3D Fabrication of Polymeric Scaffolds for Regenerative Therapy

Dec 15, 2016 - provides great potential for the freeform fabrication of 3D living organs using cellular spheroids, biocomposite nanofibers, or bioinks...
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3D Fabrication of Polymeric Scaffolds for Regenerative Therapy Greeshma Ratheesh,†,‡ Jayarama Reddy Venugopal,*,† Amutha Chinappan,† Hariharan Ezhilarasu,† Asif Sadiq,† and Seeram Ramakrishna†,§ †

Center for Nanofibers & Nanotechnology, Department of Mechanical Engineering, National University of Singapore, Singapore 117576 ‡ Science and Engineering Faculty, Queensland University of Technology, Brisbane, Australia § Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou 510632, China ABSTRACT: Recent advances in bioprinting technology have been used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. Organ printing and biofabrication provides great potential for the freeform fabrication of 3D living organs using cellular spheroids, biocomposite nanofibers, or bioinks as building blocks for regenerative therapy. Vascularization is often identified as a main technological barrier for building 3D organs in tissue engineering. 3D printing of living tissues starts with potential support of biomaterials to maintain structural integrity and degradation of certain time periods after printing of the scaffolds. Biofabrication is the production of complex living and nonliving biological products from raw materials such as cells, molecules, ECM, and biomaterials. Generally, two basic methods are used for the fabrication of scaffolds such as conventional/traditional fabrication processes and advance fabrication processes for engineering organs. A wide range of polymers and biomaterials are used for the fabrication of scaffolds in tissue engineering applications. 3D additive manufacturing is advancing day-by-day; however, there are various critical challenging factors used for fabricating 3D scaffolds. This review is aimed at understanding the various scaffold fabrication techniques, types of polymers and biomaterials used for the fabrication processes, various fields of applications, and different challenges faced in their fabrication of scaffolds in regenerative therapy. KEYWORDS: biofabrication, 3D printing, polymers, scaffolds, tissue engineering

1. INTRODUCTION The biomaterials used in the form of nanostructures and bioresorbable materials are at its maximum in healthcare. It is estimated that the global market will reach $88.4 billion by 2017 from $44.0 billion in 2012 with a compounded annual growth rate (CAGR) of 15%. Tissue engineering and regenerative products such as scaffolds, tissue implants, and biomimetic materials are expected to reach $89.7 billion by 2016 from $55.9 billion in 2010 with a CAGR of 8.4%. Furthermore, the global market for biomaterials is estimated to be more than 300 billion US dollars increasing statistically at a rate of 20% per year. Similarly, the global market for bone and cartilage tissue engineering strategies is estimated to be about $300 million. Tissue engineering aims to regenerate damaged tissues by combining cells with bioresorbable materials, hydrogels, biomimetic materials, nanostructures, and nanomaterials scaffolds for tissue regeneration. During the last 10 to 15 years, scientists have reported the presence of several sources of stem cells to regenerate the damaged tissue in tissue engineering applications. Tissue engineering scaffolds is considered as potential elements, which constitutes the basic concepts of regenerative medicine and is said to form the core for the development of regenerative therapy. A number of surgical techniques are © 2016 American Chemical Society

developed to replace or repair tissues that are damaged by diseases or trauma. Scaffolds play an important role in tissue engineering applications such as design, fabrication, 3D model, surface ligands, and molecular architecture, interaction between nanoparticles and cells. Pores of the scaffolds have been used in the regeneration of different tissues and organs in the body. The term regenerative medicine implies translational research which replaces or regenerates human cells, tissues, or organs, to restore normal functioning of organs and helps in the migration of small biomolecules in cell-based therapies. The use of stem cells has become common for tissue repair, and healing processes; likewise, cardiac stem cell therapy is used for cardiac regeneration, and functional restorative therapy. The achievement of on-demand fabrication of three-dimensional (3D) human tissues/organ structures is a challenging issue in the scientific and engineering community. The term biofabrication implies the production of complex living and nonliving biological products from cells, molecules, extracellular Special Issue: Biomimetic Bioactive Biomaterials: The Next Generation of Implantable Devices Received: June 29, 2016 Accepted: December 15, 2016 Published: December 15, 2016 1175

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ACS Biomaterials Science & Engineering matrices, and biomaterials.1 It is a combination of cells and developmental biology, material science, and mechanical engineering.1 Harrison led the foundation of modern cell culture by growing tissue in in vitro conditions for regenerative therapy.2 The basic fundamental of organ printing is the concept of tissue fusion and tissue fluidity.1 The concept of tissue fluidity and differential adhesion hypothesis was coined by Forgacs and Steinberg.3,4 All of these concepts such as cell culture, tissue fluidity, and tissue fusion make up the backbone of the biological part of biofabrication. Some of the hardships which are associated with the biological part of biofabrication are cell survival, tissue constructs, vascularization, and tissue maturation.5−10 The sensational study of growing a human ear on a mouse in the late 1990s led to a breakthrough in in vitro fabrication and tissue and organ regeneration. Biodegradable polymer scaffolds were made in the shape of a human ear and were seeded with bovine chondrocytes on the surface; the tissue engineered ear was implanted on the skin of a nude mouse.11 The seeded chondrocytes gradually produced an extracellular matrix (ECM). The mouse with the human ear study later led to many research attempts to create tissues and organs under in vitro conditions by constructing scaffolds with various biocompatible biomaterials such as natural collagen,12 synthetic polymers,13 artificially synthesized bone substitutes (calcium phosphate cement),14 and fibrin glue.15 The common approach of tissue engineering was by seeding cell suspensions on solid, preformed, biodegradable polymeric scaffolds with high porosity that facilitate the transfer of nutrients and cell incorporation to form tissues.

