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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 ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00370 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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ACS Biomaterials Science & Engineering

3D Fabrication of Polymeric Scaffolds for Regenerative Therapy Greeshma Ratheesha,b, Jayarama Reddy Venugopala, Chinappan Amuthaa, Hariharan Ezhilarasua, Asif Sadiqa, Seeram Ramakrishnaa,c a

Center for Nanofibers & Nanotechnology, Department of Mechanical Engineering, National

University of Singapore, Singapore 117576 b c

Science and Engineering Faculty, Queensland University of Technology, Brisbane, Australia

Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University,

Guangzhou 510632, China

Corresponding Author J. Venugopal/Prof. Seeram Ramakrishna Email: [email protected]/[email protected]/

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Abstract Recent advances in bioprinting technology 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 degrade certain time periods after printing of the scaffolds. Biofabrication is the production of complex living and non-living biological products from raw materials such as cells, molecules, ECM and biomaterials. Generally, two basic methods used for the fabrication of scaffolds such as conventional/traditional fabrication process and advance fabrication process 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 aimed to understand the various scaffolds fabrication techniques, types of polymers and biomaterials used for the fabrication process, various fields of applications and different challenges faced in their fabrication of scaffolds in regenerative therapy.

Keywords: Biofabrication, 3D printing, polymers, scaffolds, tissue engineering.


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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 to $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, 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 estimated to be about $300 millions. 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 developed to replace or repair tissues that are damaged by diseases or trauma. Scaffolds plays an important role in tissue engineering applications such as design, fabrication, 3D model, surface ligands and molecular architecture, interaction between nanoparticles and cells; and pores of the scaffolds are 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 process; 3 ACS Paragon Plus Environment


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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 non-living biological products from cells, molecules, extracellular matrices and biomaterials1. It is a combination of cells and developmental biology, material science and mechanical engineering1. Harrison (1907) led the foundation of modern cell culture by growing tissue expands in an in vitro condition for regenerative therapy2. The basic fundamental of organ printing is the concept of tissue fusion and tissue fluidity1. The concept of tissue fluidity and differential adhesion hypothesis was coined by Forgacs and Steinberg’s3,4. All these concepts such as cell culture, tissue fluidity and tissue fusion make up the back bone of biological part of biofabrication. Some of the hardships which are associated with the biological part of biofabrication are cell survival, tissue construct, vascularization and tissue maturation5-10. The sensational study of growing human ear on mouse in the late 1990’s, lead to a breakthrough in in vitro fabrication; tissues and organs regeneration. Biodegradable polymer scaffolds was made in the shape of a human ear and was seeded with bovine chondrocytes on the surface; the tissue engineered ear was implanted on the skin of a nude mouse11. The seeded chondrocytes gradually produced extracellular matrix (ECM). The mouse with human ear study later lead many research attempts in creating tissues and organs under in vitro conditions by constructing scaffolds with various biocompatible biomaterials such as natural collagen12, synthetic polymers13, artificially synthesized bone substitutes (calcium phosphate cement)14 and fibrin glue15. The common approach of tissue engineering was by


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seeding cell suspension on solid, preformed, biodegradable polymeric scaffolds with high porosity that facilitates the transfer of nutrients and cells incorporation to form tissues. 2. Polymers/Biomaterials for the fabrications of 3D scaffolds Biomaterials imply any material, derived from nature or man-made consisting whole or a part of living structure. It is otherwise known as device/scaffolds that creates augments or replaces a function lost by disease or injury16. In this context biomaterial 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 temporary skeleton for cell adhesion and cell infiltration17. Biodegradability, high mechanical strength and dimensional stability, high processability, high porosity, high surface to volume ratio, support cell adhesion, proliferation, induces 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, non-toxicity, ease of fabrication and handling. 3D porous scaffolds developed using natural, synthetic and composite ceramic materials18. Polymers are generally used for the fabrication of medical devices for organ and tissue engineering scaffolds. Scaffolds build by using of polymers serves various advantages such as biocompatibility, versatility and biological properties19. Biomaterials are classified into natural polymers, synthetic biodegradable polymers and ceramics based on their structural, chemical and biological properties. 2.1 Synthetic polymers



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Synthetic polymers are tailored for tissue engineering applications and thereby helps to mimic biological tissues similar to that of physiochemical and mechanical properties18,20. The degradation pattern of these polymers is usually by simple hydrolysis which remains constant for every host. Out of the big list of synthetic 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). 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 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 rapid loss in PLA-PGA copolymers. Nevertheless the co-polymer of PLA-PGA does not possess any linear relationship with PLA-PGA ratio. PLA, PGA and PLGA are commonly used polymer 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 β-galactosidase protein. These research group reports to be the first attempt in the incorporation of plasmid DNA within a polymer scaffolds by the process of electrospinning. Hacker et al.22 fabricated scaffolds that has the ability to control the unspecific protein adsorption and covalent binding protein or peptide by incorporating amine-reactive di-block copolymers, N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(Lactic acid).


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PLGA, PLA, PGA has been proven for their application in the regeneration of tissues such as skin, cartilage, blood vessels, nerves, and liver tissues23. 2.1.2 Polyanhydride polymers Polyanhydride is synthesized by the process of reaction of diacides with anhydride forming 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 polyanhydride-co-imides. Griffin et al.24 fabricated the scaffolds using aligned PLGA bioactive polyanhydride fibrous substrates by 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 blend with other polymers. The degradation pattern of PCL is through hydrolysis and the degradation process takes upto 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 composite by electrospinning technique 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 have degradation mechanism similar to that of poly(α-hydroxyester), which is controlled by crosslinking of vinyl monomers with unsaturated double bonds27. Peter et al.28 investigated the biodegradability and biocompatibility of poly(propylene fumarate) (PPF) under in vivo conditions by varying the PPF



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to N-vinyl pyrrolidone 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-cross-linked PPF scaffolds of varying porosity and pore size were implanted subcutaneously and in cranial defect rabbits to prove biocompatibility. 2.1.5 Polyethylene glycol Polyethylene glycol (PEG) is a hydrophilic polymer 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 and thereby changing the co-polymer PEG biocompatible. Comolli et al.30 studied poly(N-isopropylacrylamide)-co-poly(ethylene glycol) (PNIPAAmPEG) ability to be used as injectable scaffolds for the repair of spinal cord injury (SCI). This study suggested that PNIPAAm-PEG 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 Polyvinyl alcohol Polyvinyl alcohol (PVA) is a biocompatible polymer possesses ability of swelling and thereby holding large amount 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 their incomplete degradation properties. The polymer has a wide range of applications such as cartilage regeneration, breast augmentation and diaphragm replacement31. Aramwit et al.32 fabricated scaffolds using silk sericin (a byproduct from silk) and PVA at varying concentration with and without glycerin and genipin. The study proves that composition of scaffold plays a significant role on its physical properties.


