Stereolithography

SLA, the first commercially available 3D printing process, involves selective laser curing of photopolymers based on the slice-file information from a CAD model. ^{Reference Fisher, Dean and Mikos29} In exposed areas, the polymer cures and solidifies as a result of cross-linking, but it remains liquid at unexposed sites ( Figure 1a). This process is still very popular for soft materials, and inclusion of hard materials, mainly ceramic structures, is allowed through the use of particle-loaded photopolymers as in the LAMP process. The main application of SLA in the field of biomaterials is scaffolds for TE. ^{Reference Kang and Cho31–Reference Chia and Wu33} However, the fact that only a few biocompatible polymers are stable under exposure to laser light has become one of the major limitations for broad application of SLA in TE.

Figure 1. (a) Schematic of a stereolithography system. ^{Reference Lee, Wang, Fox, Ritman, Yaszemski and Lu6} (b–c) Scanning electron microscope images of a poly(propylene fumarate)/diethyl fumarate–hydroxyapatite (PPF/DEF–HA) scaffold processed by microstereolithography and of (d–e) pre-osteoblast cells in a PPF/DEF–HA scaffold. ^{Reference Lee, Ahn, Kim and Cho30} (a) Reproduced with permission from Reference Reference Lee, Wang, Fox, Ritman, Yaszemski and Lu6. © 2007 American Chemical Society. (b–e) Reproduced with permission from Reference Reference Lee, Ahn, Kim and Cho30. © 2009 Elsevier.

Poly(propylene fumarate) (PPF), a biodegradable polymer, has been widely used in bone TE because of its biocompatibility, injectability, and good mechanical properties. PPF is generally processed by SLA, using diethyl fumarate (DEF) as the solvent and bis(acyl)phosphine oxide as the photoinitiator. To achieve the desired structure, different parameters, such as solution viscosity, laser speed, and power, must be optimized. ^{Reference Lee, Wang, Fox, Ritman, Yaszemski and Lu6}

Localized delivery of drugs is another important application for biomedical engineering. Drugs can be incorporated into 3D structures and released in a controlled manner to increase the efficiency of treatment and decrease side effects involving other tissues. For example, Lee et al. embedded bone morphogenetic protein-2 (BMP-2), a protein with a stimulating effect for new bone formation in vitro and in vivo, in poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres suspended in a PPF/DEF photopolymer. They then used 3D printing via SLA to create a scaffold that enabled the gradual release of BMP-2. These 3D-printed scaffolds enhanced new bone formation in vivo compared to scaffolds made by the traditional particulate leaching/gas-foaming method. ^{Reference Lee, Kang, Lee, Kim, Lee and Cho7}

Chitosan, a natural polymer, is also used in medical applications because of its biodegradability and biocompatibility and is a good candidate for orthopedic applications involving cartilage and bone TE. To process chitosan using SLA, Irgacure 2959, a photoinitiator composed of unsaturated monomers and prepolymers, and poly(ethylene glycol) diacrylate were added to the polymer solution to enhance its photosensitivity. ^{Reference Akopova, Demina, Bagratashvili, Bardakova, Novikov, Selezneva, Istomin, Svidchenko, Cherkaev, Surin and Timashev8,Reference Qiu, Zhang, Kang, An and Wen9} The fabricated scaffolds exhibited in vitro cytocompatibility in the presence of fibroblast cells and enhanced bone tissue growth in vivo. ^{Reference Qiu, Zhang, Kang, An and Wen9}

Recently, custom-designed projection stereolithography (PSL) was introduced for the fabrication of multiple biomaterials for TE applications. ^{Reference Gauvin, Chen, Lee, Soman, Zorlutuna, Nichol, Bae, Chen and Khademhosseini10} In PSL, instead of the scanned laser used in SLA, a digital light-processing chip is used to create photomasks from a CAD file to produce 2D slices based on 3D structures. Collagen-based gelatin methacrylate (GelMA) hydrogels were successfully processed by PSL, with methacrylamide enabling photopolymerization of the hydrogel. GelMA hydrogels of different structures (e.g., hexagonal and woodpile) with precisely controlled pore sizes were fabricated to support adhesion and proliferation of human umbilical vein endothelial cells (HUVECs), confirming the functionality of hydrogels in TE applications. ^{Reference Gauvin, Chen, Lee, Soman, Zorlutuna, Nichol, Bae, Chen and Khademhosseini10}

