Scaffolds for. tissue fabrication by Peter X. Ma

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1 Scaffolds for tissue fabrication by Peter X. Ma Tissue engineering is an interdisciplinary and multidisciplinary field. It has shown great promise in generating living alternatives for harvested tissues and organs for transplantation and reconstructive surgery. Materials and fabrication technologies are critically important for tissue engineering in designing temporary, artificial extracellular matrices (scaffolds), which support three-dimensional tissue formation. This review briefly introduces the concept of tissue engineering, and illustrates the relationship between tissue engineering and materials science and engineering. Important scaffold design principles are described. The most frequently used materials and fabrication technologies for scaffolds are reviewed. Some exciting new developments in scaffold materials and fabrication technologies are also discussed. Department of Biologic and Materials Sciences, Department of Biomedical Engineering, Macromolecular Science and Engineering Center, The University of Michigan, Ann Arbor, MI USA Imagine that one day a patient will go to a human body shop for a prescription for a lost leg, a failing liver, or a dysfunctional heart. In addition to the physiological, medical, and genetic information of the patient, the doctor will also collect three-dimensional images of the patient s remaining leg (in the case of the lost-leg patient) with detailed anatomical structures (bone, cartilage, tendons, ligaments, blood vessels, muscles, nerves, skin, and so forth) and the external shape. The doctor may collect the patient s saliva or other body fluids to extract genetic material (DNA) and also a tiny piece of tissue or bone marrow to obtain seeding cells for expansion. After the patient leaves the body shop, a computer is used by a tissue engineer to design the structure of the mirror leg (the lost leg) based on the symmetrical remaining leg, using various materials that simulate the extracellular matrices of the tissues of the leg (known as scaffolds or templates). Then, cells from a universal cell source, specifically designed for the patient, or banked cells grown from the patient s own cells are seeded onto these scaffolds. These engineered cell-scaffold components are then grown separately and/or assembled in a special chamber (bioreactor) that provides the right nutrients, regulating molecules (such as proteins, growth factors, and differentiation factors), physical and mechanical stimuli, temperature, pressure, and mass transport conditions for cell proliferation, differentiation, and tissue/organ formation. While the tissue/organ is regenerating, the scaffolding materials degrade and disappear, leaving nothing foreign to the body. The regenerated leg or leg precursor will be 30 ISSN: Elsevier Ltd 2004

2 surgically grafted onto the patient during the second visit to the human body shop (Fig. 1). The engineered tissues will have the capacity to grow, model, and remodel in concert with the dynamic changes of the physiological environment of the body. The grafted leg will integrate into the body. The new leg will grow and age as the body s natural leg. This scenario is an example of what the field of tissue engineering is hoping to do in the future. Tissue engineering and scaffolds Tissue engineering has been defined as an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function 1. There are three approaches in tissue engineering 1,2 : (1) the use of isolated cells or cell substitutes to replace those cells that supply the needed function 3 ; (2) the delivery of tissueinducing substances, such as growth and differentiation factors, to targeted locations 4,5 ; (3) growing cells in threedimensional scaffolds 1. The use of isolated cells or tissue-inducing substances is considered when the defects are small and well contained. To engineer tissues of practical size scale and predetermined shapes, these two approaches are seriously limited. Therefore, the third approach, i.e. growing cells in three-dimensional scaffolds, has become increasingly active. In this approach, scaffolds play a pivotal role. They guide cells to grow, synthesize extracellular matrix and other biological molecules, and facilitate the formation of functional tissues and organs 2,6. There are a few basic requirements that have been widely accepted for designing polymer scaffolds 2. First, a scaffold has to have high porosity and proper pore size. Second, a high surface area is needed. Third, biodegradability is generally required, and a proper degradation rate is needed to match the rate of neotissue formation. Fourth, the scaffold must have the required mechanical integrity to maintain the predesigned tissue structure. Fifth, the scaffold should not be toxic to the cells (i.e. biocompatible). Sixth, the scaffold should positively interact with cells, including enhanced cell adhesion, growth, migration, and differentiated function. Materials for scaffolds Polymers (macromolecules) are the primary materials for scaffolds in various tissue engineering applications, including Fig. 1 