polymers, some of the commonly used polymers for the biofabrication process are PGA, PLA, PLGA, polyanhydride, poly(propylene fumarate), PCL, PEG, and polyurethane (Table 1). Table 1. Applications of Natural and Synthetic Polymers in Tissue Engineering polymers natural polymer agarose alginate chitosan collagen gelatin synthetic polymers PLA, PGA PLGA PCL PEG PVA polyurethane

biomedical applications

references

injectable, tissue engineering, drug delivery supporting matrix to cells, drug delivery, tissue engineering tissue regeneration applications, drug delivery artificial skin, cartilage and bone scaffolds, blood vessels growth factor delivery, neovascularisation

35, 36 37, 38

bone tissue engineering, artificial vessels bone tissue engineering, resorbable sutures bone and cartilage repair, bone regeneration injectable scaffolds for spinal cord injury cartilage regeneration, breast augmentation, and diaphragm replacement meniscus tissue lesions, heart valves, catheters, artificial organs

39−43 44−48 63−65

66−70 66−70 25, 26 30 31 33, 34

2.1.1. Poly(α-hydroxy esters). The most important properties of poly(α-hydroxy esters) are their biocompatibility, biodegradability, and easy processing ability. The degradation pattern of poly(α-hydroxy esters) is by hydrolytic cleavage of the ester bonds which produces the glycolic acid group. The hydrophobic characteristic of PLA helps in the reduction rate of backbone hydrolysis; on the other hand, crystalline structure of PGA plays a crucial role in the rapid loss of PLA−PGA copolymers. Nevertheless, the copolymer of PLA−PGA does not possess any linear relationship with the PLA−PGA ratio. PLA, PGA, and PLGA are commonly used polymers for the biofabrication of scaffolds. Luu et al.21 fabricated a scaffold by the process of electrospinning in which the scaffold was mainly composed of PLGA and PLA−PEG block copolymer for DNA delivery. The results suggest that the DNA released from the scaffolds are capable of cellular transfection and encode βgalactosidase protein. These research group reports to be the first attempt in the incorporation of plasmid DNA within a polymer scaffold by the process of electrospinning. Hacker et al.22 fabricated scaffolds that have the ability to control the unspecific protein adsorption and covalent binding protein or peptide by incorporating amine-reactive diblock copolymers, Nsuccinimidyl tartrate monoamine poly(ethylene glycol)-blockpoly(lactic acid). PLGA, PLA, and PGA has been proven for their application in the regeneration of tissues such as skin, cartilage, blood vessels, nerves, and liver tissues.23 2.1.2. Polyanhydride Polymers. Polyanhydride is synthesized by the process of reaction of diacides with anhydride forming an acetic anhydride prepolymer. Polyanhydride is a highly reactive and unstable synthetic polymer. The biomaterial is generally used for hard tissue engineering applications due to its ability to react with imides and thereby increasing the physical and mechanical properties resulting in polyanhydrideco-imides. Griffin et al.24 fabricated the scaffolds using aligned PLGA bioactive polyanhydride fibrous substrates by an

2. POLYMERS/BIOMATERIALS FOR THE FABRICATIONS OF 3D SCAFFOLDS Biomaterials imply any material, derived from nature or manmade consisting of whole or a part of a living structure. It is otherwise known as devices/scaffolds that create augment or replace a function lost by disease or injury.16 In this context, biomaterials act as a bridging material that combines the scaffold or matrix with living cells or bioactive molecules such as growth factors in order to promote the regeneration of damaged tissues or organs. Scaffolds play a vital role in the field of tissue engineering; they serve as a temporary skeleton for cell adhesion and cell infiltration.17 Biodegradability, high mechanical strength, dimensional stability, high processability, high porosity, high surface to volume ratio, support cell adhesion, proliferation, and induction of cellular response are ideal properties of the scaffolds. Some of the critical parameters for 3D scaffold fabrication must follow high porosity, interconnectivity, mechanical strength and stiffness, cell adhesion and proliferation, nontoxicity, and ease of fabrication and handling. 3D porous scaffolds are developed using natural, synthetic, and composite ceramic materials.18 Polymers are generally used for the fabrication of medical devices for organ and tissue engineering scaffolds. Scaffolds build by using polymers serves various advantages such as biocompatibility, versatility, and biological properties.19 Biomaterials are classified into natural polymers, synthetic biodegradable polymers, and ceramics based on their structural, chemical, and biological properties. 2.1. Synthetic Polymers. Synthetic polymers are tailored for tissue engineering applications and thereby help to mimic biological tissues similar to that of physiochemical and mechanical properties.18,20 The degradation pattern of these polymers is usually by simple hydrolysis which remains constant for every host. Out of the big list of synthetic 1176