In addition to soft biomaterials, hard biomaterials such as ceramics and ceramic/polymer composites have also been fabricated using SLA. Hydroxyapatite (HA) nanopowders embedded in poly(d,l-lactide) oligomers were processed for bone TE³⁴ using ethyl-2,4,6-trimethylbenzoylphenyl phosphinate and N-methyl-2-pyrrolidone as the photoinitiator and diluent, respectively. Small amounts of tocopherol inhibitor and Orange Orasol G dye were also added to prevent premature polymerization and to set the requisite light penetration depth, respectively. The results showed that the amounts of HA nanoparticles and diluents had significant effects on the viscosity of the resins. In addition, the modulus of elasticity of the composites increased with increasing concentration of HA nanoparticles. ^{Reference Ronca, Ambrosio and Grijpma34} Three-dimensional composites containing 7% HA nanoparticles in 70:30 PPF/DEF were found to be suitable for SLA processing and to result in enhanced bone-cell attachment and proliferation (Figure 1b–e). ^{Reference Lee, Ahn, Kim and Cho30} SLA-printed biodegradable and bioactive glass/methacrylated poly(ε-caprolactone) (PCL) composite has also shown potential in regenerative medicine, because of an increased deposition of calcium phosphate and an enhanced metabolic activity of fibroblast cells. ^{Reference Elomaa, Kokkari, Närhi and Seppälä35}

Fused deposition modeling

Similar to SLA, FDM was originally used to fabricate 3D polymeric structures. In the FDM process, a thermoplastic polymer filament passes through a heated liquefier and is then extruded through a nozzle. The extrusion head moves along the x and y axes to deposit material. ^{Reference Griffin, McMillin, Laser Sintering and Deposition36} The method was later modified to fabricate ceramic and ceramic/polymer composites, in a process termed fused deposition of ceramics. ^{Reference Agarwala, Bandyopadhyay, Danforth, Jamalabad, Langrana, Priedeman, Safari and van Weeren37}

Numerous polymeric structures have been processed using FDM; however, only a few of them are biocompatible or can be directly used in biomedical applications, such as acrylonitrile butadiene styrene (ABS), which can be sterilized with γ-radiation or ethylene oxide treatment. ^{Reference Novakova-Marcincinova and Kuric38} PCL is one of the most studied biopolymers for use in FDM. For example, Zein et al. produced 3D PCL structures with a variety of channel sizes, filament diameters, and porosities and found a significant correlation between compressive strength and porosity. ^{Reference Zein, Hutmacher, Tan and Teoh11} Korpela et al. processed poly(lactic acid) (PLA), PCL, PCL/bioactive glass, and poly(l-lactide-co-ε-caprolactone) (PLC) copolymer using FDM. Processing of both PCL and PLA was simple; however, the addition of bioactive glass increased the viscosity of PCL, and the FDM of PLC was interrupted frequently because of high viscosity and filament buckling. All compositions supported cell attachment and proliferation, but fibroblast proliferation was the highest for PLA scaffolds. ^{Reference Korpela, Kokkari, Korhonen, Malin, Nӓrhi and Seppӓlӓ12}

Three-dimensional poly(ethylene glycol terephthalate)/poly(butylene terephthalate) block copolymer scaffolds with varying porosities and pore sizes were also successfully produced for cartilage TE. The deposited scaffolds supported chondrocyte attachment and distribution and encouraged the deposition of articular cartilage extracellular matrix in a nude mouse model. ^{Reference Woodfield, Malda, de Wijn, Peters, Riesle and van Blitterswijk15}