Schematic diagram showing the tissue engineering concept using a hypothetical example of leg regeneration. Scaffolding materials (temporary synthetic extracellular matrices) are designed as a three-dimensional mirror image, on which cells grow and regenerate the needed tissues. Because the scaffolding materials are biodegradable, they will resorb after fulfilling the template function and leave nothing foreign in the patient. bone and other mineralized tissues. A limited number of inorganic materials are used in bone and mineralized tissue engineering research. Here, the most commonly used and a few new materials in tissue engineering are reviewed. Materials for porous solid-state scaffolds The materials discussed in this category can form solid, stable porous structures to serve as predesigned three-dimensional scaffolds. They generally do not dissolve or melt under in vitro tissue culture conditions (in an aqueous medium) or when implanted in vivo. Linear aliphatic polyesters Poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and their copolymers poly(lactic acid-co-glycolic acid) (PLGA) are a family of linear aliphatic polyesters, which are most frequently used in tissue engineering 2,7,8. These polymers degrade through hydrolysis of the ester bonds 9. PGA is one of the most widely used scaffolding polymers 10. Because of its relatively hydrophilic nature, PGA degrades rapidly in aqueous solutions or in vivo, and loses mechanical integrity between two and four weeks 10,11. It has been processed into nonwoven fibrous fabrics as one of the most widely used scaffolds in tissue engineering today. PLA is also widely used for scaffold fabrication. The extra methyl group in the PLA repeating unit (compared with PGA) makes it more hydrophobic, reduces the molecular affinity to water, and leads to a slower hydrolysis rate. It takes many months or even years for a PLA scaffold or implant to lose 31

3 mechanical integrity in vitro or in vivo 12,13. To achieve intermediate degradation rates between PGA and PLA, various lactic and glycolic acid ratios are used to synthesize PLGAs. These polymers (PLA, PGA, and PLGAs) are among the few synthetic polymers approved by the US Food and Drug Administration (FDA) for certain human clinical applications. There are other linear aliphatic polyesters, such as poly(ε-caprolactone) (PCL) 14 and poly(hydroxy butyrate) (PHB) 15, which are also used in tissue engineering research. PCL degrades at a significantly slower rate than PLA, PGA, and PLGA 16. The slow degradation makes PCL less attractive for general tissue engineering applications, but more attractive for long-term implants and controlled release applications. PCL-based copolymers have recently been synthesized to improve degradation properties 17. PHB is made by microorganisms via fermentation 18,19. PHB and PHB-based copolymers degrade very slowly because of their hydrophobic nature and, therefore, are less popular compared with PGA, PLA, and PLGA for tissue engineering applications. Other important synthetic biodegradable polymers Poly(propylene fumarate) (PPF) can degrade through hydrolysis of the ester bonds similar to glycolide and lactide polymers 20. Tyrosine-derived polymers have shown promising biocompatibility 21. These polymers have been used for bone tissue engineering research 20,22. Segmented polyurethanes allow the structural variations to achieve a range of mechanical properties 23. Recent efforts have been focused on the development of biodegradable urethane-based polymers using less toxic diisocyanates Polyphosphoesters 27 and polyphosphazenes 28,29 have been frequently used for controlled release applications, and have recently been explored for certain tissue engineering applications Similarly, polyanhydrides and poly(ortho esters) are primarily designed for controlled drug delivery applications because of their surface erosion properties 33,34. Nevertheless, they have also been explored for tissue engineering scaffolding applications 35,36. This is not surprising because there are some similar requirements on biocompatibility and biodegradability in both controlled release matrices and tissue engineering scaffolds. Natural macromolecules Natural polymers, such as proteins and polysaccharides, have also been used for tissue engineering applications. Collagen is a fibrous protein and a major natural extracellular matrix component. It has been used for various tissue regeneration applications, especially for soft tissue repair 37,38. On one hand, collagen as a natural extracellular component has useful biological properties desirable for tissue engineering applications. Therefore, collagen-glycosaminoglycan (GAG) copolymers are fabricated into scaffolds for tissue engineering 39,40. Denatured collagen (gelatin) is also processed into porous materials for tissue repair 41. On the other hand, there are concerns over collagen because of the potential pathogen transmission, immune reactions, poor handling and mechanical properties, and less controlled biodegradability. Another category of well-known natural fibrous proteins is silk. Silkworm silk has been used in textile production for