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2.1.7 Polyurethane The unique properties of polyurethane such as durability, elasticity, elastomer-like characteristics, fatigue resistance, compliance and also important substrate in the field of biomedical tissue engineering. The biomaterials possess a wide range of degradation pattern namely thermal and enzymatic, hydrolysis and oxidation. Verdonk et al.33 studied on tissue ingrowth in polyurethane scaffolds implanted in partial meniscal lesions patients. The data such as DCE-MRI, biopsy/histologic analysis prove consistent regeneration of tissue in polyurethane scaffolds. Hejikants et al.34 constructed porous scaffolds with high 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. Polynucleotide, which are made up of chains of nucleotides; polyamides, that consist of chains of proteins and lastly 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 scaffolds fabrication due to its potential mechanical properties, bioactivity, structures 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



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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 normal stratified layer and stromal keratocytes proliferated rapidly. Moreover the endothelial monolayers possess a pattern similar to that of normal corneal endothelium. Roman et al.36 employed 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 thereby improve the graft performance. The study suggests that the materials possess various advantages such as easily injectable, reinforced hydrogels behavior and drug release profile which is highly dependent on the composition of materials. 2.2.2 Alginates Alginates, otherwise known as alginic acid is an anionic polysaccharide derived from algae. The polysaccharide is made up of two repeating monosaccharides namely L-glucuronic acid and Dmannuronic 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 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.


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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 semi-crystalline nature of the polymer makes it biocompatible under in vivo condition39. Chitosan used for wide range of applications in food, cosmetics, biomedical and pharmaceuticals40,41. The degradation pattern of chitosan is controlled by the residual acetyl functional groups19. Ma et al.42 constructed collagen and chitosan porous scaffolds by freeze drying process, then treated with glutaraldehyde crosslinking to provide good biocompatibility, biostability and play an prominent role in dermal equivalents for wound healing. VandeVord et al.43 investigated the biocompatibility of chitosan scaffold transplanted in mice. Various studies such as macroscopic inspection, histological assessment, gram staining and limulus assay, angiogenic activity test, lymphocyte proliferation assay, ELISA was carried out and proved that chitosan is potential implantable biomaterials for tissue regeneration43. 2.2.4 Collagen Collagen is a most abundant protein present in mammals and type I, IV collagens are commonly used for tissue engineering applications. Various properties like mechanical stability, strength and toughness of collagen is 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 recognition44. All these properties are important for various applications and processed into several patterns such as gels, solutions, filamentous, tubular, and composite matrix45- 48. Ma et al.49 produced 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 was improved by the collagen layer.



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Zhong et al.50 employed electrospinning method for fabricating nanofibrous collagen scaffolds with well-defined architecture to mimic native ECM. The study suggests that the alignment of the collagen fibrous scaffold play a significant role in cell adhesion, orientation, proliferation and interaction between the cells and fibers.

2.2.5 Fibrin Fibrin is a non-globular protein plays a significant role in blood clotting. 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 had a wide range of application especially in cartilage tissue engineering51. 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 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, skeletal myoblast in BSA was injected into the left ventricle of rat which was occluded in myocardial infarction. The immunohistochemistry studies suggested that the myoblast cells spreading was significantly high in fibrin glue and has a potent ability to improve cellular cardiomyoplasty and myocardial infarction.


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2.2.6 Gelatin Gelatin is a biodegradable polymer derived from collagen and also greatest advantages is the minimal immune response and could be easily fabricated as scaffolds. They are made up of large number of residues namely glycine, proline and 4-hydroxyproline. Chong et al.54 designed a dermal wound healing construct which is cost effective in skin tissue engineering. The constructs was 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 10-25ºC. Gelatin scaffolds cross-linked with genipin, which possess varying pore size from 50 to 500 µm used to study the cell metabolism and proliferation. Cell growth highly depends on the fiber morphology, wettability, 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 skeletal system. Bioceramic 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 each material to the other materials56. 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 environment. The group of calcium phosphates such as tetra-calcium phosphate, amorphous calcium phosphate, alpha tricalcium phosphate, beta



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tricalcium phosphate, and hydroxyapatite generally used for bone tissue engineering. Tissue compatibility and porous structure make it as a potent candidate as 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 homogenous composites of PCL and calcium phosphate by injection molding method. The study suggests that PCL and PCL-CaP scaffold is 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, 1220 µm respectively. The results proved that scaffolds with 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, hence has a wide range of application in bone repair and regeneration. Generally used as coating of material in titanium and titanium alloy due to its bioactive properties56. Deville et al.59 synthesized porous hydroxyapatite scaffold by freeze casting with high compression and porosity by which demerits of hydroxyapatite lack the load bearing ability. Leukers et al.60 employed 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.


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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 tissues and thereby stimulate bone growth61. Fu et al.62 constructed porous scaffolds by polymer foam replication technique using 13-93 bioactive glass which possess microstructure similar to that of human trabecular bone. The in vitro study proves that 13-93 bioactive glass scaffold is a potent candidate in bone repair and regeneration. Table 1. Applications of natural and synthetic polymers in Tissue engineering Polymers

Biomedical applications

References

Natural polymer Agarose

Injectable, tissue

35, 36

engineering, drug delivery Alginate

Supporting matrix to cells,

37,38

drug delivery, tissue engineering Chitosan

Tissue regeneration

39-43

applications, drug delivery Collagen

Artificial skin, cartilage and

44-48

bone scaffolds, blood vessels Gelatin

Growth factor delivery,

63-65

neovascularisation Synthetic polymers PLA, PGA

Bone tissue engineering, artificial vessels


66-70


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PLGA

Bone tissue engineering,

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66-70

resorbable sutures PCL

Bone and cartilage repair,

25,26

bone regeneration PEG

Injectable scaffolds for

30

spinal cord injury PVA

Cartilage regeneration,

31

breast augmentation and diaphragm replacement Polyurethane

Meniscus tissue lesions,

33, 34

heart valves, catheters, artificial organs

3. PROCESS FOR SCAFFOLDS FABRICATION 3.1 Conventional methods Conventional scaffolds fabrication techniques are process that creates scaffolds with continuous, uninterrupted porous structures which lacks long range of channeling micro-architecture. The commonly used conventional scaffold fabrication processes are solvent casting and particulate leaching, phase separation, gas foaming, melt molding and freeze drying71. Table 2 gives information about the advantages and disadvantages of conventional process for fabricating scaffolds. 3.1.1 Solvent casting and particulate leaching Solvent casting and particulate leaching is a process 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 into the scaffolds. The salt is later leached out by immersing the matrix in water thereby producing porous structure of the 16 ACS Paragon Plus Environment

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scaffolds72. The processes produce porous scaffolds with porosity value upto 93%. A disadvantage of this technique is to produce only thin membranes upto 3 mm thick73, 74. Figure 1 shows the schematic representation of the solvent casting process for developing scaffolds. Sin et al.75 employed solvent casting/particulate leaching method to prepare 3D porous polyurethane scaffolds for cardiac tissue engineering. This method was modified by employing centrifugation process for improving pore uniformity and pore interconnectivity of the scaffolds. A mixture of solvent namely dimethyl formamide (DFM) and tetrahydrofuran (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.

