Introduction
Three-dimensional (3D) printing, which is also known as additive manufacturing, solid freeform fabrication, or rapid prototyping, is a layer-by-layer fabrication method for creating structures directly from computer-aided design (CAD) files. In 3D printing techniques, a 3D CAD model is first converted into a standard tessellation language (.STL) file format and then sliced in a virtual environment to create stacked two-dimensional (2D) sections along the height. A 3D printer builds each 2D layer based on the slice-file information, starting from the base and continuing to build layer-by-layer on top of previously built layers until the final product is printed.
Three-dimensional printing first emerged in 1986 through a stereolithography (SLA) technique devised by Hull. Reference Hull1 In 1991, Stratasys 2 and Helisys Reference Das, Madras, Dasgupta and Umarji3 commercially introduced fused deposition modeling (FDM) and laminated object manufacturing, respectively. Later, in 1992, DTM Corporation introduced a selective laser sintering (SLS) machine. Reference Lou and Grosvenor4 Sachs et al. invented inkjet 3D printing on a powder bed and patented this method in 1993. Reference Sachs, Haggerty, Cima and Williams5 Over the past 25 years, 3D printing has seen several commercial implementations, but the FDM-based concept still leads the world in terms of purchased machines, broader utilization, and applications. Some other techniques have recently found commercial success for the fabrication of soft materials, such as multijet fusion by HP and large-area maskless photopolymerization (LAMP) by DDM Systems (Atlanta, Ga).
Three-dimensional printing of biomaterials has become an area of intense research (see the February 2015 issue of the MRS Bulletin devoted to this topic). It offers the chance to fabricate parts with complex geometries for flexible biomedical devices tailored to a patient’s specific needs while avoiding multistep processing approaches. Over the past two decades, the use of 3D-printed biomaterials for tissue-engineering (TE) applications has offered significant advantages by employing a large variety of materials for the printing of one-of-a-kind structures. Also, incorporation of living cells during processing adds another advantage over other scaffold fabrication approaches.
3D printing technologies for biomaterials and soft materials
This section summarizes some of the key 3D printing technologies and their applications in biomaterials and soft materials (see Table I ). Reference Lee, Wang, Fox, Ritman, Yaszemski and Lu6–Reference Wu, Luo, Cuniberti, Xiao and Gelinsk28
* Acronyms: 3DP, three-dimensional printing; DEF, diethyl fumarate; FDM, fused deposition melting; HA, hydroxyapatite; LAB, laser-assisted bioprinting; MSC, multipotent stromal cell; PBT, poly(butylene terephthalate); PCL, poly(ε-caprolactone); PDLLA, poly(d,l-lactic acid); PEEK, poly(ether ether ketone); PEGT, poly(ethylene glycol terephthalate); PHBV, poly(hydroxybutyrate-co-hydroxyvalerate); PLA, poly(lactic acid); PLGA, poly(d,l-lactic-co-glycolic acid); PP, polypropylene; PPF, poly(propylene fumarate); PVA, poly(vinyl alcohol); SLA, stereolithography; SLS, selective laser sintering; (β-)TCP, (β-)tricalcium phosphate.
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.
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
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
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
Current challenges and future directions
Three-dimensional printing offers various advantages in the fabrication of biomaterials and has become a versatile and popular manufacturing tool for applications involving soft materials. However, different 3D printing techniques have certain advantages and drawbacks, and a good understanding of each process is needed before selection for a specific application. For 3D printing of biomaterials, a sterile fabrication environment is a major issue for some processes. Maintaining cell viability and printing complex 3D parts adds an extra set of challenges that is still being researched. Finally, part accuracy and reproducibility are continuing issues with most 3D printers. Reference Bandyopadhyay, Bose and Das55
The field of soft-material 3D printing is quite advanced. Large FDM-based printers that can print objects as substantial as cars are now commercially available. However, surface finish can be an issue for large parts, for which slice thickness is usually kept large to reduce the build time.
Future directions for 3D printing lie with multimaterial and gradient parts that cannot be created using conventional manufacturing. Three-dimensional printing offers designers capabilities that were not previously available. Therefore, full utilization of 3D printing will come when parts are designed specifically to capitalize on the strengths of the technique and not merely because they are not easily fabricated using conventional tools. The addition of polymers with ceramics to aid in drug delivery, variation in composition to tailor cell-materials interactions, and mechancical properties are some of the concept that will evolve as the next-generation 3D printed scaffolds. Reference Tarafder and Bose56,Reference Bose, Tarafder, Banerjee, Davies and Bandyopadhyay57 Simultaneous deposition of multiple materials offering unique properties and functionalities will lead to a manufacturing revolution in the coming years with the help of 3D printing.
Acknowledgments
The authors thank the W.M. Keck Foundation, M.J. Murdock Charitable Trust, National Institutes of Health (NIH, Grant AR066361-01A1), and Life Sciences Discovery Fund (LSDF) for their financial support.