FDM has been used directly and indirectly to make scaffolds for bone TE applications. ^{Reference Bose, Sugiura and Bandyopadhyay39–Reference Kalita, Bose, Bandyopadhyay and Hosick41} Alumina 3D-printed scaffolds have been processed by FDM to support attachment and proliferation of human osteoblast cells. ^{Reference Darsell, Bose, Hosick and Bandyopadhyay42,Reference Bose, Darsell, Kintner, Hosick and Bandyopadhyay43} FDM-processed PLGA is another promising material for bone TE applications because of its interaction with the L-929 fibroblast cell line and increased bone formation in an in vivo rabbit tibia model. ^{Reference Park, Park, Shin, Kang, Kim, Yoon and Shin44} PLGA/β-tricalcium phosphate (β-TCP) composites with simple and complex structures were processed by FDM and coated with HA. After 12 weeks postimplantation, with or without HA coating, samples were biocompatible and supported bone deposition. ^{Reference Kim, McBride, Tellis, Alvarez-Urena, Song, Dean, Sylvia, Elgendy, Ong and Hollinger18} Complex TCP/polypropylene (PP) microstructures (see Figure 2a) have been investigated for bone TE applications because their mechanical properties are similar to those of cancellous bone, the spongy inner part of bone, and because they have the ability to support attachment and proliferation of human osteoblast cells. ^{Reference Kalita, Bose, Hosick and Bandyopadhyay19}

Figure 2. (a) Fused deposition modeling-processed porous tricalcium phosphate/polypropylene scaffolds with (1) complex and (2,3) gradient-controlled porosity. ^{Reference Kalita, Bose, Hosick and Bandyopadhyay19} (b) Scaffolds produced by selective laser sintering: (1) poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), (2) calcium phosphate/PHBV, (3) poly(l-lactic acid) (PLLA), (4) carbonated hydroxyapatite/PLLA. ^{Reference Duan, Wang, Zhou, Cheung, Li and Lu21} (c) Bioplotted collagen/alignate/silica scaffold. ^{Reference Lee, Kim, Kim and Kim24} (d) 3D-printed hydroxyapatite scaffolds fabricated using direct ink writing. ^{Reference Simon, Michna, Lewis, Rekow, Thompson, Smay, Yampolsky, Parsons and Ricci16} (e) 3D-printed bionic ear. ^{Reference Mannoor, Jiang, James, Kong, Malatesta, Soboyejo, Verma, Gracias and McAlpine45} (a) Reproduced with permission from Reference Reference Kalita, Bose, Hosick and Bandyopadhyay19. © 2003 Elsevier. (b) Reproduced with permission from Reference Reference Duan, Wang, Zhou, Cheung, Li and Lu21. © 2010 Elsevier. (c) Reproduced with permission from Reference Reference Lee, Kim, Kim and Kim24. © 2014 Royal Society of Chemistry. (d) Reproduced with permission from Reference Reference Simon, Michna, Lewis, Rekow, Thompson, Smay, Yampolsky, Parsons and Ricci16. © 2007 Wiley. (e) Reproduced with permission from Reference Reference Mannoor, Jiang, James, Kong, Malatesta, Soboyejo, Verma, Gracias and McAlpine45. © 2013 American Chemical Society.

Selective laser sintering

SLS uses a powder bed in which each layer is built by sintering using a laser directed by a CAD model. This technique is commonly used for manufacturing both metallic and nonmetallic parts. Common nonmetallic biomaterials include ceramics such as TCP, HA, alumina, and zirconia and polymers such as PCL, PP, poly(ether ether ketone) (PEEK), and polyamide 12. PCL scaffolds, ^{Reference Williams, Adewunmi, Schek, Flanagan, Krebsbach, Feinberg, Hollister and Das13} scaffolds made of PEEK/HA biocomposite blends, ^{Reference Tan, Chua, Leong, Cheah, Cheang, Abu Bakar and Cha20} and interconnected porous scaffolds using calcium phosphates and polymers such as PLA and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) (see Figure 2b) ^{Reference Duan, Wang, Zhou, Cheung, Li and Lu21} have been fabricated using SLS for TE-related applications. Recent advances in SLS have allowed the production of scaffolds for craniofacial and joint defect applications, as well as for the fabrication of low-stiffness porous scaffolds for soft-tissue applications such as cardiac tissues using PCL and poly(vinyl alcohol) (PVA) with HA. ^{Reference Yeong, Sudarmadji, Yu, Chua, Leong, Venkatraman, Boey and Tan14,Reference Chua, Leong, Tan, Wiria and Cheah22,Reference Partee, Hollister and Das46}