centuries, and has been used as nondegradable sutures for decades because of its excellent tensile mechanical properties 42. This natural macromolecular material has recently been introduced into the field of tissue engineering 43. Although silk is often characterized as a nondegradable material, it can degrade in vivo via enzymatic mechanisms. However, the degradation rate is very slow. There is also some concern over cytotoxicity 44. There is research into the chemical modification of silk materials to enhance biocompatibility. There are also other types of silks, such as spider silks and even genetically engineered silks 45, which may be considered in certain applications. Polysaccharides are another class of natural polymers. For example, alginate 46, chitosan 47, and hyaluronate 48 have been used as porous solid-state tissue engineering scaffolds. In addition to the use of relatively pure natural macromolecules extracted from an animal or plant tissue source, processed extracellular matrix (decellularized) materials with multiple natural macromolecules are also used as scaffolds for tissue engineering or repair applications. One such example is small intestinal submucosa (SIS), which contains type I collagen, GAGs, and some growth factors. SIS has been used in the reconstruction of several tissue types 49,50. Similarly, other decellularized tissues, such as urinary bladder submucosa, porcine heart valves, and human dermis have been used for tissue engineering or repair Again there are concerns over pathogen transmission and immune rejection 52. Inorganic materials In addition to the large variety of polymeric (macromolecular) materials, certain inorganic compounds have also been studied for bone and other mineralized tissue 32

4 engineering research 54. These materials can be categorized as porous bioactive glasses 55 and calcium phosphates 56. Within the calcium phosphates, β-tricalcium phosphate (β-tcp), hydroxyapatite (HAP) and its derivatives, and their combinations are the most frequently used These inorganic materials are widely considered to be osteoconductive (their surface properties support osteoblastic cells adhesion, growth, and differentiation). They are also reported to be osteoinductive (capable of inducing bone formation) resulting from their capacity to bind and concentrate bone morphogenetic proteins (BMPs) in vivo 56. However, these inorganic materials are often difficult to process into highly porous structures and are mechanically brittle. To overcome these disadvantages, composite materials with synthetic or natural polymers have been explored for bone tissue engineering research Highly porous composite scaffolds with strong mechanical properties have been demonstrated 60. Such scaffolds support threedimensional new bone tissue formation 61. Materials for hydrogel scaffolds Hydrogels are cross-linked hydrophilic polymers that contain large amounts of water without dissolution. Injectable hydrogels are attractive candidates for certain tissue engineering applications because of the ability to fill irregularly shaped tissue defects, the allowance of minimally invasive procedures such as arthroscopic surgeries, and the ease of incorporation of cells and bioactive agents Among the synthetic hydrogels, poly(ethylene glycol) (PEG) is frequently studied. PEG-diacrylamide has been synthesized, and a photopolymerization step can be performed in contact with cells 70,71. There remains some concern over the toxicity of cross-linking agents. However, a major limitation of PEG hydrogel for tissue engineering scaffolds is its lack of degradability. Efforts are being made to impart degradability to PEG by either formulating copolymers with PLA, PGA, and PPF, or by introducing enzymatically degradable linkages into the PEG backbone Semi-interpenetrating polymer networks consisting of poly(n-isopropylacrylamide) and linear poly(acrylic acid) chains have been developed as synthetic hydrogel scaffolds 77. Diblock copolypeptide amphiphiles containing charged and hydrophobic segments have recently been reported to form hydrogel scaffolds with promising thermal stability and mechanical properties 78. More studies are needed to evaluate these new synthetic hydrogels for tissue engineering applications. Alginates, polysaccharides from seaweeds, cross-linked using calcium sulfate (CaSO 4 ), have been used as cell delivery vehicles for in vivo tissue engineering research However, one of the major disadvantages of using CaSO 4 is that gelation kinetics are difficult to control, and the resulting structure is not uniform. A method has recently been developed to control the gelation rate so that cross-linking occurs uniformly to form structurally regular and mechanically strong three-dimensional alginate gels 66. Collagen, in addition to porous foam, has also been used as a hydrogel for a variety of tissue repair and regeneration studies, including blood vessel, skin, cartilage, ligaments, and tendons Collagen has also been compounded with other natural polymers, such as chitosan, to form composite hydrogel scaffolds 91. Fibrin, involved in clotting, is another gel-forming fibrillar protein that has been