Figure 1. Schematic representation of solvent casting and particulate leaching process for developing porous scaffolds. 3.1.2 Gas foaming Gas foaming is a process molded for biodegradable polymers is subjected to high pressure with gas foaming agents such as CO2, nitrogen, water or fluroform76-78. Later the polymer is subjected to 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 17 ACS Paragon Plus Environment


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growth of gas bubbles with a size range of 100-500 µm. The advantage of gas foaming technique is that the process is free of organic solvents. However the process carries certain drawback such as the yield of structure with huge unconnected pores and nonporous external surface72, 79. The graphical representation of the process for gas foaming method presented in figure 2. Yoon et al.80 fabricated PLGA scaffolds incorporated with dexamethasone by 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 dexamethasone-releasing PLGA scaffolds used as antiinflammatory porous prosthetic device or as a temporal biodegradable stent for the reduction of intimal hyperplasia in restenosis.

Figure 2. Schematic representation of gas foaming process for preparing porous scaffolds. 3.1.3 Phase separation Phase separation process polymer dissolved in molten phenol or naphthalene and biologically active molecules such as alkaline phosphatase81. The solution is quenched to produce a liquidliquid phase separation, polymer rich and polymer poor phase. The polymer poor phase is removed mechanically and polymer rich phase solidifies leaving behind a porous scaffold which is embedded with bioactive molecules on its surface. Schematic for the process of fabricating porous scaffolds is shown in figure 3. This process is carried out at low temperatures, and helpful for the incorporation of bioactive molecules into the scaffolds. Using phase separation 18 ACS Paragon Plus Environment

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techniques, nanoscale fibrous structure could be formed; this mimics natural ECM architecture and provides a better environment for cell adhesion and proliferation82. 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) technique for tissue engineering and drug delivery applications. The binary composition of solvent and non-solvents were dioxane and water. The quenching temperature helped to tune the pore size of the scaffolds. Amorphous polymer with a slow cooling rate produced macropores and semicrystalline polymer with fast cooling rate 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.

Figure 3. Schematic representation of phase separation method for developing porous scaffolds. 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 porogen is leached out of the scaffolds leaving behind a porous structure with specific surface. Similar to gas foaming technique, it is a non-solvent fabrication process presented in the schematic representation of figure 4. The drawback of melt molding includes the possibility of



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residual porogen and high processing temperatures that preclude the ability to incorporate bioactive molecules84, 85. Oh et al.86 fabricated PLGA/PVA scaffolds by using blend of PLGA and PVA helps in improving the hydrophilicity and cell compatibility. The scaffolds analyzed by in vitro (human chondrocytes) and in vivo condition (skull defect of rabbit), the study proved that the scaffolds made up of PLGA/PVA blend had better cell adhesion and growth under in vitro condition for bone in-growth and new bone formation inside the scaffolds under in vivo condition 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.

Figure 4. Schematic representations of melt molding process for preparing porous scaffolds. 3.1.5 Freeze drying Freeze drying technique polymer solution is cooled down to a certain critical temperature leads to frozen state and 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 and thereby creating interconnected porous scaffolds87,

88

. The porosity of the scaffolds depends on the

concentration of the polymer solution, pore size distribution is affected by the freezing temperatures89. The greatest advantage of freeze drying technique is that, it neither requires high temperature nor separate leaching step. Some of the demerits are smaller pore size and long 20 ACS Paragon Plus Environment

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processing time for freeze drying methods90. Graphical representation of the process of freeze drying method for fabricating biocomposite scaffolds showing 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 50-100 µm and 100-500 µm by freeze drying process for tissue engineering applications.

Figure 5. Schematic representation of freeze drying process for preparing porous scaffolds. Table 2: Advantages and disadvantages of conventional process for fabricating scaffolds. Techniques

Advantages

Disadvantages

Solvent casting and

Produce highly

Only produce

particulate leaching porous scaffolds (93%)

References 72-74

membrane with 3 mm thickness, limited mechanical property

Gas foaming

Organic solvent free

Yields structure with

72, 76-79

process, porosity and large unconnected pore size can be

pore and non-porous

controlled in

external surface,

scaffolds

limited mechanical property

Phase separation

Process conducted at

Solvent selection

low temperature -

and processing


73, 81, 82


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Melt molding

incorporation of

temperature is

bioactive molecules

crucial

Morphology and

Possibility of

shape can be tuned

residual porogen and

for the scaffolds

high processing

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73, 84, 85

temperature Freeze drying

No separate leaching Long processing process is required

temperature and

for the fabrication of

small pore size

73, 89, 90

scaffolds

3.2 Advances in biofabrication techniques 3.2.1 Electrospinning 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 able to produce nanofibers with diverse forms, such as core-shell fibers, hollow fibers (Figure 6B) and three dimensional fibers. Electrospun nanofibers are generally used for tissue engineering applications more than a decade and gained lot of interest in neural tissue engineering92-94. Yoshimoto et al.95 employed electrospinning technique to fabricate PCL scaffolds for seeding neonatal rat mesenchymal stem cells (MSCs) and cultured under dynamic culture. The results suggest that the surface of cell-polymer constructs was covered with multilayers of cells, expression of type I collagen and mineralization of cells. Electrospinning has a prominent role for 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 were seeded with human 22 ACS Paragon Plus Environment

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skin fibroblasts showed a significant cell growth on the fiber matrix for wound healing. Yang et al.97 employed electrospinning technique to fabricate PLLA nanofibrous scaffolds which could serve as a cell carrier in neural tissue engineering.