Bioplotting/3D plotting

Bioplotting is an extrusion-based 3D dispensing technique that uses a pressure-controlled dispenser that can be moved in three dimensions to fabricate materials layer by layer on an aqueous medium, thus achieving buoyancy compensation without a support structure. ^{Reference Landers and Mülhaupt47} Bioplotting can be employed for a wide range of materials, mainly in the form of hydrogels, which facilitates the fabrication of complex internal substructures. The main advantage of this technique is its use of natural polymers, such as chitosan, alginate, and collagen, as fabrication materials. Other biomaterials commonly used for this process include polymers such as PCL, PVA, and poly(l-lactic acid) (PLLA) and ceramics such as HA, TCP, bioglass, and their composites.

Haberstroh et al. demonstrated the bioplotting of composite scaffolds made of collagen-treated TCP, TCP/chitosan/collagen hydrogel, and PLGA for ovine critical-sized calvarial defects. ^{Reference Haberstroh, Ritter, Kuschnierz, Bormann, Kaps, Carvalho, Mülhaupt, Sittinger and Gellrich23} Fabrication of collagen/alginate/silica composite scaffolds (see Figure 2c) using a low-temperature bioplotting process for hard-tissue regeneration ^{Reference Lee, Kim, Kim and Kim24} and of 3D hydrogel scaffolds composed of alginate and gelatin for application in pancreas TE²⁵ has also been achieved. Thus, scaffolds fabricated using 3D bioplotting can incorporate living cells and facilitate the growth of tissues. Efforts are being made to improve the process and make it more widely applicable in TE. For example, Kim et al. modified a 3D bioplotting system by introducing a piezoelectric transducer that generated vibrations while fabricating PCL scaffolds, resulting in a rougher surface finish for improved cell attachment. ^{Reference Kim and Son48}

Direct ink writing

Direct ink writing or direct write assembly is a broad term describing fabrication methods that use computer-aided programs to print 3D structures using a nozzle to form droplets on a powder bed. Specifically, this process is similar to binder jetting, which selectively deposits powder material using a liquid bonding agent. A variety of materials, including HA (see Figure 2d ^{Reference Fielding, Bandyopadhyay and Bose16} ), TCP, PCL, and many other polymers, can be processed using this method. ^{Reference Fielding, Bandyopadhyay and Bose17,Reference Bose, Vahabzadeh and Bandyopadhyay26,Reference Seyednejad, Gawlitta, Kuiper, de Bruin, van Nostrum, Vermonden, Dhert and Hennink27,Reference Bose and Fielding49} This process controls the composition of printed parts by using predetermined volumes of the appropriate binder, thus resulting in patterned parts with the desired densities and uniformities. Therefore, with direct ink writing, complex structures can be printed with programmed porosities, as seen in Figure 3a–c. ^{Reference Fielding, Bandyopadhyay and Bose17,Reference Bose, Vahabzadeh and Bandyopadhyay26,Reference Bose and Fielding49,Reference Tarafder, Dernell, Bandyopadhyay and Bose50}

