3-dimensional bioprinting for tissue engineering applications
- Bon Kang Gu†1,
- Dong Jin Choi†1,
- Sang Jun Park1,
- Min Sup Kim1,
- Chang Mo Kang1 and
- Chun-Ho Kim1Email author
© Gu et al. 2016
Received: 4 December 2015
Accepted: 12 April 2016
Published: 25 April 2016
The 3-dimensional (3D) printing technologies, referred to as additive manufacturing (AM) or rapid prototyping (RP), have acquired reputation over the past few years for art, architectural modeling, lightweight machines, and tissue engineering applications. Among these applications, tissue engineering field using 3D printing has attracted the attention from many researchers. 3D bioprinting has an advantage in the manufacture of a scaffold for tissue engineering applications, because of rapid-fabrication, high-precision, and customized-production, etc. In this review, we will introduce the principles and the current state of the 3D bioprinting methods. Focusing on some of studies that are being current application for biomedical and tissue engineering fields using printed 3D scaffolds.
ASTM standard terminology for additive manufacturing technologies
Additive Manufacturing (ASM F2792)
• Stereolithography (SLA)
• Digital light processing (DLP)
• Multi-jet modeling (MJM)
• Fused deposition modeling (FDM)
Powder bed fusion
• Electron beam melting (EBM)
• Selective laser sintering (SLS)
• Selective heat sintering (SHS)
• Direct metal laser sintering (DMLS)
• Powder bed and inkjet 3D printing (PBIH)
• Plaster-based 3D printing (DMLS)
• Laminated object manufacturing (LOM)
• Ultrasonic consolidation (UC)
Directed energy deposition
• Laser metal deposition (LMD)
Recently, the aim of tissue engineering is regeneration, restoration, or replacement of defective or injured functional living organs and tissues [24–26]. In order to achieve this aim, biomedical scaffolds made of natural or synthetic polymers have been commonly used in biomedical and tissue engineering applications [27, 28]. The major focus of these scaffolds is to replace or regenerate the native tissues functionally and structurally. In general, the scaffolds for use as tissues and organs have a several mandatory functions: it should provide internal pathways for the cell attachment and migration, it must transfer various growth factors and waste products, and it should keep its shape while the cells are growing, and have adequate mechanical properties. . To achieve these functions, biomedical scaffolds for tissue engineering require a highly porous 3D structure that allows cell affinity such as proliferation, migration, attachment, and differentiation, even enables nutrients and oxygen transport [30, 31]. Therefore, 3D bioprinting technology is one of the most appropriate methods for producing a 3D structure for use as biomedical scaffolds, tissues, and organs. The 3d bioprinting is the technique for controlling a cell pattern to be retained functionality and viability of the cells within the printed 3D structure. In tissue engineering, development of the appropriate scaffold using a 3D printing has already been studied by many researchers [32, 33]. Advances introduced by 3D bioprinting have importantly enhanced the ability to control pore size distribution, pore volume, and pore interconnectivity of scaffolds. Furthermore, 3D bioprinting accredit to important advances in tissue engineering field by the study of biomaterials or bio-ink. Development of biomaterials in 3D bioprinting is an important prerequisite to a direct effect on cell growth. Some 3D printing processes to contain living cells and bioactive molecules in biomaterials (hydrogels) made successfully 3D structures at room temperature without any significant effect on the cell viability. For applications using 3D bioprinting technologies in tissue engineering, researchers should be considered the biomaterials (bio-ink) as well as the 3D structure (design).
Among additive manufacturing technologies, several methods such as SLA [34, 35], FFF [36, 37], SLS [38, 39] and inkjet 3D printing [40, 41], etc. have been applied in tissue engineering field. These methods have been used in various sectors as architectural modeling, art, and lightweight machines and also 3D structures from biomaterials is used for tissue engineering and regenerative medicine. 3D bioprinting is to produce a 3D structure of the desired shape by combining the living cells and biomaterials. Researchers are developing various methods to fabricate 3D unique structure with biological and mechanical properties suitable for regeneration of native tissue. In this review, we describe the four different type of 3D bioprinting technology for fabrication of 3D structure and its application in tissue engineering and regenerative medicine fields.
3D bioprinting for tissue engineering application
Advantages and disadvantages of various 3D bioprinting methods for tissue engineering applications
• Manufactured simple and complex
• Expensive equipment and materials
PEG, PCL, PEG-co-PDP, PEGDA.
• Fast and good resolution
• Only photopolymers
• No need for support materials
• Cytotoxicity of uncured photoinitiator
• Easy to use
• Materials limited to thermoplastics
• Good mechanical properties
• Filament required
• Solvent not required
• Cannot used with cells
• No need for support materials
• Rough surface
PCL/HA, PCL, HA/PEEK, Titanium.