Figure 6. Graphical representation of electrospinning process. (A) Basic electrospinning set-up, (B) Core shell/hollow fiber set-up. 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 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 electrospinning technique. Li et al.100 studied the ability of PCL nanofibrous scaffolds to support chondrogenesis of MSCs under in vitro condition for cartilage tissue engineering. The ability of MSCs in the presence of TGF-β1 to differentiate into chondrocyte phenotype and the level of chondrogenesis was compared with that 23 ACS Paragon Plus Environment


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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 advancement 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 scaffolds fabrication technique. It helps in early detection and correction of design flaws. Rapid prototyping technique binds liquid, powder and sheet material to form complex parts101. 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 heat for 3D scaffolds fabrication by slicing layer by layer for printing objects102. In this set-up, 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 scaffolds103. The melting temperature is a factor depends on the process temperature and materials used for FDM are PLA, ABS and nylon103. Zein et al.102 produced scaffolds with PCL, which possess a honeycomb-like pattern with interconnected channel 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


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which PLA was used as a feed material105,106. Tellis et al.107 developed a 3D CAD model of canine trabecular bone scaffolds of varying pore structure by micro CT technique exported to FDM which in-turn produced polybutylene terephthalate (PBT) trabeculae scaffolds along with four other scaffolds. Schematic of FDM process and Photographic image of the FDM-1650 building a scaffold presented in figure 7107.

Figure 7. Fused deposition model for the fabrication of tissue engineering scaffolds. (A) Schematics of FDM process, (B) Photographic image of the FDM-1650 building a scaffold107. 3.2.2.2 Selective laser sintering Selective laser sintering (SLS) commonly used approach for 3D scaffolds fabrication and high power laser used for additive manufacturing process. Small particles of plastic, metal, ceramic and glass powder are fused with carbon dioxide laser into a desired 3 dimensional shapes. The SLS technique helps in rendering complex porous scaffolds. Figure 8 shows the schematic of SLS method for developing porous scaffolds. Zhou et al.108 employed SLS method to prepare porous scaffolds by the use of bio-nanocomposite microsphere made up of carboxy hydroxyapatite (CHAp) nanosphere in the poly(L-lactide) (PLLA) matrix. The micro and nanospheres are prepared by emulsion technique, these CHAp nanostructures were embedded with PLLA microspheres thereby forming nanocomposites. SLS-Sinterstation® 2000 machine



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used to fabricate the porous scaffolds of PLLA microspheres and PLLA/CHAp nanocomposite microspheres.

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 = 45x bar = 500 µm; (C) magnification = 100x, bar = 200 µm 112. Williams et al.109 computationally designed PCL scaffolds and fabricated the scaffolds using SLS techniques. The scaffolds produced through 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 process for the fabrication of polyetheretherketone (PEEK) and hydroxyapatite (HA). The study suggested that high melting point polymer can be laser sintered in a much lower temperature to incorporate 200µm

bioactive material (hydroxyapatite) into the polymer to develop potential scaffolds for tissue engineering. Chua et al.111 created tissue engineering scaffolds by administrating SLS technique 26 ACS Paragon Plus Environment

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in which a biocomposite blend of polyvinyl alcohol and hydroxyapatite was subjected to lasersintering process for bone tissue engineering. 3.2.2.3 Stereolithography Additive manufacturing process is stereolithography (SLA), a technique works by polymer resin materials for the fabrication of scaffolds. The process employs a single laser beam (UV) to polymerize or crosslink photopolymer resin103, 113. This process liquid monomer used to solidify layer by layer fashion. The UV spot beam draws a pre-programmed design on the photopolymer resin surface which solidifies and forms layer, this process is continued until the desired 3D structure of the scaffold is formed114. Figure 9 shows the schematic representation of SLA process for fabricating fibrous scaffolds. Cooke et al.115 utilized biodegradable resin mixture of diethyl fumarate (DEF), poly(propylene fumarate) (PPF) and photoinitator bis-acylphospine oxide (BAPO) for the construction of scaffolds. Ravoos et al.116 constructed a flexible and elastic poly(trimethylene carbonate) (PTMC) scaffolds for cartilage tissue engineering. These scaffolds constructed with 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 were built with photocrosslinkable 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 helps to understand the cell behavior in complex microenvironment and helps in engineering complex hybrid tissue structures for tissue engineering.



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Figure 9. (A) Schematic representation of stereolithography process, (B) SEM image of a scaffolds prepared by stereolithography process, showing (a) a single-layer scaffold, and (b) a multilayered scaffolds117. 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 factors103. Fused deposition manufacturing (FDM) used high temperature to melt the material which can cause adverse effect on cells and biomolecules. The cells survival and co-printing with material to form suitable scaffolds, printing of scaffolds incorporated with biomolecules such as growth factors is very critical in the FDM. These major limitations of FDM were overcome by Liquid frozen deposition manufacturing technique (LFDM), which possess 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 LFDM technique, the scaffolds possess similar mechanical strength of native articular cartilage. Hsu et al.119 employed LFDM process for the fabrication of chitosan scaffolds and treated with air plasma (AP) for cell attachment and proliferation. This study proved reduced


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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 manufactured by LFD and treated with AP suitable candidate for bone tissue engineering applications.

Figure 10. Schematic representation of liquid frozen deposition manufacturing process for developing scaffolds. 3.2.2.5 Direct metal laser sintering Direct metal laser sintering (DMLS) technique employed for printing 3D objects for metal fabrication for regenerative therapy. The technique builds up component in layers by depositing metal materials120. A thin layer of materials placed on the building platform, a laser beam with the help of computer guidance fuses the powder into desired 3D structures and the process is repeated for the fabrication of scaffolds120. DMLS method works by injection molding process in which aluminum mold is required for the fabrication of scaffolds. Moreover, the production of complicated shapes and structures are very difficult in DMLS. These methods biomaterials used not suitable for human cells as high temperature in the chamber of injection molding could



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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 uses laser to cure polymers (Figure 11). The digital mirror device (DMD) an array of micro mirrors rotating independently to controls the laser beam in curing polymers. Dean et al.121 fabricated scaffolds using resorbable polymer poly(propylene fumarate) (PPF) by 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.

Figure 11. Schematic representation of digital light processing for fabricating constructs. 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 is the bioprinting technology which is the key element of organ printing123, 124. Healthy human cells are injected through a very thin nozzle on the bed surface. However the cell density, vascularization and tissue maturation remains a challenge125-127. The process involves successive layering of materials under computer guidance128. One of the challenging and most widely


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studied areas in tissue engineering is the 3D organ printing. Organ printing is evolving into a promising approach for engineering new tissues and organs129. Organ printing follows three basic steps; firstly pre-processing which involves the development of blueprint for organs, secondly processing which involves the actual organ printing and lastly the post-processing process which involves the organ conditioning and accelerated organ maturation129.