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interconnectivity with moduli of 40−400 kPa and porosity of 72−87% using polyurethane Estane 5701-F1 by the process of freeze-drying of dioxane and water in combination with salt leaching for tissue engineering applications. 2.2. Natural Polymers. Natural polymers are classified into three main categories: polynucleotides, which are made up of chains of nucleotides; polyamides, that consist of chains of proteins; and last polysaccharides that are made up of chains of sugars. One of the greatest advantages of natural polymers is its good cell interaction with minimal inflammatory response for organ regenerative therapy. It is a good candidate for scaffold fabrication due to its potential mechanical properties, bioactivity, structure, and biodegradability. Some of the commonly used natural polymers for biofabrication are collagen, gelatin, silk, fibrin, alginate, agarose, chitosan, and glycosaminoglycans (Table 1). 2.2.1. Agarose. Agarose isolated from seaweed and has a wide range of applications in controlled release of drugs. One of the unique characteristics of agarose in biofabrication is the ability to tune mechanical properties for tissue engineering applications. Alaminos et al.35 engineered rabbit cornea by seeding three different types of cells, namely, epithelial, stromal, and endothelial cells on gels of human fibrin and 0.1% agarose. The studies suggested that cultured endothelial cells expressed COL8 gene and epithelial cells expressed KRT12. Microscopic evaluation suggests that the epithelial cells tend to form a normal stratified layer and that the stromal keratocytes proliferated rapidly. Moreover, the endothelial monolayers possess a pattern similar to that of normal corneal endothelium. Roman et al.36 employed a thermal gelation process for the fabrication of biodegradable scaffolds of β-TCP and agarose. Multiple concentrations of agarose and ceramic vancomycin was added in order to facilitate inclusion during scaffold preparation and thereby improve the graft performance. The study suggests that the materials possess various advantages such as ease of injection, reinforced hydrogel behavior, and drug release profile, which is highly dependent on the composition of materials. 2.2.2. Alginates. Alginates, otherwise known as alginic acid, are an anionic polysaccharide derived from algae. The polysaccharide is made up of two repeating monosaccharides, namely, L-glucuronic acid and D-mannuronic acid. The polysaccharide has a wide range of application in cartilage and bone tissue engineering. The degradation pattern of calcium alginate can be enhanced by the use of chelating agents such as EDTA or enzymes. Li et al.37 developed scaffolds with chitosan and alginate which possess high biological and mechanical properties for bone tissue engineering. The osteoblasts attached to the scaffolds thereby proliferate and deposit a calcified matrix. Dar et al.38 engineered alginate scaffolds to achieve 3D high density cardiac constructs for cardiac tissue engineering. The alginate scaffolds possess highly interconnected pores with porosity greater than 90% to mimic the natural extracellular matrix of the native tissues. 2.2.3. Chitosan. Chitosan is a linear polysaccharide produced by the process of deacetylation of chitin, in which chitin is treated with alkali sodium hydroxide. The semicrystalline nature of the polymer makes it biocompatible under in vivo conditions.39 Chitosan is used for a wide range of applications in food, cosmetics, biomedicals, and pharmaceuticals.40,41 The degradation pattern of chitosan is controlled by the residual acetyl functional groups.19 Ma et al.42 constructed collagen and chitosan porous scaffolds by a freeze-drying process, then

electrospinning technique to biomimic the native nerve structure for neuronal tissue engineering. 2.1.3. Polycaprolactone. PCL is a semicrystalline polymer with low melting point and has the ability to form a blend with other polymers. The degradation pattern of PCL is through hydrolysis, and the degradation process takes up to 24 months. One of the promising characteristics of PCL is its biocompatible nature. Zhang et al.25 fabricated fibrous scaffolds of gelatin fibers and gelatin/PCL composites by electrospinning techniques for engineering three-dimensional tissues. Oh et al.26 developed PCL cylindrical scaffolds by centrifugation method to investigate the pore size effect on cells and tissue interactions. 2.1.4. Poly(propylene fumarate). Poly(propylene fumarate) is a unsaturated linear polyester and has a degradation mechanism similar to that of poly(α-hydroxyester), which is controlled by cross-linking of vinyl monomers with unsaturated double bonds.27 Peter et al.28 investigated the biodegradability and biocompatibility of poly(propylene fumarate) (PPF) under in vivo conditions by varying the PPF to N-vinylpyrrolidone ratio and PPF to β-tricalcium phosphate. The study proves that the lack of β-tricalcium phosphate resulted in weak and insufficient mechanical properties for bone replacement therapy. Fisher et al.29 studied tissue response of photo-crosslinked PPF scaffolds of varying porosity and pore size that were implanted subcutaneously and in cranial defect rabbits to prove biocompatibility. 2.1.5. Polyethylene Glycol. Polyethylene glycol (PEG) is a hydrophilic polymer that has the ability to minimize adverse immune response by preventing other proteins getting adhered to the scaffold surface. It has the ability to control the attachment of cells, thereby changing the copolymer PEG to be biocompatible. Comolli et al.30 studied the ability of poly(Nisopropylacrylamide)-co-poly(ethylene glycol) (PNIPAAmPEG) to be used as an injectable scaffold for the repair of spinal cord injury (SCI). This study suggested that PNIPAAmPEG scaffolds possess similar mechanical properties to that of native neuronal tissue and biocompatible with bone marrow stromal cells for tissue engineering applications. 2.1.6. Poly(vinyl alcohol). Poly(vinyl alcohol) (PVA) is a biocompatible polymer that possesses the ability of swelling and thereby holding large amounts of water. The reactive alcohol group can be easily modified by physical or chemical crosslinking to transform into hydrogels. However, one of the greatest limitations of PVA is its incomplete degradation properties. The polymer has a wide range of applications such as cartilage regeneration, breast augmentation, and diaphragm replacement.31 Aramwit et al.32 fabricated scaffolds using silk sericin (a byproduct from silk) and PVA at varying concentrations with and without glycerin and genipin. The study proves that composition of scaffold plays a significant role in its physical properties. 2.1.7. Polyurethane. The unique properties of polyurethane such as durability, elasticity, elastomer-like characteristics, fatigue resistance, and compliance also make it an important substrate in the field of biomedical tissue engineering. The biomaterials possess a wide range of degradation patterns, namely, thermal and enzymatic hydrolysis and oxidation. Verdonk et al.33 studied tissue ingrowth in polyurethane scaffolds implanted in partial meniscal lesions patients. The data from DCE-MRI and biopsy/histologic analysis prove consistent regeneration of tissue in polyurethane scaffolds. Hejikants et al.34 constructed porous scaffolds with high 1177