explored for tissue engineering applications Fibrin gels are typically prepared by combining fibrinogen and thrombin solutions to entrap cells and form tissue engineering constructs. The fibrin gel has promising properties 93, but its degradation rate is rapid, which could be challenging for certain applications. There are also artificial proteins, such as elastin-mimicking polypeptides developed with recombinant DNA technology, which may serve as biomimetic hydrogel scaffolds This novel synthetic technology may also allow the design of chemical structure for functionalities, potentially for synthetic hydrogels or solid-state scaffolds. Scaffold fabrication technologies In the body, tissues are organized into three-dimensional structures as functional organs and organ systems 6. To engineer functional tissues and organs successfully, the scaffolds have to be designed to facilitate cell distribution and guide tissue regeneration in three dimensions. Textile technologies Earlier tissue engineering scaffolds comprising fibrous biodegradable polymer fabrics were produced using textile technologies. PGA, PLA, and other semicrystalline polymers can be processed into fibers using textile technologies. One of these scaffolds widely used in tissue engineering research is PGA nonwoven scaffold (Fig. 2). These PGA scaffolds have been used either alone or combined with other biodegradable 33

5 polymers for the engineering of cartilage , tendon 101, ureter 102, intestine 103, blood vessels 104,105, heart valves 106, and other tissues. However, there are several limitations of PGA nonwoven scaffolds, such as low mechanical strength, fast degradation rate, difficulty in controlling pore shape, and limited fiber diameter variations. Particulate-leaching techniques Particulate leaching is another technique that has been widely used to fabricate scaffolds for tissue engineering applications 107,108. Briefly, salt is first ground into small particles and those of the desired size are transferred into a mold. A polymer solution is then cast into the salt-filled mold. After the evaporation of the solvent, the salt crystals are leached away using water to form the pores of the scaffold (Fig. 3). The process is easy to carry out. The pore size can be controlled by the size of the salt crystals and the porosity by the salt/polymer ratio. However, certain critical variables such as pore shape and inter-pore openings are not controlled. To overcome these shortcomings and those of textile technologies, new techniques are being developed. Phase separation A homogeneous multicomponent system, under certain conditions, becomes thermodynamically unstable and tends to separate into more than one phase in order to lower the system free energy. A polymer solution separates into two phases, a polymer-rich phase and a polymer-lean phase. After the solvent is removed, the polymer-rich phase solidifies. Phase-separation techniques have been used to fabricate porous membranes for filtration and separation 109. However, the pores formed using such techniques usually have Fig. 2 Scanning electron micrograph of a PGA nonwoven scaffold with a fiber diameter of approximately 15 µm. (From Ma John Wiley & Sons.) Fig. 3 Scanning electron micrograph of a poly(l-lactic acid) (PLLA) foam fabricated using the salt-leaching technique. (From Ma John Wiley & Sons.) diameters on the order of a few to tens of microns and are often not uniformly distributed, which is not suitable for tissue engineering applications. Controlled phase separation processes, primarily thermally induced phase separation, have recently been explored for scaffold fabrication 7. Solid-liquid phase separation Phase separation can be achieved by lowering the temperature to induce solvent crystallization from a polymer solution. We define this process as a solid-liquid phase separation (solid phase formation in a liquid phase). After the removal of the solvent crystals (sublimation or solvent exchange), the space originally taken by the solvent crystals becomes pores. This technique can be used to fabricate scaffolds from many types of polymers and polymeric composite materials (Fig. 4) 60,61. By manipulating the phase separation conditions, various pore structures can be achieved. For example, many tissues (such as nerve, muscle, tendon, ligament, dentin, and so on) have oriented tubular or fibrous bundle architectures. To facilitate the organization and regeneration of such tissue types, a scaffold with a high porosity and an oriented array of open microtubules may be desirable. To achieve this goal, a novel phase separation technique has been developed to grow oriented rod-shaped crystals from a polymer solution. After the removal of these rods, a parallel array of microtubules is formed (Fig. 5a) 110. This oriented tubular scaffold has anisotropic mechanical properties similar to fibrillar and tubular tissues, and has been shown to facilitate cell organization into oriented tissues (Fig. 5b)