Table 3. Different rapid prototyping technique: Advantages and Disadvantages. Technique

Materials

Materials

Advantages

Disadvantages

Application

Reference

Type SLA

Liquid based

Polymers, wax or wax

Mechanical

Toxic (reactive resins),

Bone, cartilage,

compounds

strength, easy to

high cost

heart valves

Eg. Poly 1500, Tusk

remove support

2700, protogen white,

material, easy to

flex 70B, NeXt etc.

achieve small

114-116

features SLS

FDM

Powder-based

Solid-based

Metals,

Mechanical

High temperatures,

Bone, Cartilage

ceramics,

Strength, high

uncontrolled porosity,

bulk

accuracy,

rough surface

polymers

broad range of

Eg. PEEK-HA, PCL

materials, fast

etc.

processing

Thermoplastic

Low costs,

polymers/Ceramics,

good mechanical

Adipose tissue,

Eg.polyphenylsulfone

Strength, solvent

Cartilage

(PPSF),

not required

High temperature

Bone,

109-111

102-107

polycarbonate (PC), PCL-HA etc. D printing

Digital light

Powder-based

Liquid based

Powder of

Fast processing;

Material are powder

bulk

low costs;

form; weak bonding

polymers;

no toxic

between powder

Ceramics

components;

particles; rough

Bone

123-129

Eg. PLGA, starch

water used as

surface; might require

based polymers

binder

post-processing

High resolution,

Cost, toxic resin

Bone

121, 122

Freeze drying

Cartilage

118,119

Photopolymer resin

fast processing,

processing

less shrinkage Liquid frozen deposition

Liquid based

Polymers

Low temperature, biomolecules



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3.2.2.8 Organ Printing: Pre-Processing The first step in the organ printing process is the design of blue print in the form of a computeraided design for designing the organ. It is defined as computer compatible precise spatial information about the localization of cells in 3D organ or otherwise known as ‘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 bio-imaging and ultrasound make it possible to find the gross anatomical characteristics of organs. This method gives information about the patients specific anatomy of the organs. The resolution of this method not reached to cellular level and cell redistribution cannot identify in the process of organ printing. Another method is based on computer-aided reconstruction is the histological section. Some of the advantage of this approach is that it provides information about the size and shape of the organ, and most importantly it gives information about the composition130. However this method possess a great disadvantage for human organs are available for this sort of inspection only after death, moreover this process is time consuming and is not patient specific. The design of digital model of human organ or organ blueprint is based on the combination of all the above described approaches, for instance the clinical imaging gives information about the anatomical feature; serial histological section gives information about the structural functional units; and lastly mathematical modeling and computer simulation helps to combine macro- and micro-anatomical structural information together to form desired digital model of human organ or organ blueprint for 3D bioprinting131.


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3.2.2.9 Organ Printing: Processing Bioprinting process of organs and tissues, liquid media containing cells and nutrients are loaded in the cartridge for developing the live tissues. The organ and tissues are bioprinted based on the organ blueprint and is printed by layer-by-layer assembly of the cells in order to produce artificial organs. The various components like computer-aided design, computer simulation, mathematical modeling, virtual reality methods and informational technologies play an important role for organ printing technologies and industrial scale biofabrication process engineering132. 3.2.2.10 Organ Printing: Post-Processing Post-processing the viability and survival of 3D bioprinted organ constructs fusion tissue spheroids into integrated constructs and its accelerated maturation. The most important step in modeling tissue maturation is the identification of structural determinants of material properties of tissues and organs. The materials properties of vascular wall into the large diameter blood vessels determined by ECM proteins collagen and elastin. This approach used to identify the concentration of collagen, diameter of collagen microfibrils and level of collagen cross-linking which is the potent structural determinant of material properties of heart valves133. The 3D printing technique used for delivering various bioactive molecules134 and drugs. Yi et al.135 demonstrated 3D printing of bioabsorbable implants containing anti-cancer drugs could be a powerful tool for an effective local delivery of chemotherapeutic agents for the treatment of cancers. Pozzoli et al.136 fabricated novel drug delivery device with the aid of 3D printing alginate used as shell and PLGA as core, the fabricated tubes showed sequential release of distinct fluorescent dyes incubated with the human embryonic kidney cell lines. Ahlfeld et al.137 proved extrusion-based 3D plotting for the fabrication of calcium phosphate cement (CPC) scaffolds incorporated with biological components to increase the capability to restore functional



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tissues. A new strategy was developed for loading growth factors based on multichannel plotting with a biphasic scaffolds design combining CPC with VEGF-laden. The blend of alginate and an alginate-gellan gum used to exhibit favourable VEGF release profile. Lim et al.138 incorporated anti-epileptic drug Carbamazepine in 3D printed scaffolds with variation in pore positions, number of pores, and pore diameters. These research groups justifies that the drug release rate depends on the pore diameter of the scaffolds in tissue engineering. 4. ARCHITECTURE DESIGN OF THE SCAFFOLDS Scaffolds architecture plays an important role in 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 suggests that the optimum pore size suitable for neovascularization is 5 µm; a pore size of 5-15 µm is beneficial for fibroblast ingrowth, 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 µm139,140. Compared to the conventional biofabrication technique, advanced biofabrication technique 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 by advanced technique called freeze casting, which produces linearly oriented architecture. In this process the polymer slurry is subjected to a temperature gradient allows nucleation and growth of the ice crystals. This technique is appropriate for the fabrication of nerve conduits with a porous structure that has ability to guide axon growth in neural tissue engineering. 34 ACS Paragon Plus Environment

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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 scaffolds architecture and subsequent reaction of partially heparinized. The study suggests that the blood reaction depends on the diameter of fiber and scaffolds roughness. Scaffolds with thin fibers (diameter < 1µm) possess low coagulation and no platelet adhesion and scaffolds with fiber diameter (2-3µm) trigger thrombin formation and platelet adhesion. Ranella et al.142 investigated the adhesion and viability of fibroblasts on a high rough 3D silicon surface. The optimal cell adhesion was obtained for less roughness ratio independent of the surface wettability for the adhesion of fibroblasts on surface energy. Surface modification of the scaffolds helps to improve its biocompatibility. Plasma treatment is a common techniques carried out to modify the surface of polymer nanofibers. Sanders et al.143 fabricated polyurethane fibers by electrospinning in different surface charges were introduced by the process of plasma induced surface polymerization of negatively or positively charged monomers. The tissue compatibility of the constructs was evaluated by implanting in rat subcutaneous dorsum results the negatively charged surface facilitate vessel in-growth into fibroporus biomaterials. Even though the scaffolds possess porous structures with interconnected pores to be good enough for cell infiltration and growth; surface characteristics such as hydrophilicity/ hydrophobicity must be satisfactory for cell adhesion, migration and proliferation144, 145. The surface property of the scaffolds plays a vital role in cellular interaction for biomimicking with naturally derived biomolecules are immobilized on the surface of the scaffolds. This in-turn helps in cell adhesion and growth or sustained release of growth factors to facilitate tissue regeneration146. Proteins such as collagens, fibronectin and laminin coated with silk fibroin nanofiber surface to promote cell adhesion and proliferation145-148. Jia et al.147 fabricated catalytic 35 ACS Paragon Plus Environment


