of the final scaffold in terms of pore symmetry, and material properties due to polymer exposure to solvents. An advantage of indirect casting is the production of discrete composite scaffolds in which material regions are mechanically interdigitated (Fig. 5), as well as the incorporation of microporosity within the scaffold material which allows for preconditioning with bioactive agents and manipulation of the surface roughness.

Fig. 5. (A) Biphasic PLA/HA scaffold (top=PLA, bottom=HA). PLA global pores are 600m, HA global pores are 500m. (B) Biphasic PLA/PGA scaffold (top=PGA, bottom=PLA), 800m orthogonal pores (22).

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3.2 Mechanobiology/Tissue Engineering Scaffold Criteria With regard to tissue engineering investigations, it is necessary to fabricate scaffolds which meet the following criteria; (i) regular internal geometries, (ii) complete interconnectivity, (iii) independent control over pore size and porosity, (iv) scaffolds for use within strain environments, (v) facilitate the creation of scaffolds from a broad range of materials without loss of pore definition, and (vi) allow for the incorporation and manipulation of microporosity within the scaffold material. Some of these criteria cannot be easily met with the existing techniques as outlined previously. Regular architecture scaffolds permit cells to be seeded in the core much more readily than random architecture scaffolds and create environments which encourage uniform conditions for promoting cell viability. The added advantage of developing regular architecture scaffolds is that they permit parametric analyses to be conducted, which is essential in scientific investigations of how scaffolds perform as a function of their physical characteristics. Finite element diffusion/perfusion studies are more feasible when regular architecture scaffolds are employed. As a first step in addressing these issues, we have developed a regular orthogonal fibre stacking (ROFS) technique to fabricate scaffolds. 4. Process Overview The process is essentially a lost mould process, in which layers with parallel unidirectional nylon fibres of equal spacing create the mould. The layers are created by winding nylon fibre around a plate with defined notches of equal spacing. The mould architecture forms a 3D mesh-like structure. This mould is infiltrated with either a ceramic slip or polymer solution. Once the infiltrate has been cured/sintered, the nylon fibre mould is removed either by pyrolisation (ceramic infiltrate) or by physical extraction of the fibres (polymer infiltrate) (Fig. 6).

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Infiltration of mesh with ceramic slip

Thermal mould removal

Sintering

Creation of Regular Orthogonal Stacked mesh

Ultrasonic Bath Treatment

Infiltration of mesh with polymer via solvent/melt casting

Physical extraction of fibers

Regular Architecture Scaffold sheet

Fig. 6. Process flow chart for creation of regular orthogonal polymer/ceramic scaffold.

4.1 Materials and Methods Poly(dimethylsiloxane) (PDMS) is widely used in microfabrication for biological applications and offers biocompatibility, optical transparency, permeability to gases, flexibility, and durability. PDMS produced by Dow Corning, Sylgard 184 was obtained from R W Greef (Glasgow, UK). The notched recessed plate (Fig. 7) was fabricated using a diamond saw (STRUERS ACCUTOM 50) and 400 m blade (METPREP 1012-50), with a notch spacing of 300 m. Nylon fibre of diameter 250, 300 and 350 m (Climax, Germany) was used in this study. PDMS infiltrate was mixed using a 10:1 weight ratio of base to curing agent, and degassed under vacuum for 1hr. The polymer solution was then infiltrated using a 10ml surgical syringe into the fibre mesh matrix and cured for 4hrs at 60C. After curing, the fibres were then extracted from the polymer to reveal a slab (50mm x 50mm x 3mm) with orthogonal oriented channels (Fig 9.).

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Fig. 7. 400m notched aluminium plate, with notch spacing of 300m for creation of the 3D orthogonal stacked mesh.

4.2 Results Each layer of the unidirectional fibres is stacked orthogonal to the previous layer creating a 3D mesh-type structure (Fig. 8). Once the mould is infiltrated and the fibres are removed, macro pores with nonrandom interconnectivity are created in the x, y and z planes. The pores created in the z direction are a direct result of the contact points between the fibres in the x and y directions. The pores generated in the z direction are typically around 100m (when 350m fibres are employed), but this is dependent on the viscosity of the polymer infiltrate.

Fig. 8. (A) Layer with fibres of 200m diameter and spacing of 300m. (B) Stacked layers showing orthogonal mesh structure.

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Fig. 9. (A) Approx 5cm square slab of PDMS scaffold (B) & (C) Magnified structure of regular PDMS scaffold 350m pore size and 300m pore spacing.

5. Discussion Scaffolds have been successfully fabricated according to the method of preparation described. The resulting scaffold slab is approximately 5cm

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square, with regular orthogonal geometry in all three directions. Figure 9 shows images of the regular geometrical structure obtained with a PDMS infiltrate. As can been seen the structure is highly regular and repeatable. A limitation with this technique, is that the resulting scaffold is anisotropic (between xy and z direction), and this is due to penetration of the polymer around the point of contact of the fibres. A higher infiltrate viscosity results in larger sized pores in the z direction. However when viscous solutions are employed, higher pressures are required to successfully infiltrate the mesh mould, which can result in spreading of the fibres. Thermal treatment of the mould prior to infiltration may improve contact area, but this may only be applicable when fabricating ceramic scaffolds, since fusion of the fibres may occur, and make the process of extracting fibres form a polymer matrix difficult. Another possibility in improving the contact area includes using fibres of square or hexagonal cross sectional area, to create the mesh mould. However this may not provide an optimum substrate for cells, in terms of pore curvature. Since the notched plate was created using a 400m blade and the fibre employed was 350m in diameter, it is possible for the resulting pore spacing to vary between 300m and 400m. Using fibres with diameters closer to the notch width will further reduce the variation between pore spacing. Other methods of creating a notched plate can be employed in order to increase the range of pore sizes, and increase macroporosity. Possible methods include the use of electro-discharge machining (EDM) or SFF techniques to fabricate notched plates for the creation of the orthogonal mesh through fibre winding. This technique also allows independent control over pore size and porosity, which is essential for parametric analyses. Indirect casting techniques allow for a much wider range of materials to be employed, and also facilitates the incorporation of microporosity into the scaffold material, which may have added advantages with regard to nutrient delivery and cellular response. Traditional fabrication methods such as particulate leaching, emulsion-solvent diffusion and gas foaming could technically impart microporosity to scaffolds. However, control over the actual micro-pore size may be difficult to achieve and control. Further studies will determine the optimum technique to be used.

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Another advantage of developing regular architecture scaffolds is that they permit parametric analyses to be conducted in terms of nutrient concentration gradients and local strain environments, which is essential in identifying and predicting optimal cell environments in order to develop scaffolds for preliminary analysis and eventual implantation. 6. Conclusions and Outlook A process has been described which allows regular orthogonal repeating unit scaffolds from a wide range of materials to be produced in large quantities with independent control over pore size and porosity, with complete interconnectivity. Although PDMS is a non-bioresorbable material, it is biocompatible and could serve as a delivery vehicle as well as a model for future work on bioresorbable scaffolds such as PLA, PGA and their copolymers. Regular architectures allow mechanical and diffusion/perfusion finite element analyses to be carried out, which permit quantification of suitable mechanical and chemical microenvironments for bone cells, aiding in the development of the next generation of tissue engineered scaffolds. Acknowledgments This work was supported by the HEA under the Programme for Research in Third Level Institutions (PRTLI) Cycle III. The authors would also like to thank Mr. Tony Tansey of the Department of Mechanical Engineering, IT Tallaght, Dublin 24 for producing the RP scaffolds. References
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