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fabricated by electrospinning PCL/gelatin nanofibers on polyurethane membrane for wound dressing applications. Lien et al.55 proposed a novel approach in constructing gelatin scaffolds by varying cross-linking temperature from 10 to 25 °C. Gelatin scaffolds cross-linked with genipin, which possesses varying pore size from 50 to 500 μm used to study cell metabolism and proliferation. Cell growth highly depends on the fiber morphology, wettability, and pore size of the scaffolds in articular cartilage tissue engineering. 2.3. Bioceramics. The biocompatible bioceramic is used for the repair and reconstruction of diseased and damaged parts of the skeletal system. Bioceramics possess various advantages such as bioinert, resorbable, bioactive, and porous scaffolds for tissue ingrowth. They have various medical applications, and commonly used as bioceramics are alumina, zirconia, tricalcium phosphate, hydroxyapatite, bioactive glasses, glass-ceramics, and hydroxyapatite-coated metals. The degradation process of bioceramics depends on the composition which may vary from material to material.56 2.3.1. Calcium Phosphate. Calcium phosphate ceramics are of various kinds; most of the materials are resorbable and tend to dissolve under exposure to physiological environments. The group of calcium phosphates such as tetra-calcium phosphate, amorphous calcium phosphate, alpha tricalcium phosphate, beta tricalcium phosphate, and hydroxyapatite are generally used for bone tissue engineering. Tissue compatibility and porous structure make it a potent candidate as a bone substitute. The formation of apatite-like mineral phase of bone and teeth make it suitable in their applications. Mondrinos et al.57 fabricated scaffolds with polycaprolactone or homogeneous composites of PCL and calcium phosphate by an injection molding method. The study suggests that PCL and PCL-CaP scaffolds are suitable for hard tissue repair due to its cytocompatibility and mechanical property. Doernberg et al.58 studied the in vivo behavior of calcium phosphate scaffolds with varying pore size of 150, 260, 510, and 1220 μm. The results proved that scaffolds with a pore size 510 μm has a highly resorbable ability when compared to 150 and 260 μm pores, which was due to lower bone content and high content of soft tissues for bone tissue engineering. 2.3.2. Hydroxyapatite. Hydroxyapatite shows chemical similarity to that of mineral components of the bone. It is considered thermodynamically stable at physiological pH and also considered as an active participant in forming very strong chemical bonds with the surrounding bone tissue and hence has a wide range of application in bone repair and regeneration. It is generally used as coating of material in titanium and titanium alloys due to its bioactive properties.56 Deville et al.59 synthesized porous hydroxyapatite scaffolds by freeze casting with high compression and porosity by which hydroxyapatite lacks the load bearing ability. Leukers et al.60 employed a 3D printing technique to fabricate hydroxyapatite based scaffolds with complex internal structures and high resolution which was seeded with MC3T3- E1 cells. The study proved that the cells were able to possess close contact with hydroxyapatite granules by proliferating deep into the scaffold structure for forming tissues in bone tissue engineering. 2.3.3. Bioactive glass. Bioactive glasses are ceramic materials that are different from conventional glasses. The calcium and phosphate content of bioactive glass is similar to that of bone hydroxyapatite. Bone graft scaffolds, coating materials for dental implants, are some of the common applications of bioactive glasses. These grafts have the ability to bind bone and soft

treated the scaffolds with glutaraldehyde cross-linking to provide good biocompatibility and biostability. This plays a prominent role in dermal equivalents for wound healing. VandeVord et al.43 investigated the biocompatibility of chitosan scaffolds transplanted in mice. Various studies such as macroscopic inspection, histological assessment, gram staining and limulus assay, angiogenic activity test, lymphocyte proliferation assay, and ELISA were carried out and proved that chitosan is a potentially implantable biomaterial for tissue regeneration.43 2.2.4. Collagen. Collagen is a most abundant protein present in mammals, and types I and IV collagens are commonly used for tissue engineering applications. Various properties like mechanical stability, strength, and toughness of collagen are similar to that of tendons and ligaments, skin, cornea, bone, and dentin. Moreover, collagen has the ability to regulate cell attachment and proliferation by providing cellular recognition.44 All these properties are important for various applications and are processed into several patterns such as gels, solutions, filamentous, tubular, and composite matrixes.45−48 Ma et al.49 produced a stable collagen layer on the surface of 3D PLLA scaffolds along with fibroblast growth factor (bFGF) by modifying the grafting and coating method in which Fe2+/-OOH was used instead of UV induced graft polymerization. The study suggests that the cell spreading, growth, and distribution of chondrocytes were improved by the collagen layer. Zhong et al.50 employed an electrospinning method for fabricating nanofibrous collagen scaffolds with welldefined architecture to mimic native ECM. The study suggests that the alignment of the collagen fibrous scaffold plays a significant role in cell adhesion, orientation, proliferation, and interaction between the cells and fibers. 2.2.5. Fibrin. Fibrin is a nonglobular protein that plays a significant role in blood clotting. A mixture of fibrinogen and thrombin forms fibrin glue which has a potent role as a carrier. The degradation process of fibrin is by hydrolytic or enzymatic cleavage. Fibrin has a wide range of applications especially in cartilage tissue engineering.51 The main advantages of fibrin are its biocompatibility, biodegradability, and hemostasis properties. Bensaid et al.52 investigated the possibility of using fibrin gel as a delivery system for human mesenchymal stem cells. The study suggests that the optimal concentration of fibrinogen and thrombin activity for cell spreading and proliferation is 18 mg/mL and 100 IU/mL, respectively. The in vivo study proved that hMSCs migrated from fibrin gels and invaded calcium carbonate based ceramic scaffolds, which proves that fibrin gel is a potent candidate for hMSCs delivery. Christman et al.53 proved the ability of fibrin glue to improve cell transplant retention, reduce infarct expansion, and induce neovasuclature formation. In this study, bovine serum albumin (BSA), fibrin glue, skeletal myoblast in fibrin glue, and skeletal myoblast in BSA were injected into the left ventricle of rats which was occluded in myocardial infarction. The immunohistochemistry studies suggested that the myoblast cell spreading was significantly high in fibrin glue and has a potent ability to improve cellular cardiomyoplasty and myocardial infarction. 2.2.6. Gelatin. Gelatin is a biodegradable polymer derived from collagen, and also, great advantages are the minimal immune response and the ability to be easily fabricated as scaffolds. They are made up of large numbers of residues, namely, glycine, proline, and 4-hydroxyproline. Chong et al.54 designed a dermal wound healing construct which is costeffective in skin tissue engineering. The constructs was 1178

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ACS Biomaterials Science & Engineering Table 2. Advantages and Disadvantages of Conventional Processes for Fabricating Scaffolds techniques solvent casting and particulate leaching gas foaming phase separation melt molding freeze-drying

advantages

disadvantages

produce highly porous scaffolds (93%) organic solvent free process, porosity, and pore size can be controlled in scaffolds process conducted at low temperature - incorporation of bioactive molecules morphology and shape can be tuned for the scaffolds no separate leaching process is required for the fabrication of scaffolds

refs

only produce membrane with 3 mm thickness, limited mechanical property yields structure with large unconnected pore and nonporous external surface, limited mechanical property solvent selection and processing temperature is crucial possibility of residual porogen and high processing temperature long processing temperature and small pore size

72−74 72, 76−79 73, 81, 82 73, 84, 85 73, 89, 90

Figure 1. Schematic representation of solvent casting and the particulate leaching process for developing porous scaffolds.