• Various of biomaterials
• Expensive and cumberstone equipment
• Cells and hydrogel printed
• Limited biomaterials suite
Collagen/PDL, Fibrin, Gelatin.
• Incorporation of drug and biomolecules
• Low resolution
• Low mechanical properties
Vat photopolymerization method
The main advantages of vat photopolymerization method in tissue engineering applications are that fabrication of simple, complex designs, fast processing, high resolution, and no need for support material. The disadvantages are that expensive equipment, expensive curing materials as photoinitiator, and cytotoxicity of uncured photoinitiator.
Fused filament fabrication method
FFF printers in material extrusion method use a thermoplastic filament. This filament is heated to the melting point and then extruded to prepare a 3D structure. These thermoplastic filaments are deposited through an extrusion nozzle during printing. The nozzle melts the filaments and then extrudes onto the substrate for fabricating 3D structure (FFF method). The nozzle and substrate are controlled by a computer that translates the dimensions of a structure into X, Y and Z coordinates during printing. A schematic of material extrusion method is shown in Fig. 2(b). FFF method is a thermal-heating technique for use 3D scaffolds fabrication in tissue engineering applications. Many researchers were reported using FFF method for tissue engineering. Pati et al. reported that to enhance the biological properties of extracellular matrix (ECM)-ornamented 3D printed scaffolds with cells using FFF bioprinting . They developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells. Lee et al. fabricated melt-plotted/in situ plasma-treated PCL scaffolds coated with chitosan of various molecular weights in a layer-by-layer manner . They evaluated the effects of the chitosan coating on various physical and cellular activities, including water wetting ability, cell proliferation, ALP activity, and calcium deposition using the osteoblast-like MG63 cell line. Hong et al. fabricated solid freeform fabrication based 3D PCL/PLGA scaffolds that provide functionalized surfaces through a simple but efficient coating of mussel adhesive proteins without any surface modification procedures .
The main advantages of FFF method in tissue engineering applications are that easy to use, a variety of biomaterials, good mechanical properties, and the solvent not required. The disadvantages are material restriction related to thermoplastic polymers. In addition, it cannot be printed with the cells due to the high manufacturing temperature.
Selective laser sintering method
SLS is a technique that uses the laser as a power source to form solid 3D structures. This method uses a high power laser for powder sintering to form a scaffold. This method is produced by selective laser printing from 3D modeling software in the part on the surface of a powder bed. This process may be printed from several of materials such as ceramics, metals, and polymers. A schematic of SLS is shown in Fig. 2(c). SLS of polymer powder has been evaluated by several groups for tissue engineering application and drug delivery system [52–55]. Moreover, the SLS has been used to tissue engineering application as scaffolds from polymeric biomaterials and their composites [56–58]. Du et al. fabricated a novel protocol to produce SLS-derived bone scaffolds using the PCL microspheres and polycaprolactone/hydroxyapatite (PCL/HA) composite microspheres as the basic building materials . The biocompatible evaluation of the SLS-derived scaffolds was investigated using rat MSCs and the results showed both pure PCL scaffolds and PCL/HA composite scaffolds can well support cell adhesion, proliferation, and growth. Williams et al. used SLS to process PCL to produce parts with controlled pore sizes in the range 1.75 ~ 2.5 mm and designed porosities from 63.1 % to 79 %, but met with limited success in terms of accurately achieving the required porosity levels . Particle size and thermodynamic variations were found to play critical roles. Tan et al. demonstrated the ability of SLS to fabricate physically blended hydroxyapatite/poly(ether-ether-ketone) composites for tissue scaffold development and observed micropores on the scaffold surface . Chen et al. showed that PCL scaffolds manufactured by SLS were surface modified by immersion coating with either gelatin or collagen for cartilage tissue engineering . Ciocca et al. reported a technique to design and manufacture a customized titanium mesh for minimal bone augmentation of an atrophic maxillary arch, guided by the final position of the prosthesis and according to the implants necessary for its support .
The main advantages of this process for tissue engineering applications are a wide range of biomaterials that can be used. Powder bed is used as a support, therefore, no need for secondary support structures. Also, unused powders may be recycled. The disadvantage of SLS is that the detail is not as crisp and sharp when compared with other processes, such as SLA and FFF. Another disadvantage is that the SLS bioprinters tend to be large, cumbersome, and expensive.