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nanofibers by immobilizing enzymes onto hydroxyl-containing polystyrene (PS) nanofibers for biomedical applications. 5. APPLICATION OF 3D PRINITNG IN REGENERATIVE THERAPY 3D printing has a wide range of applications in the regenerative therapy. The past few decades 3D printing has spread itself in the fabrication of living tissues for various applications. The process creates a revolutionary impact in the field of medical science and pharmaceuticals. Some of the common applications of 3D printing techniques are as follows: 5.1 Bone Bone and cartilage are one of the most widely studied areas in the application of tissue engineering149 and 3D printing. Chou et al.150 fabricated biodegradable bone cage using fused deposition modeling (FDM) type 3D printing; in which biodegradable polymers are used to convert corticocancellous bone chips into a structured strut grafts for segmental bone defects. In vivo analysis was done on New Zealand white rabbits in which group A was implanted with 3D printed cage, implantation of corticocancellous chips and fixation of K-wire; where as in group B bone chips implantation and K-wire fixation was done. The results suggest that group A rabbits had an increase in the activity and decrease in anterior cortical disruptions, higher leg length ratio and improved final bony ingrowth within the bone defects (Figure 12 (1), (2)). Gao et al.151 studied bioactive ceramic nanoparticles (NPs) in order to stimulate osteogenesis of printed hMSCs in poly(ethylene glycol) dimethacrylate (PEGDMA) scaffolds. Inkjet bioprinter was used to fabricate PEGDMA. MSCs were co-printed with NPs of bioactive glass (BG) and hydroxyapatite. The construct showed high cell viability (86.62 ± 6.02%) and high compressive modulus (358.91 ± 48.05 kPa) after 21 day culture in vitro. The results suggest that HA is more 36 ACS Paragon Plus Environment

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effective when compared to BG for hMSCs osteogenesis in bioprinted bone constructs. Fedorovich et al.152 fabricated heterocellular tissue constructs of MatrigelTM and alginate hydrogel by bioprinting process. Cells such as endothelial progenitor and multipotent stromal cells was bioprinted and the constructs was implanted subcutaneously in immune deficient mice. The study suggests that the incorporation of osteoinductive biphasic calcium phosphate microparticles led to the differentiation of stromal cells into osteogenic lineage and improved bone formation. 5.2 Cartilage Bioprinting has gained a lot of interest in tissue engineering of cartilage which can imitate native tissues with differentiated cells and ECM. The major cell source of cartilage is the chondrocytes for encapsulation in various designs of bioink in-order-to mimic shapes from simple to complex. Abbadessa et al.153 investigated UV cross-linked hydrogel based on thermos-sensitive methacrylated pHPMA-lac-PEG triblock copolymer was laden with chondrocytes, mechanical properties of long term stability and printability of the hydrogels. Porous structures developed by 3D bioprinting of hydrogels containing methacrylated hyaluronic acid (MHA) laden with chondrocytes without affecting the cell viability. The study suggests that MHA hydrogel are a potent system for the design of 3D cell-laden constructs for cartilage regeneration (Figure 12 (3)). Izadifar et al.154 developed 3D printing based biofabrication process for producing hybrid constructs from polymer (PCL) and hydrogel (alginate) incorporated with cells (embryonic chick cartilage). The study proved that there was an increase in cell viability, cell proliferation and cartilage differentiation in the hybrid constructs and thereby confirming 3D hybrid constructs of PCL and cell impregnated hydrogel to be a promising approach for cartilage tissue engineering.



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5.3 Cardiac tissue Cardiovascular disease is considered as one of the major causes of morbidity and mortality worldwide with an approximate of 20 million death reported annually155. Ischemic disease is one of the major causes of death in CVD, of all CVD death 42% is reported to be ischemic disease. Gaetani et al.156 bioprinted alginate with human cardiac-derived cardiomyocytes progenitor cells. The construct was reported to possess high cell viability (92% and 89% at 1 and 7 days of culturing), the constructs retained the cardiac lineage and showed enhanced gene expression of early cardiac transcription factor, sarcomeric protein. The study also suggests that cells were able to migrate from the construct matrix and form tube-like structure by colonizing on the Matrigel layer (Figure 12 (4, 5)). Gaebel et al.157 fabricated cardiac patch by using laser-induced-forwardtransfer (LIFT) cell printing technique with human umbilical vein endothelial cells and hMSCs on PEUU for cardiac tissue. The in vivo study revealed that the transplantation of LIFT-tissue engineered cardiac patch had an increased vessel formation; significant improvement in infarcted heart enhanced capillary density and integration of the cells into the vessels of murine vascular system. The group suggests that LIFT based tissue engineering of cardiac patch is a promising approach in the treatment of myocardial infarction (Figure 12 (7)). 5.4 Heart valves Cardiac tissue engineering of heart valves is also critical as they do not have the ability to regenerate the valves. Duan et al.158 bioprinted alginate/gelatin hydrogels with direct incorporation of dual cells, aortic root sinus smooth muscle cells (SMCs) and aortic valve leaflet interstitial cells (VIC). The group suggest that the cells were viable within the conduit over 7 days in culture; moreover the hydrogel showed reduced modulus, ultimate strength and a peak


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strain slightly reducing over 7 day culture maintaining the tensile biomechanics of cell-laden hydrogels. The same group in another study159 bioprinted hybrid hydrogels of methacrylated hyaluronic acid (Me-HA) and methacrylated gelatin (Me-Gel) encapsulated with human aortic valvular interstitial cells (HAVIC). The constructs represents tri-leaflet valve shape and increase in Me-Gel concentration facilitated cell spreading and thereby maintaining HAVIC fibroblastic phenotype. The HAVIC cells incorporated within the constructs express alpha smooth muscle actin (α-SMA) and vimentin thereby remodeling the ECM due to the deposition of collagen and glycosaminoglycan for vascular tissue engineering. 5.5 Neural tissue Bioprinting technology in neural tissue fabrication has tremendous application for diseases, aged or injured patients. Lee et al.160 bioprinted neural cells (astrocytes and neurons from embryonic rat) in a 3D multilayered collagen gel acts as a scaffold for cells. The cell density in hydrogel was adjusted by the change in the printing resolution. Fluorescent imaging revealed that only fewer cells were observed in the lowest neuron ring in the single-layered collagen. They suggest that this might be attributed by the decreased level of perfusion. Owens et al.161 biofabricated a fully biological graft with Schwann cells (SCs) and MSCs pellet was expel in the 3D printed agarose molds. The regenerative capacity of neural tissue grafts was compared with that of autologous grafts and hollow collagen tubes. The study suggests that bioprinting is a promising approach in the fabrication of nerve grafts. Lee et al.162 bioprinted an artificial neural tissue with murine neural stem cells (C17.2), collagen hydrogels and VEGF releasing fibrin gels. The results suggest that there was an increase in the cell viability upto 92.89 ± 2.32%; growth factor induced changes in morphology and cell migration towards the VEGF releasing fibrin gels for neural tissue engineering. 39 ACS Paragon Plus Environment