Figure 2. Schematic representation of the gas foaming process for preparing porous scaffolds.

tissues and thereby stimulate bone growth.61 Fu et al.62 constructed porous scaffolds by a polymer foam replication technique using 13−93 bioactive glass which possesses microstructure similar to that of human trabecular bone. The in vitro study proves that the 13−93 bioactive glass scaffold is a potent candidate in bone repair and regeneration.

into the scaffolds. The salt is later leached out by immersing the matrix in water, thereby producing porous structure of the scaffolds.72 The processes produce porous scaffolds with porosity values up to 93%. A disadvantage of this technique is to produce only thin membranes up to 3 mm thick.73,74 Figure 1 shows the schematic representation of the solvent casting process for developing scaffolds. Sin et al.75 employed the solvent casting/particulate leaching method to prepare 3D porous polyurethane scaffolds for cardiac tissue engineering. This method was modified by employing a centrifugation process for improving pore uniformity and pore interconnectivity of the scaffolds. A mixture of solvent, namely, dimethylformamide (DFM) and tetrahydrofuran, was (THF) used in this method for casting scaffolds. The pores of the scaffolds were coated with type I collagen acidic solution. Polyurethane scaffolds coated with type I collagen showed uniform distribution of cells, when seeded and cultured with human aortic endothelial cells (HAECs) for cardiac tissue engineering. 3.1.2. Gas Foaming. Gas foaming is a process molded for biodegradable polymers subjected to high pressure with gas foaming agents such as CO2, nitrogen, water, or fluoro-

3. PROCESS FOR SCAFFOLD FABRICATION 3.1. Conventional Methods. Conventional scaffold fabrication techniques are processes that create scaffolds with continuous, uninterrupted porous structures which lack longrange channeling microarchitecture. The commonly used conventional scaffold fabrication processes are solvent casting and particulate leaching, phase separation, gas foaming, melt molding, and freeze-drying.71 Table 2 gives information about the advantages and disadvantages of conventional processes for fabricating scaffolds. 3.1.1. Solvent Casting and Particulate Leaching. Solvent casting and particulate leaching are processes in which a polymer in an appropriate solvent is uniformly mixed with salt particles of a specific diameter. The solvent evaporates, thereby leaving behind a matrix of polymer with salt particles embedded 1179

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Figure 3. Schematic representation of the phase separation method for developing porous scaffolds.

Figure 4. Schematic representations of the melt molding process for preparing porous scaffolds.

form.76−78 Later, the polymer is subjected to a sudden decrease in the solubility of gas. This sudden decrease in the solubility of gas in the polymer brings the pressure of CO2 back to atmospheric level resulting in nucleation and the growth of gas bubbles with a size range of 100−500 μm. The advantage of the gas foaming technique is that the process is free of organic solvents. However, the process carries certain drawbacks such as the yield of structure with huge unconnected pores and nonporous external surface.72,79 The graphical representation of the process for the gas foaming method is presented in Figure 2. Yoon et al.80 fabricated PLGA scaffolds incorporated with dexamethasone by the gas foaming/salt leaching method for burst release of drugs. The drug release was also confirmed by in vitro cell culture in which dexamethasone dramatically suppressed the proliferation of lymphocytes and smooth muscle cells in vitro. The study proved that dexamethasone-releasing PLGA scaffolds were used as the anti-inflammatory porous prosthetic device or as a temporal biodegradable stent for the reduction of intimal hyperplasia in restenosis. 3.1.3. Phase Separation. The phase separation process polymer was dissolved in molten phenol or naphthalene and biologically active molecules such as alkaline phosphatase.81 The solution is quenched to produce a liquid−liquid phase separation, polymer rich and polymer poor phase. The polymer poor phase is removed mechanically, and the polymer rich phase solidifies leaving behind a porous scaffold which is embedded with bioactive molecules on its surface. The schematic for the process of fabricating porous scaffolds is shown in Figure 3. This process is carried out at low temperatures and is helpful for the incorporation of bioactive molecules into the scaffolds. Using phase separation techniques, nanoscale fibrous structure could be formed; this mimics natural ECM architecture and provides a better environment for cell adhesion and proliferation.82 The selection of solvent and phase separation temperature is a critical parameter for this method for fabricating the scaffolds. Nam and Park83 fabricated porous biodegradable scaffolds with PLA and its copolymer PLGA by employing thermally induced phase separation (TIPS) techniques for tissue engineering and drug delivery applications. The binary composition of solvent and non-