6 REVIEW FEATURE (a) Fig. 4 Scanning electron micrograph of a PLLA scaffold fabricated using solid-liquid phase separation. (From Ma et al John Wiley & Sons.) Liquid-liquid phase separation Lowering the temperature can induce the liquid-liquid phase separation of a polymer solution with an upper critical solution temperature. When such a process leads to the formation of a bicontinuous structure (both the polymer-rich and polymer-lean phases are continuous), a scaffold with an open-pore structure is formed after the removal of the solvent. For example, a mixture of dioxane and water has been used for liquid-liquid phase separation to fabricate PLA and PLGA scaffolds (Fig. 6) Design of three-dimensional pore architecture One of the common shortcomings of the fabrication technologies discussed above is the lack of precise control of the three-dimensional pore architecture of the scaffolds. To tackle this problem, computer-assisted design and manufacture (CAD/CAM) are being adopted114. Such a technique was initially explored at Massachusetts Institute of Technology115,116. One of the solid, free-form fabrication (aka rapid prototyping) techniques, called threedimensional printing, was used. With such a technique, complex-shaped objects were designed using CAD software. Scaffolds were fabricated by ink-jet printing a binder onto sequentially laid polymer powder layers117. However, the smallness of the powder particles and the binder drops (pixels) are limited (to a few hundred microns). The accuracy of positioning the printing nozzle is also limited. Therefore, the preciseness of the technology is seriously limited2. Similarly, other rapid prototyping techniques such as fused deposition modeling (FDM)118 and stereo (b) Fig. 5 (a) Scanning electron micrograph of a PLLA scaffold with oriented microtubular architecture, (b) MC3T3-E1 cells cultured on the PLLA scaffold for four weeks in vitro (von Kossa s silver nitrate staining). (From Ma and Zhang John Wiley & Sons.) Fig. 6 Scanning electron micrograph of a porous scaffold prepared from a 10% solution of PLGA (85/15) in a mixture of dioxane and H2O. (From Ma and Zhang John Wiley & Sons.) 35

7 (a) (b) (c) (d) Fig. 7 Scaffold fabrication using rapid prototyped negative replica: (a) a computer-generated, three-dimensional negative replica of a scaffold with a cubical pore shape, (b) the negative replica of the scaffold fabricated using a rapid prototyping machine, (c) a photograph of the polymer scaffold fabricated using the negative replica, and (d) a scanning electron micrograph of the internal pore structure of the scaffold. (From Ma John Wiley & Sons.) lithography are also being explored for scaffold fabrication Rapid prototyping techniques have inherent shortcomings such as limited material selection and inadequate resolution. In addition, the resulting constructs have structural heterogeneity because of the pixel assembly nature of the fabrication process. To overcome this shortcoming, a reverse fabrication technique has been developed to fabricate a negative replica of the scaffold 2. A polymer solution is cast into such a mold and solidified after the removal of the solvent. The mold is then dissolved away to form the polymer scaffold with the designed three-dimensional pore network (Fig. 7). The scaffold is more homogeneous, but the feature resolution is not improved. To achieve higher resolution for scaffolds with wellcontrolled interconnected spherical pores, paraffin spheres are fabricated by a dispersion method 123. These paraffin spheres are then transferred into a three-dimensional mold of the designed shape. The spheres are bonded together through a heat treatment process. A polymer solution is cast into the paraffin assembly in the mold. After removal of the solvent, the paraffin sphere assembly is dissolved away. In this way, an interconnected spherical pore structure is created (Fig. 8). Importantly, the features generated have significantly better resolution than those achievable with current rapid prototyping techniques. In addition, investment in expensive equipment is not required, which allows the technology to be easily adapted to a research, as well as an industrial, setting. Nano-featured and bioactive scaffolds Scaffolds serve as temporary, artificial extracellular matrices to accommodate cells and support three-dimensional tissue regeneration. Therefore, it is often beneficial to mimic certain (a) (b) Fig. 8 Scanning electron micrographs of polymer scaffolds with interconnected spherical pore structures prepared using paraffin spheres (porogen): (a) PLLA scaffolds, paraffin spheres: µm, 100x; (b) PLGA (85/15), paraffin spheres: µm, 50x. (From Ma and Choi Mary Ann Liebert. ) features of a natural extracellular matrix in scaffold design. It is now well known that many biologically functional molecules, extracellular matrix components, and cells interact 36