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5.6 Liver tissue Liver tissue bioprinting is one of the promising studies as liver failure is associated with failure of multiple organs163. Jones et al.164 reported first time to bioprint human induced pluripotent stem cells and their post printing differentiation into hepatocyte like cells (HLCs). Human induced pluripotent stem cells and human embryonic stem cells derived HLCs were bioprinted using valve based printing and examined for the presence of hepatic markers. Alginate hydrogel matrix was used for bioprinting of hESCs derived HLCS and examined for its viability and albumin secretion. The results observed that 21st day of differentiation and albumin secretion has reached its peak in 3D printed constructs. The study thereby concludes that valve based printing process is effective in printing human pluripotent stem cells (hPSCs) (Figure 12 (6)). 5.7 Skin tissue Skin tissue engineering by bioprinting process also plays a vital role for the therapy of chronic diseases especially for burns. Lee et al.165 bioprinted keratinocytes and fibroblasts cells which represented the epidermis and dermis; the dermal matrix was constructed with collagen. The constructs was cultured in a submerged media, later exposed to air-liquid interface in-order-to promote maturation and stratification. Histology and immunofluorescence analysis revealed that the 3D construct was a representative of human skin tissue both morphologically and biologically. Koch et al.166 first attempted the 3D arrangement of vital cells by laser assisted bioprinting (LaBP) technique. Fibroblasts and keratinocytes incorporated into collagen were printed for skin tissue engineering. The study revealed that LaBP is an excellent tool for the generation of multicellular 3D constructs (Figure 12 (8)). Michael et al.167 bioprinted fully cellularized skin substitute using fibroblasts and keratinocytes by positioning on top of a


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stabilizing matrix (Matriderm®). In vivo (nude mice) analysis revealed the multilayer epidermis formation and the presence of blood vessels in the wound bed and edges in the direction of printed cells (Figure 12 (9)).

Figure 12. 3D printing in tissue engineering applications. (1) Leg specimens for leg-length ratio study. (a) A rabbit in group A showed good bone healing with conservation of femoral length; (b) A rabbit in group B showed plenty bridging callus formation but shortening of left femoral length150; (2) H and E staining of coronal section on femsite at 12 weeks; (b) At 24 weeks good bone callus formation was observed around the entire defect site150; (3) 3D printed porous cartilage construct based on MHA. (a) Top view, (b) top-side view, (c) Top corner view, (d) Top view showing a homogeneous distribution of encapsulated green fluorescent beads153; (4) Porous cardiac scaffold printed with hCMPCs in alginate156; (5) Migration assay of printed hCMPCs in



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Matrigel scaffold after 3 weeks of culture156; (6) Liver tissue: 3D printed alginate ring structures with 40 layers printed with blue dye for visual clarity: (a) top view and (b) side view (scale bar 2 mm)164; (7) In vitro acceleration of tube forming by LIFT: Immunofluorescent micrographs of PECAM stained patches treated with co-culture of HUVEC/hMSC 8 days after cell seeding. (a) Vessel formation in the simulated printed pattern. (b) Proliferation of cells but marginal vessel formation157; (8) Skin tissue: Schematic of the laser printing setup. The cell–hydrogel mixture is propelled as a jet by the pressure of a laser-induced vapour bubble and thereby forming a 3D cell pattern. A printed grid structure (top view) of ﬁbroblasts (green) and keratinocytes (red) demonstrates micropatterning capabilities of the laser printing technique. Bottom: Seven alternating colour-layers of red and green keratinocytes (left; detail view in right). Each colourlayer consists of four printed sublayers. The structure has a height of about 2 mm and a base area of 10 mm x 10 mm166; (9) Tissue engineered skin construct in the dorsal skin fold chamber in nude mice. The skin construct is inserted into the wound directly after the implantation (left) and on day 11 (right)167. (Received copyright for all figures). 6. CHALLENGES IN ORGAN PRINING Currently, better advancement and increased interest in tissue engineering, certain critical challenges need to be addressed in regenerative therapy. Some of the challenges include type of materials used, organ blueprint, scaffolds architecture, biopaper, cell viability, vascularization and tissue maturation. 6.1 Type and properties of materials There are various parameters that have to be taken into consideration for the type of materials used namely:


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i. Material processability: The choice of material is a critical parameter used for different rapid prototyping techniques. Specific form of input material is required for each technique such as powder, filament, solid pellet or solution. ii. Degradation pattern: The degradation patterns of polymer materials used in the process are different from one another. In a usual scenario the scaffolds will be resorbed and gradually be replaced by newly formed ECM and differentiated cells. There are various factors that governs the degradation rate and degradation pattern of the polymer such as; hydrophilicity, degree of crystallinity, catalysts, porosity and surface area. iii. Degradation product: One of the major challenges in tissue engineering process is the product produced due to the degradation of polymers. In a usual scenario the degradation products of biodegradable polymer is known to be non-cytotoxic, but there is some possibility of acid byproduct formation which depends on the degradation rate168. Sung et al.169 investigated the effect of scaffolds degradation rate on 3D cell growth and angiogenesis and suggested that fast degradation of polymer affects cell growth and migration under in vitro and in vivo condition. This is due to the acidic environment created by degradation of biocompatible polymers. iv. Mechanical strength: The mechanical property of the scaffolds plays a vital role in tissue engineering. Research studies suggest that the cells have the ability to detect the mechanical properties of the adhesion substrate in order to regulate the integrin binding and construction of focal adhesion plaque and cytoskeleton170. For instance if the adhesion substrate is too rigid and non-deformable cells, reorganization and the construction of focal adhesion plaque is affected. Likewise if the construct is too compliant, cell anchorage is affected in tissue engineering.