solvents was dioxane and water. The quenching temperature helped to tune the pore size of the scaffolds. An amorphous polymer with a slow cooling rate produced macropores and semicrystalline polymer with fast cooling rate to produce microporous scaffolds. In vitro cell culture study was conducted by using these scaffolds for recombinant human growth hormone (rHGH) to test the cell delivery to the tissues. 3.1.4. Melt Molding. Melt molding process polymer mixed with a suitable porogen is filled in a mold and heated above the glass transition temperature while applying pressure to the mixture. The polymer tends to bind together forming a scaffold with specific surface morphology. Then the mold is removed, and the porogen is leached out of the scaffolds leaving behind a porous structure with specific surface. Similar to the gas foaming technique, it is a nonsolvent fabrication process presented in the schematic representation of Figure 4. The drawback of melt molding includes the possibility of residual porogen and high processing temperatures that preclude the ability to incorporate bioactive molecules.84,85 Oh et al.86 fabricated PLGA/PVA scaffolds by using a blend of PLGA, and PVA helps in improving the hydrophilicity and cell compatibility. The scaffolds were analyzed by in vitro (human chondrocytes) and in vivo conditions (skull defect of rabbit), and the study proved that the scaffolds made up of the PLGA/ PVA blend had better cell adhesion and growth under in vitro conditions for bone in-growth and new bone formation inside the scaffolds under in vivo conditions when compared to control PLGA scaffolds. The hydrophilicity and cell compatibility of the scaffolds improved by the addition of 5% PVA to PLGA during the fabrication of PLGA/PVA blend scaffolds. 3.1.5. Freeze-Drying. Freeze-drying technique polymer solution is cooled down to a certain critical temperature and leads to a frozen state; the solvent forms ice crystals, then the polymer molecules are forced to aggregate into the interstitial spaces. The solvent removal is accomplished by sublimation in which a pressure lower than the equilibrium vapor pressure of the frozen solvent is applied, thereby creating interconnected porous scaffolds.87,88 The porosity of the scaffolds depends on the concentration of the polymer solution, and pore size distribution is affected by the freezing temperatures.89 The 1180

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Figure 5. Schematic representation of the freeze-drying process for preparing porous scaffolds.

scaffolds which could serve as a cell carrier in neural tissue engineering. Mobarakeh et al.98 fabricated random and aligned biocomposite nanofibrous scaffolds of PCL/gelatin suitable for nerve tissue engineering applications. This study suggests that in vitro analysis such as the MTS assay and SEM studies on the scaffolds with PCL/gelatin concentration of 70:30 seeded with C17.2 nerve stem cells enhanced cell proliferation, differentiation, and neurite outgrowth parallel to aligned fibers. Li et al.99 fabricated scaffolds using silk fibroin fibers incorporated with BMP-2 and/or nanoparticles of nHAP by the electrospinning technique. Li et al.100 studied the ability of PCL nanofibrous scaffolds to support chondrogenesis of MSCs under in vitro conditions for cartilage tissue engineering. The ability of MSCs in the presence of TGF-β1 to differentiate into the chondrocyte phenotype and the level of chondrogenesis was compared with that of MSCs maintained as cell aggregates or pellets. The result suggests that PCL nanofibrous scaffolds is a practical carrier for MSCs transplantation and has greater importance in cartilage repair and regeneration. 3.2.2. Rapid Prototyping. Rapid prototyping is advancing in the field of scaffold fabrication process used for quick fabrication on computer assisted/aided design (CAD) and manufacturing techniques (Table 3). This method creates parts in an additive in a layer-by-layer fashion. Rapid prototyping gives better control of external macro-shape and internal microstructure when compared to the conventional scaffold fabrication technique. It helps in early detection and correction of design flaws. The rapid prototyping technique binds liquid, powder, and sheet material to form complex parts.101 Some of the commonly used methods are as follows. 3.2.2.1. Fused Deposition Modeling. Fused deposition modeling (FDM) is a rapid prototyping technology used to heat 3D scaffolds for fabrication by slicing layer by layer for printing objects.102 In this setup, the liquefier is fed with the filament of desired material and melted by heat before extrusion from the nozzle. The polymer melt is extruded from the nozzle and deposited on a layer by layer process in order to create scaffolds.103 The melting temperature is a factor that depends on the process temperature and materials used for FDM are PLA, ABS, and nylon.103 Zein et al.102 produced scaffolds with PCL, which possess a honeycomb-like pattern with interconnected channels and controlled porosity and channel size. Hutmacher et al.104 used FDM technology to design and fabricate bioresorbable 3D scaffolds with a fully interconnected pore network. The study showed that cells like fibroblast and osteoblast can proliferate, differentiate, and produce a cellular tissue in the interconnected 3D PCL matrix. Hsu et al. and Yen et al. fabricated scaffolds with fiber stacking orientation in which PLA was used as a feed material.105,106 Tellis et al.107 developed a 3D CAD model of canine trabecular bone scaffolds of varying pore structure by a micro CT technique exported to FDM, which in turn produced polybutylene terephthalate

greatest advantage of the freeze-drying technique is that it neither requires high temperature nor a separate leaching step. Some of the disadvantages are smaller pore size and long processing time for freeze-drying methods.90 Graphical representation of the process of the freeze-drying method for fabricating biocomposite scaffolds are shown in Figure 5. Wu et al.91 fabricated the porous scaffolds with microtubules orientation structure with high porosity, 98% with a width and length from 50 to 100 μm and 100−500 μm by a freezedrying process for tissue engineering applications. 3.2. Advances in Biofabrication Techniques. 3.2.1. Electrospinning. In the electrospinning process, high voltage is applied to a polymer solution which produces electrostatic force at the tip of the needle, thereby forming a Taylor cone which elongates into a fluid jet; this charged fluid jet is collected on a grounded collecting device (Figure 6A). Electrospinning is

Figure 6. Graphical representation of the electrospinning process. (A) Basic electrospinning setup and (B) Core−shell/hollow fiber setup.

able to produce nanofibers with diverse forms, such as core− shell fibers, hollow fibers (Figure 6B), and three-dimensional fibers. Electrospun nanofibers have been generally used for tissue engineering applications more than a decade and have gained lot of interest in neural tissue engineering.92−94 Yoshimoto et al.95 employed an electrospinning technique to fabricate PCL scaffolds for seeding neonatal rat mesenchymal stem cells (MSCs) cultured under dynamic culture. The results suggest that the surface of the cell−polymer constructs was covered with multilayers of cells, expression of type I collagen, and mineralization of cells. Electrospinning has a prominent role in fabricating porous nanofibrous membranes in skin tissue engineering. Kumar et al.96 fabricated PLGA nanofibers with varying fiber diameters, porosity, and tensile strength moduli within the range of normal human skin. The scaffolds seeded with human skin fibroblasts showed significant cell growth on the fiber matrix for wound healing. Yang et al.97 employed an electrospinning technique to fabricate PLLA nanofibrous 1181