Inkjet 3D printing
Inkjet 3D printing method is a rapid prototyping and layered manufacturing technology for making structures described by 3D modeling data. Inkjet 3D printing is closely related to Inkjet head printing. Lately, inkjet 3D printing method has been significant developments in the use of polymeric bio-ink printing for applications in biological and tissue engineering fields. A schematic of inkjet 3D printing is shown in Fig. 2(d). Inkjet bioprinters are the most commonly used type of printer for both non-biological and biological applications. Many researchers were reported using inkjet head 3D bioprinting method for tissue engineering. Sanjana et al. reported on the use of inkjet bioprinting to create neuron adhesive patterns as islands and other pattern using PEG (cell-repulsive material) and collagen/poly-D-lysine mixture (cell-adhesive material) . Xu et al. use the inkjet bioprinting technology for the fabrication of 3D scaffolds, based on fibrin gel . Fibrin has been used as a printable hydrogel for building a 3D neural construct. Lee et al. reported the printing of a growth factor-releasing fibrin gel containing murine neural stem cells (NSCs) to construct an artificial neural tissue and then examined the effects of the growth factor-releasing fibrin gel on the survival of the murine NSCs . Lorber et al. printed retinal glia cells with cell culture media and subsequently assessed the survival of these cells in culture . Pati et al. have focused on bioprinting of dome-shaped adipose tissue constructs using human decellularized adipose tissue matrix bio-ink that encapsulates human adipose tissue-derived mesenchymal stem cells through biomimetic approach for evaluation of their efficacy in adipose tissue regeneration . Irvine et al. reported on the development of printable gelatin as the bio-ink with cell-encapsulated. They were fabricated patterned 3D structure by using inkjet bioprinter and then confirmed excellent cell affinity .
The advantages of inkjet 3D bioprinting method for tissue engineering applications are that patient-customized fabrication, rapid production, low cost of production, and easy to incorporate both drug and biomolecules. In addition, it can be a printing with the cells. The disadvantages are that limitation of size and biomaterials, low resolution, and negligible mechanical properties.
Current and future direction for 3D bioprinting
The technology for 3D bioprinting has a lot of advantages, but it still has many challenges that remain to be overcome. Heretofore, several types of research about 3D bioprinting have conducted in the lab of universities and companies. For example, Organovo’s exVive3D™ Liver bioprinted human tissue models with collagen are created using proprietary 3D bioprinting technology . The resulting tissues contain accurate and reproducible 3D structure that can remain completely functional and reliable over 40 days. Also, Atala group was succeeded in scaffold production for the human kidney using 3D bioprinting technology . Cornell university researchers reported that 3D printed ears similar to human ear using 3D bioprinting and collagen gels with living cells . So far, as mentioned above, patient-customized 3D bioprinting was studied only in a few laboratories. However, 3D bioprinting in the future has to be the development of various models in many laboratories.
Bio-inks for 3D bioprinting
exVive3D™ Human Liver Models
Wake Forest Univ.
Kidney cell, nephron
Collagen, Calcium phosphate
Acrylonitrile butadiene styrene (ABS)
The future of 3D bioprinting is not limited to inanimate structures. 3D printed medical implants will be able to enhance the quality of human life. 3D bioprinting is currently used for prosthetic limbs, orthodontic devices, and bone implants because it can be matched to the correct body shape of the patient. Printing of soft tissue is progress, and can be used immediately in veins and arteries printing operations. Today, medical applications of 3D bioprinting have developed a nano-medicine, pharmaceuticals, and organs such as human health fields. Finally, direct organ fabrication using 3D bioprinting technology is the ultimate goal in tissue engineering and regenerative medicine. There is a possibility of printing a complete organ that could be directly transplanted into the human body.
In the recent years, a lot of 3D bioprinting method and design has been developed for tissue engineering. Especially, computer-aided 3D printing techniques have a great potential to fabricate complex 3D structures with highly porosity architecture. It can be achieved great strides in biomedical application fields, especially infusion of medical imaging techniques such as CT and MRI. However, the low resolution and using only one technology for fabricating a native tissue similar 3D structure, there is a limit. Thus, using more than two 3D printing technologies or combination of 3D printing technologies with other scaffold fabrication technologies can overcome the limitations and fabricate a multifunctional 3D structure. In the recent, only a few of the research groups have been deeply characterized though extensive in vitro and in vivo studies and results are mostly limited to a restricted number of biomaterials. Thus, development of materials (bio-ink) is one of the most important goals in 3D printing. It has enabled to directly create implantable devices such as biodegradable tissue engineering scaffolds.
This research was supported by the National R&D Program through the Korea Institute of Radiological and Medical Sciences funded by the Ministry of Science, ICT & Future Planning (1711021779) and the Technology Innovation Program (10053595, Development of functionalized hydrogel scaffold based on medical grade biomaterials with 30 % or less of molecular weight reduction) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea).
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