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6.2 Organ blueprint 3D cells and organ printing requires a description and representation of details of organ anatomy, morphology, tissue heterogeneity and vascular system at different tissue/organ organization scales171. A blueprint is nothing but a human organ computer-aided design in a stereolithographic file. The specific function of a blueprint are (i) to describe anatomy, geometry and internal architecture of an organ including the tissue heterogeneity, tissue geometry and the boundary distinction within the organ of interest. (ii) It helps in defining the vascular network and 3D topology. (iii) Provide database on organ/tissue geometry, heterogeneity and vascular network which could be used for tool path generation of 3D cells and organ printing172. 6.3 Scaffold architecture The design of biofabrication process is a challenging task for mechanical engineers. The fabrication of various complex structures such as kidney will require several steps and a broad spectrum of specially designed equipments173. 6.3.1 Pore size: The pore size is a very critical parameter which determines the cell growth and vascularization. The pore size differs with the type of tissue. The optimal size range of pores still remains a question174, 175 6.3.2 Scaffold morphology: The morphology of the constructed scaffolds might affect the cell adhesion and migration. Indolfi et al.176 investigated on the role of 3D microarchitecture of scaffolds in regulating the endothelial cells behavior in matrix-embedded endothelial cells (MEECs) constructs. The study suggests that the scaffold microtopology is a potent regulator of MEECs function and has the ability to tune the immunosuppressive properties of these cells177, 178.


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6.3.3 Surface topography: The scaffolds topography mainly the surface roughness has a potent role in cell matrix interaction. Topography enhances the differentiation of progenitor cells into their programmed pathways. The rough powder surface formed from powder-based rapid prototyping technique might enhance cell adhesion168. Surface morphology of micro and nanoscale structures enhances the stimulation of cell alignment, polarization, elongation, migration, proliferation, gene expression etc. 178 - 180. 6.4 Biopaper Biopaper defined as processable and biomimetic tissue fusion-permissive hydrogels specially designed for bioprinting process126, 181. An ideal hydrogel used for organ printing should follows certain characteristics such as biocompatible, biodegradable, stimuli sensitive, fast solidification, naturally derived hydrogels, low cost etc. Xu et al.182 used computer-aided inkjet printing of viable mammalian cells namely Chinese hamster ovary cells and rat embryonic motor neurons cells as bioink; in which cells were printed on biopaper made from soy agar and collagen gels. Lee et al.183 experimented on new harvesting, transfer and assembly technique used to fabricate laminated tissue composites of the biopaper such as hepatic hydrogel sheet modules with augmented liver function for stratified 3D hepatic tissue reconstruction. The hepatic hydrogel sheet modules embedded with HepG2 cells were fabricated either with hexagonal microarchitecture or without any microarchitecture. 6.5 Cell viability and vascularization Ensuring the uniform distribution of the seeded cells on the scaffolds is a significant challenge in tissue engineering. The use of petri-dish for seeding cells fails to deliver cells deep inside the scaffolds with uniform distribution184-186. In order to overcome this issue, cellularization of 3D scaffolds accomplished by the advances of bioreactor technologies. Ensuring the viability and 45 ACS Paragon Plus Environment


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vascularization is very critical and challenging issue in tissue engineering. The viability of printed tissue construct follows several aspects such as pre-processing survival which is the viability of cells during cell loading in the bioprinter cartridge; secondly, cell viability during processing and finally the tissue constructs survival during post processing. The primary consideration in engineering large tissue construct is sufficient vascularization of the scaffolds for maintaining adequate perfusion. One of the commonly used approaches in order to achieve high vascularization is the incorporation of growth factors such as vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, platelet- derived growth factor and transforming growth factor. The incorporation of these growth factors into the scaffolds promotes the formation of new vascular beds from endothelial cells present within the tissues for vascular tissue engineering187. 7. CONCLUSIONS AND FUTURE PERSPECTIVES Research studies show day-to-day advances in various fields of tissue engineering. Several new approaches have been studied in 3D scaffolds fabrication for easy, quick and accurate processing in regenerative therapy. 3D printing is a novel transforming approach in the field of tissue engineering which has potential for surpassing traditional solid scaffold-based tissue engineering. The process is used to fabricate scaffolds with defined shapes, with controlled and interconnected porous structures. A wide range of polymers are used for the construction of these scaffolds which are grouped under natural and synthetic. There is great deal of promises in the advancement of technology; there are many challenges that have to be addressed in the fabrication of 3D scaffolds in tissue engineering applications. The future development in the rapid prototyping in the field of tissue engineering will require the use of wide range of biomaterials, optimization of scaffold designs, and good knowledge about cells and 46 ACS Paragon Plus Environment

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developmental biology so as to overcome the various challenges in the fabrication of 3D scaffolds in organ tissue engineering. The selection of a suitable type of materials, which determines the material processability, degradation pattern, scaffolds degradation and mechanical property. Secondly the scaffolds architecture is a very important parameter which determines the pore size, morphology and topography of the scaffolds. Finally, printing live cells in which most of the challenges are limited to survivability of the cells and vascularization. A promising future approach for regenerative therapy could be a hand held bioprinting device that will help the delivery of cells into various tissues such as skin or cartilage. Moreover, in future biodegradable and biocompatible materials development will have a great impact in tissue engineering applications. Acknowledgement This study was supported by the National Research Foundation Singapore (WBS R-265-000554-592), Campus for Research Excellence and Technological Enterprise (CREATE) programme, Department of Mechanical Engineering, National University of Singapore, Singapore.



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Abbreviations: ECM

-

Extracellular matrix

PLGA

-

Poly(lactide-co-glycolide)

PLA-PEG -

Poly(D,L-lactide)- poly(ethylene glycol)

PPF

-

Poly(propylene fumarate)

SCI

-

Spinal cord injury

DCE-MRI -

Dynamic contrast-enhanced magnetic resonance imaging

BSA

-

Bovine serum albumin

DFM

-

Dimethyl formamide

THF

-

Tetrahydrofuran

HAECs

-

Human aortic endothelial cells

TIPS

-

Thermally induced phase separation

rHGH

-

Recombinant human growth hormone

HSF

-

Human skin fibroblast

CAD

-

Computer assisted/ aided design

PEEK

-

Polyetheretherketone

HA

-

Hydroxyapatite

PEGDMA -

Photocrosslinkable poly(ethylene glycol) dimethacrylate

DMD

-

Digital mirror device

PPF

-

Poly (propylene fumarate)

AM

-

Additive manufacturing



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HAVIC

-

Human aortic valvular interstitial cells

SCs

-

Schwann cells

HLCs

-

Hepatocyte like cells

hPSCs

-

Human pluripotent stem cells


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