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118, 119 cartilage freeze-drying low temperature, biomolecules

liquid based liquid based digital light processing liquid frozen deposition

photopolymer resin

(PBT) trabeculae scaffolds along with four other scaffolds. Schematic of FDM processes and photographic image of the FDM-1650 building a scaffold presented in Figure 7.107 3.2.2.2. Selective Laser Sintering. Selective laser sintering (SLS) is a commonly used approach for 3D scaffold fabrication, and a high power laser is used for the additive manufacturing process. Small particles of plastic, metal, ceramic, and glass powder are fused with carbon dioxide laser into desired 3dimensional shapes. The SLS technique helps in rendering complex porous scaffolds. Figure 8 shows the schematic of the SLS method for developing porous scaffolds. Zhou et al.108 employed the SLS method to prepare porous scaffolds by the use of a bionanocomposite microsphere made up of carboxy hydroxyapatite (CHAp) nanospheres in the poly(L-lactide) (PLLA) matrix. The micro and nanospheres are prepared by emulsion techniques, and these CHAp nanostructures were embedded with PLLA microspheres thereby forming nanocomposites. SLS-Sinterstation 2000 machine was used to fabricate the porous scaffolds of PLLA microspheres and PLLA/CHAp nanocomposite microspheres. Williams et al.109 computationally designed PCL scaffolds and fabricated the scaffolds using SLS techniques. The scaffolds produced through the SLS technique possess porous architecture and sufficient mechanical properties suitable for bone tissue engineering. Tan et al.110 studied the fabrication of solvent free porous polymeric and composite scaffolds using SLS processes for the fabrication of polyetheretherketone (PEEK) and hydroxyapatite (HA). The study suggested that a high melting point polymer can be laser sintered in a much lower temperature to incorporate bioactive material (hydroxyapatite) into the polymer to develop potential scaffolds for tissue engineering. Chua et al.111 created tissue engineering scaffolds by administrating the SLS technique in which a biocomposite blend of poly(vinyl alcohol) and hydroxyapatite was subjected to laser-sintering process for bone tissue engineering. 3.2.2.3. Stereolithography. The additive manufacturing process is stereolithography (SLA), a technique that works by using polymer resin materials for the fabrication of scaffolds. The process employs a single laser beam (UV) to polymerize or cross-link photopolymer resin.103,113 This process used a liquid monomer to solidify the scaffold in a layer by layer fashion. The UV spot beam draws a preprogrammed design on the photopolymer resin surface which solidifies and forms layer, and this process is continued until the desired 3D structure of the scaffold is formed.114 Figure 9 shows the schematic representation of the SLA process for fabricating fibrous scaffolds. Cooke et al.115 utilized a biodegradable resin mixture of diethyl fumarate (DEF), poly(propylene fumarate) (PPF) and photoinitiator bis-acylphospine oxide (BAPO) for the construction of scaffolds. Ravoos et al.116 constructed a flexible and elastic poly(trimethylene carbonate) (PTMC) scaffold for cartilage tissue engineering. These scaffolds constructed with a methacrylate PTMC macromer possessed a pore diameter of 350 ± 12 μm and porosity of 54.0 ± 2.2%. Mapili et al.117 developed scaffolds by simple layer by layer stereolithography method that could carry ligands, ECM components, and growth factors. The structural component of the scaffolds was built with photocross-linkable poly(ethylene glycol) dimethacrylate (PEGDMA). Cell attachment was improved by incorporation of adhesive peptide or ECM components to the scaffolds. The study suggests that such scaffolds help to understand the cell

polymers

121, 122

materials are in powder form; weak bonding between powder particles; rough surface; might require postprocessing high resolution, fast processing, less shrinkage cost, toxic resin powderbased D printing

FDM

SLS

bone

123−129

102−107

bone, adipose tissue, cartilage bone fast processing; low costs; no toxic components; water used as binder

high temperatures, uncontrolled porosity, rough surface

high temperature

109−111

114−116

bone, cartilage, heart valves bone, cartilage toxic (reactive resins), high cost

mechanical strength, easy to remove support material, easy to achieve small features mechanical strength, high accuracy, broad range of materials, fast processing low costs, good mechanical strength, solvent not required

polymers, wax, or wax compounds, e.g., poly 1500, tusk 2700, protogen white, flex 70B, NeXt etc. metals, ceramics, bulk polymers, e.g., PEEK-HA, PCL etc. thermoplastic polymers/ceramics, e.g., polyphenylsulfone (PPSF), polycarbonate (PC), PCL-HA etc. powder of bulk polymers; ceramics, e.g., PLGA, starch based polymers liquid based powderbased solidbased SLA

advantages materials materials type technique

Table 3. Different Rapid Prototyping Technique: Advantages and Disadvantages

disadvantages

application

reference

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Figure 7. Fused deposition model for the fabrication of tissue engineering scaffolds. (A) Schematics of the FDM process and (B) photographic image of the FDM-1650 building a scaffold.107 Reprinted with permission from ref 107. Copyright 2008 Elsevier.