8 at the nanoscale. Hence, nano-featured synthetic scaffold design is one of the exciting new areas in tissue engineering. Nano-fibrous scaffolds Collagen is a major natural extracellular matrix component, and possesses a fibrous structure with fiber bundles varying in diameter from nm 124,125. To mimic the nanofibrous architecture, a few technologies have been developed to engineer nano-fibrous scaffolds. Electrospinning Electrospinning was first introduced in early 1930s 126 to fabricate industrial or household nonwoven fabric products. The technique has been rejuvenated over the past decade to process biodegradable and/or biocompatible polymers (macromolecules) into fibrous fabrics with an average fiber diameter at micrometer or nanometer scales for tissue engineering scaffolds. To form such fibers using electrospinning, a polymer solution is forced through a capillary, forming a drop of polymer solution at the tip. A high voltage is applied between the tip and a grounded collection target. When the electric field strength overcomes the surface tension of the droplet, a polymer solution jet is initiated and accelerated towards the collection target. As the jet travels through the air, the solvent evaporates and a nonwoven polymer fabric is formed on the target. To generate preferential orientation or/and tubular structure, an electrically grounded rotating drum is used as the collection target. A few synthetic polymers (e.g. PGA, PLGA, PCL, and synthetic polypeptides) and natural macromolecules (e.g. collagen and fibrinogen) have been processed into fibrous nonwoven scaffolds for tissue engineering research However, there are challenges in using this technique to fabricate complex three-dimensional scaffold shapes and internal pore networks. In addition, the average fiber diameter is usually on the larger side of the extracellular matrix fibers, sometimes falling in the micrometer range. Self-assembly Self-assembly is another exciting research area, especially where nano-sized and/or patterned biological structures are concerned. Self-assembly is now loosely defined as the autonomous organization of components into patterns or structures without human intervention 135. Such a strategy has been used in developing nano-fibrous materials with potential as tissue engineering scaffolds 136. To emulate the triple helical structure of collagen, peptideamphiphiles (PAs) have been synthesized A PA consists of a collagen sequence peptide head group connected to a long-chain ester lipid. The peptide head groups form a triple helical structure like collagen, while the lipid tails associate via hydrophobic interactions to induce and/or stabilize the three-dimensional structure. However, supramolecular fiber formation has not been demonstrated. To form supramolecular nano-fibers, more complex PAs have been synthesized to contain several structural domains 140,141. These PAs can form nano-fibers 5-8 nm in diameter and over 1 µm in length, which are actually cylindrical micelle tubes with hydrophobic tails in the center and hydrophilic heads on the outside. The structure is stabilized by disulfide bonds 140. Ionic self-complementary oligopeptides have been synthesized 142,143 containing alternating segments of ionic and hydrophobic amino acids. These peptides have been used to form fibrous structures with a fiber diameter of ~10 nm, on the lower side of extracellular matrix fibers. The fibers have a β-sheet structure instead of helical one. Self-assembled nano-fiber systems are, so far, limited to hydrogel format. To develop such hydrogel scaffolds further, production cost, mechanical properties, degradability, and macropore structure need to be addressed. Phase separation The phase separation of a polymer solution can also be considered a self-assembly process. Instead of assembling small molecules, large molecules are aggregated into a new phase from an initially homogeneous one-phase system. A novel phase separation technique has been developed to fabricate nano-fibrous materials from synthetic biodegradable polymers 7,111,144. For example, a poly(l-lactic acid) (PLLA) solution is induced to phase separate and gel, forming a polymer-rich nano-fibrous network. The solvent is removed afterwards to fabricate the desired porous, solidstate, nano-fibrous material (Figs. 9a and 9b) 111, which has a fiber diameter of nm, similar to that of collagen. To improve the three-dimensional structure of nanofibrous scaffolds for tissue engineering, techniques have been developed to build predesigned macropore networks in the nano-fibrous matrices 6,144. For example, larger watersoluble fibers (of diameter 100 µm to 1 mm) are prepared from sugar as a geometrical porogen element and assembled into various three-dimensional structures. A PLLA solution is then cast into this three-dimensional assembly and is thermally induced to phase separate for nano-fibrous 37