Figure 3. (a) Schematic of the inkjet 3D printing process. ^{Reference Bose, Vahabzadeh and Bandyopadhyay26} (b) Photographs of pure and SrO/MgO-doped tricalcium phosphate (TCP) scaffolds. ^{Reference Tarafder, Dernell, Bandyopadhyay and Bose50} (c) Compressive strengths of pure and SiO₂/ZnO-doped TCP scaffolds with different pore sizes. (d) Human osteoblast cell adhesion on pure and SiO₂/ZnO-doped TCP scaffolds. ^{Reference Fielding, Bandyopadhyay and Bose17} (e) Masson Goldner’s trichrome staining of pure and doped TCP scaffolds (gray/brown), showing mineralized implants (blue) and osteoid formation (orange). (f) von Willebrand factor staining of pure and SiO₂/ZnO doped TCP scaffolds, where the dark red spots within the sections are blood vessels. ^{Reference Bose and Fielding49} (a) Reproduced with permission from Reference Reference Bose, Vahabzadeh and Bandyopadhyay26. © 2013 Elsevier. (b) Reproduced with permission from Reference Reference Tarafder, Dernell, Bandyopadhyay and Bose50. © 2015 Wiley. (c–d) Reproduced with permission from Reference Reference Fielding, Bandyopadhyay and Bose17. © 2012 Elsevier. (e–f) Reproduced with permission from Reference Reference Bose and Fielding49. © 2013 Elsevier.

Calcium phosphate scaffolds for early-stage osteogenesis have been fabricated using this process ^{Reference Bose, Vahabzadeh and Bandyopadhyay26} (see Figure 3d–f). Hydroxyl-functionalized PCL structures for TE applications ^{Reference Seyednejad, Gawlitta, Kuiper, de Bruin, van Nostrum, Vermonden, Dhert and Hennink27} and 3D-printed mesoporous bioactive glass scaffolds with improved mechanical properties and controlled pore architecture for bone regeneration applications ^{Reference Wu, Luo, Cuniberti, Xiao and Gelinsk28} have also been reported. Although direct ink writing allows the fabrication of complex parts with designed porosities and interconnected porous structures, the difficulty of selecting the appropriate binder during processing and the inability to print porous structures with pore sizes smaller than 300 µm are major drawbacks of this technique. Additional post-processing techniques might also be required, such as sintering, which could lead to part shrinkage, thus illustrating the need for process optimization.

Laser-assisted bioprinting

Laser-assisted bioprinting (LAB) is a fabrication process for hydrogel structures that affords the direct incorporation of cells. In LAB, a laser pulse is used to control the deposition of material onto transparent quartz disks, called ribbons. A wide variety of materials such as HA; PLA; PCL; zirconia; and hydrogels with incorporated living cells such as human osteoblast cells, human umbilical vein smooth cells, and multipotent stromal cells (MSCs) (an example of which can be seen in Figure 2e) ^{Reference Mannoor, Jiang, James, Kong, Malatesta, Soboyejo, Verma, Gracias and McAlpine45} can be fabricated. Material processing at ambient temperatures and direct incorporation of cells with a homogeneous distribution are two of the main advantages of LAB. Low mechanical stiffness and the requirement for homogeneous ribbons are potential disadvantages of this process. 3D-printed PLLA/PLGA scaffolds with a mixture of hepatocytes and endothelial cells have been investigated for organogenesis of liver tissue. ^{Reference Griffith, Wu, Cima, Powers, Chaignaud and Vacanti51} Also, high cell viability was observed for MSCs embedded in a printed alginate/hydrogel composite, demonstrating extracellular matrix formation both in vitro and in vivo. ^{Reference Fedorovich, Schuurman, Wijnberg, Prins, van Weeren, Malda, Alblas and Dhert53} Guillemot et al. reported the fabrication of sodium alginate/HA composites with human endothelial cells for TE applications using high-throughput laser bioprinting. ^{Reference Guillemot, Souquet, Catros, Guillotin, Lopez, Faucon, Pippenger, Bareille, Rémy, Bellance, Chabassier, Fricain and Amédée54} HUVECs embedded in glycerol/sodium alginate solution at various viscosities and cell densities were produced using LAB for tissue fabrication. ^{Reference Guillotin, Souquet, Catros, Duocastella, Pippenger, Bellance, Bareille, Rémy, Bordenave and Amédée52,Reference Guillemot, Souquet, Catros, Guillotin, Lopez, Faucon, Pippenger, Bareille, Rémy, Bellance, Chabassier, Fricain and Amédée54}