Figure 8. (A) Schematic representation of selective laser sintering process, (B) scanning electron microscope images of the microstructure of PCL scaffolds prepared by SLS at a magnification = 45×, bar = 500 μm; (C) magnification = 100×, bar = 200 μm.112 Panels B and C were used with permission from ref 112. Copyright 2013 Creative Commons.

which possesses a low temperature platform which helps to maintain the temperature. Figure 10 shows the schematic representation of LFDM model for the fabrication of scaffolds. Yen et al.118 fabricated precision scaffolds with PLGA by employing the LFDM technique; the scaffolds possess mechanical strength similar to that of native articular cartilage. Hsu et al.119 employed LFDM processes for the fabrication of chitosan scaffolds and treated them with air plasma (AP) for cell attachment and proliferation. This study proved that reduced contact angle, increased surface charge and nanometric roughness, increase in nitrogen and oxygen within the scaffolds, and thereby increase the proliferation and bone mineral deposition. The study proved that the chitosan scaffolds

behavior in complex microenvironments and help in engineering complex hybrid tissue structures for tissue engineering. 3.2.2.4. Liquid Frozen Deposition Manufacturing. The fused deposition manufacturing process possesses various limitations, among which temperature plays a very critical role in cells and other biomolecules such as growth factors.103 Fused deposition manufacturing (FDM) uses high temperature to melt the material which can cause adverse effects on cells and biomolecules. The cell survival and coprinting with material to form suitable scaffolds and printing of scaffolds incorporated with biomolecules such as growth factors are very critical in the FDM. These major limitations of FDM were overcome by a liquid frozen deposition manufacturing technique (LFDM), 1183

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Figure 9. (A) Schematic representation of the stereolithography process, (B) SEM image of scaffolds prepared by the stereolithography process, showing (a) a single-layer scaffold and (b) multilayered scaffolds.117 Reprinted with permission from ref 117. Copyright 2005 Wiley Periodicals, Inc.

Figure 10. Schematic representation of the liquid frozen deposition manufacturing process for developing scaffolds. Figure 11. Schematic representation of digital light processing for fabricating constructs.

manufactured by LFD and treated with AP are suitable candidates for bone tissue engineering applications. 3.2.2.5. Direct Metal Laser Sintering. Direct metal laser sintering (DMLS) is a technique employed for printing 3D objects for metal fabrication for regenerative therapy. The technique builds up components in layers by depositing metal materials.120 A thin layer of materials is placed on the building platform, and a laser beam with the help of computer guidance fuses the powder into the desired 3D structures, and the process is repeated for the fabrication of scaffolds.120 The DMLS method works by an injection molding process in which an aluminum mold is required for the fabrication of scaffolds. Moreover, the production of complicated shapes and structures is very difficult in DMLS. These biomaterials methods are not suitable for human cells as high temperature in the chamber of injection molding could damage the cells. All these limitations make this process unsuitable for printing human spare parts in healthcare. 3.2.2.6. Digital Light Processing. Digital light processing (DLP) technique is a 3D additive manufacturing process that uses a laser to cure polymers (Figure 11). The digital mirror device (DMD) has an array of micro mirrors rotating independently to control the laser beam in curing polymers. Dean et al.121 fabricated scaffolds using a resorbable polymer poly(propylene fumarate) (PPF) by a continuous digital light processing technique. Lu et al.122 utilized PEGDA hydrogels for scaffolds fabrication through successful encapsulation of murine bone marrow derived cells within the fabricated scaffolds. 3.2.2.7. 3D Bioprinting. The term 3D printing or additive manufacturing (AM) is the process of synthesizing three-

dimensional objects. 3D bioprinting is another promising approach in the biofabrication process, and bioprinting technology is the key element of organ printing.123,124 Healthy human cells are injected through a very thin nozzle on the bed surface. However, the cell density, vascularization, and tissue maturation remain a challenge.125−127 The process involves successive layering of materials under computer guidance.128 One of the challenging and most widely studied areas in tissue engineering is 3D organ printing. Organ printing is evolving into a promising approach for engineering new tissues and organs.129 Organ printing follows three basic steps; first, preprocessing which involves the development of blueprint for organs, second, processing which involves the actual organ printing, and third, the postprocessing process which involves organ conditioning and accelerated organ maturation.129 3.2.2.8. Organ Printing: Pre-processing. The first step in the organ printing process is the design of a blue print in the form of a computer aided design for designing the organ. It is defined as computer compatible precise spatial information about the localization of cells in 3D organs or otherwise known as the addresses of each cellular or extracellular component to build tissues or organs. There are several ways obtain the information about the anatomy, histological structure, composition, and topology of human organs for 3D printing; for instance, the recent advancement in clinical bioimaging and ultrasound make it possible to find the gross anatomical characteristics of organs. This method gives information about the patient’s specific anatomy of the organs. The resolution of 1184

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4. ARCHITECTURE DESIGN OF THE SCAFFOLDS Scaffold’s architecture plays an important role in the diffusion of nutrients and metabolic wastes, spatial organization of cell growth, and most importantly to develop specific biological function in tissues. The porosity of a scaffold is a very critical parameter to determine the diffusion of nutrients and metabolic wastes, cell migration, and neovascularization. The high surface area to volume ratio of the scaffolds helps in cell adhesion, migration, and proliferation. Studies suggest that the optimum pore size suitable for neovascularization is 5 μm; a pore size of 5−15 μm is beneficial for fibroblast ingrowth, and the pore size required for fibrovascular tissue is greater than 500 μm, and the regeneration of adult mammalian skin is effective at a pore size of 20−125 μm. 139,140 Compared to the conventional biofabrication technique, advanced biofabrication techniques are able to design and control the architecture of scaffolds, which can build scaffolds with reproducible morphology and microstructure. The porous structure created by the conventional lyophilization technique is of random orientation. To overcome this, an advanced technique called freeze casting was used, which produces linearly oriented architecture. In this process, the polymer slurry is subjected to a temperature gradient which allows nucleation and growth of the ice crystals. This technique is appropriate for the fabrication of nerve conduits with a porous structure that has the ability to guide axon growth in neural tissue engineering. Another important parameter is the surface roughness of the scaffolds. Milleret et al. 141 investigated the processing parameters of electrospun Degrapol (DP) and PLGA fibrous scaffold architecture and subsequent reaction of partially heparinized scaffolds. The study suggests that the blood reaction depends on the diameter of fibers and scaffold roughness. Scaffolds with thin fibers (diameter