9 (a) (b) (c) (d) Fig. 9 Scanning electron micrographs of nano-fibrous PLLA matrices: (a) nano-fibrous matrix prepared from PLLA/THF solution via phase separation, 500x; (b) nano-fibrous matrix prepared from PLLA/THF solution via phase separation, x; (c) PLLA nano-fibrous scaffold with helicoidal tubular macropore network, 35x; (d) PLLA nano-fibrous scaffold with helicoidal tubular macropore network, 250x. (From Ma and Zhang John Wiley & Sons; Zhang and Ma John Wiley & Sons.) matrix formation. After solvent removal, the sugar fiber assembly is dissolved away using water to achieve nanofibrous scaffolds with predesigned helicoidal tubular pore network (Figs. 9c and 9d) 6. Similarly, an interconnected spherical pore network has been combined with phaseseparation techniques to generate nano-fibrous scaffolds with interconnected spherical macro pores 145. Such macroporous and nano-fibrous scaffolds enhance protein adsorption and cell adhesion 146. Nanocomposite scaffolds Polymer/inorganic composite materials have been developed for mineralized tissue engineering applications. To mimic the size scale of mineral crystals in bone and other mineralized tissues, nano-sized hydroxyapatite (nano-hap) has been compounded with synthetic polymers or natural macromolecules to fabricate nano composite scaffolds 62,113,147. Nano-HAP/polymer composite scaffolds have not only improved the mechanical properties of polymer scaffolds, but also significantly enhanced protein adsorption over micro-sized HAP/polymer scaffolds 113. Enhanced protein adsorption improves cell adhesion and function 146. Bioactive scaffolds The ideal tissue engineering scaffold should positively interact with cells, including enhanced cell adhesion, growth, migration, and differentiated function. To achieve these positive cell-scaffold interactions, surface or bulk modifications of the polymers are often employed The surface properties can be varied by either bulk or surface modification. Bulk modification is typically realized by copolymerization or functional group attachment to the polymer chains before scaffold fabrication 148,151,152, and usually changes the mechanical and processing properties of the polymers. Surface modification can be carried out after a porous scaffold has been fabricated. For example, plasma treatment alone or followed by chemical modification has been used to modify polymer thin films and porous scaffolds 153,154. Such techniques are most effective on twodimensional film surfaces or very thin three-dimensional constructs. In a complex porous three-dimensional scaffold, the surface is not just the outside surface, but also the internal three-dimensional surfaces. A simulated body fluid has been used to modify the chemical composition of the internal three-dimensional pore surfaces of polymer scaffolds 150,155. This biomimetic process has been shown to be effective at introducing nano-sized, bone-like apatite into the internal pore surfaces in situ, and may lead to improved scaffolds for bone tissue engineering 150,156. More threedimensional surface modification techniques are needed. To program scaffolds with biological instructions, delivery of bioactive molecules and genes has been integrated into Fig. 10 Schematic of a biomimetic nano scaffold. The scaffold combines the novel nanofibrous architecture of a interconnected pore network with microspheres for controlled release of putative regenerative factors. The nano-fibrous scaffolding design uses the architectural features of collagen, providing a high surface area for cell attachment and new matrix deposition, and an open structure allowing an interactive environment for cell-cell, cell-nutrient, and cell-signal molecule interactions. The bone mineral mimicking apatite enhances the osteoconductivity of the scaffold. The biodegradable microspheres release regenerative factors in a controlled fashion in a targeted local environment. 38

10 the scaffold design for tissue engineering A model of a bioactive scaffold (from our laboratory) integrating nanofibrous architecture, three-dimensional biomimetic surface modification, and controlled factor release capacity is illustrated in Fig. 10. Conclusions In summary, tissue engineering is one of the most exciting interdisciplinary and multidisciplinary research areas today, and is growing exponentially over time. Scaffold materials and fabrication technologies play a pivotal role in tissue engineering, and are fast evolving. This review is intended to illustrate the important roles of materials science and engineering in the field of tissue engineering. It covers the most commonly used and certain new materials, as well as fabrication technologies. It is an overall low resolution picture of the materials for tissue engineering scaffolds. The references allow interested readers to zoom in for specific high resolution details of interest. The author can only predict that better scaffolds will be engineered in the future. MT Acknowledgments The author wishes to acknowledge financial support from the National Institutes of Health (DE14755 and DE15384, Biomaterials and Organogenesis Training Grants T32-DE07057 and T32-HD07505), DuPont Young Professor Award, Whitaker Foundation (RG ), Nano Materials Initiative Grant (U of M), Center for Biomedical Engineering Research (U of M), and current and past collaborators, students, and postdoctoral fellows. REFERENCES 1. Langer, R., and Vacanti, J. P., Science (1993) 260, Ma, P. X., Tissue Engineering. In Encyclopedia of Polymer Science and Technology, 3 rd Edition, Kroschwitz, J. I., (ed.), John Wiley & Sons, NJ, (2004) 3. Peterson, L., et al., Am. J. Sports Med. (2002) 30, 2 4. Bloch, J., et al., Exp. Neurol. (2001) 172, King, G. N., et al., J. Dent. Res. (1997) 76, Zhang, R., and Ma, P. X., J. Biomed. Mater. Res. (2000) 52, Zhang, R., and Ma, P. 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