3D Bioprinting of Artificial Tissues: Construction of Biomimetic Microstructures

Yongxiang Luo,* Xin Lin, and Peng Huang*

constructs with precisely designed overall structures.[12] However, the construction of microstructure biomimicking native tissues is crucially important to realize their biological functions. Specifically, the fabrication of vessel-like networks for ini-tial nutrient and oxygen supply to cells, and the arrangement of multiple types of cells for creating lamellar/complex tissues have been widely studied in the field of 3D bioprinting.

In this review, we will summarize the current advances in 3D bioprinting of arti-ficial tissues/organs from the view of con-struction of biomimetic microstructures. The 3D bioprinting approaches mainly

include laser-assisted bioprinting, inkjet bioprinting, and microextrusion-based bioprinting. The fabrication of micro-structure will be discussed by the tissue-types such as lamellar, vascular networks, and complex tissues. In the end, we will elaborate on the conclusion and outlook of 3D printing of func-tional microstructures.

2. The Approaches of 3D Bioprintingof Tissues/Organs

Common 3D printing approaches include the fused deposition modeling/manufacturing (FDM),[13] selective laser sintering (SLS),[14] and 3D powder printing.[15] In general, these methods need high temperatures to melt the printed materials (such as FDM and SLS) during printing or to sinter the constructs achieving strong mechanical stability in postprocessing (such as 3D powder printing). Therefore, only selected materials were able to be printed by these approaches, such as thermoplastic polymers, metals, and ceramics. For 3D printing of bioinks containing living cells or biological factors, there is a high demand for the processing conditions such as low tempera-ture and without use of organic solvent. According to different biofabrication systems, several selections of 3D bioprinting approaches are available and can be mainly classified as laser-assisted bioprinting, inkjet bioprinting, and microextrusion-based bioprinting (Figure 1).

2.1. Laser-Assisted Bioprinting

Laser-assisted bioprinting refers to laser-induced forward transfer that, generally, comprises of three main components: the pulsed laser, donor slide (covered with a laser energy

It is promising that artificial tissues/organs for clinical application can be produced via 3D bioprinting of living cells and biomaterials. The construc-tion of microstructures biomimicking native tissues is crucially important to create artificial tissues with biological functions. For instance, the fabrication of vessel-like networks to supply cells with initial nutrient and oxygen, and the arrangement of multiple types of cells for creating lamellar/complex tis-sues through 3D bioprinting are widely reported. The current advances in 3D bioprinting of artificial tissues from the view of construction of biomimetic microstructures, especially the fabrication of lamellar, vascular, and complex structures are summarized. In the end, the conclusion and perspective of 3D bioprinting for clinical applications are elaborated.

1. Introduction

3D printing, so called additive manufacturing, generates 3D products in layer-by-layer pattern based on computer-assisted design (CAD) and computer-assisted manufacturing. 3D printing technology has been widely reported to manufac-ture implants and scaffolds for biomedical applications.[1] So far, various biomaterials including metals,[2] bioceramics,[3] and biopolymers[4] have been fabricated as scaffolds for tissue engineering. However, 3D bioprinting of artificial tissues/organs replicating the hierarchical structures of native tissues in cell-laden manner is the most promising strategy to benefit biomedical application. In this strategy, stem cells and hydrogel-based bioinks as the two pivotal factors have been investigated deeply.[5,6] Several excellent reviews have summarized the advances of hydrogel-based bioinks, recently.[7–10] Additionally, mimicking the microstructures of native tissues is also the key step to create artificial tissues with biological functions via 3D bioprinting for biomedical application.[11]

One of the typical merits of 3D printing is the capability of precise design and control of structures, which distinguishes this technique as being far superior to other traditional tech-niques in the field of tissue engineering and regenerative medicine. To date, 3D printing can produce human-scale tissue

absorbing layer and a layer of cell-containing bioinks), and receiving substrate.[16] Once the laser is focused onto the donor slide, the material is evaporated and thus generates high gas pressure to propel the bioinks (with cells) toward the receiving substrate. By controlling the movement of the donor slide and/or the receiving substrate, 3D objects with predesigned struc-tures can be manufactured (Figure 1a,b). This method is a nozzle-free approach without nozzle clogging problem. Mean-while, the droplet building manner displays high resolution with printed droplet volume as small as 0.5 pl.[17] Furthermore, it is possible to control the multicellular distribution accurately over the ejected drops.[17] However, the high resolution is gener-ally based on the expenses of printing efficiency, which implied that long time was required for creating a large-scale object. Therefore, the laser-assisted bioprinting is a time-consuming process on the constructions of human-scale tissues for clinical applications. In addition, the laser emission might have risk to damage the printed cells.[18]

2.2. Inkjet Bioprinting

Inkjet bioprinting is a noncontact approach that builds 3D objects through depositing bioink droplets or filaments con-taining cells in layer-by-layer fashion.[19] Generally, thermal or piezo-electric system was equipped to generate pulse or shock wave that expelled the drops or filaments of cell-laden bioinks from the print nozzle. Controlling the movement of nozzle and/or the receiver platform, 3D constructs with designed morphologies can be generated (Figure 1c,d). The advantages of this approach include high resolution (up to 20 µm)[20] and affordable commercially.[21] The resolution of the printed con-structs depends on the droplet or filament size through the nozzle. Generally, the size of the nozzle used in this approach is in the range of 20–30 µm, and microheat or piezo-electricity always generates bioink drops in small volumes.[22] However, inkjet bioprinting has relatively high demand for the viscosity of the bioinks.[23] For example, the printed bioink drops with low viscosity are prone to spread on the substrates,[24] while the bioinks with high viscosity often cause the problem of nozzle clogging. Furthermore, the fabrication of 3D complex con-structs with clinically relevant size using this approach is still a challenge due to the slow printing speed and the potential harmful effects on the embedded cells from the shear fore or thermal stress.

2.3. Microextrusion-Based Bioprinting

Microextrusion-based bioprinting, so called robotic dis-pensing, can produce large-scale 3D objects in a short pro-cessing time.[25] In this approach, 3D objects were fabricated by extruding continuous filaments via a nozzle either by pneu-matic, piston, or screw driven. Hydrogels containing living cells were loaded in syringes equipped with nozzles. 3D objects with predesigned structures and dimensions are fabricated by con-trolling the movement of the syringes and/or the receiver plat-form (Figure 1e,f). The resolution of the extruded filaments and final printed objects is dependent on the inner diameter of the

nozzles, dosing pressure, and the printing speed.[26] In general, higher resolution is achieved by applying a smaller nozzle, lower dosing pressure, and faster printing speed. However, nozzles with inner diameter smaller than 100 µm are usually inapplicable in this technique due to the problem of clogging. Meanwhile, the dosing pressure is directly proportional to the printing speed, which means that faster printing speed requires higher dosing pressure to achieve homogeneous and contin-uous filaments. Furthermore, the printed bioink filaments also exhibited spreading behavior on the receiver in this approach. Therefore, the resolution of the printed 3D objects is generally limited in the order of 200 µm,[27] which is considerably lower than that prepared by laser- and inkjet-based approaches. The

dosing pressure is another important issue in this approach. For laser and inkjet methods, the dosing pressure is generated via the burst of the evaporated air bubbles, while the dosing pressure for extrusion-based bioprinting is generated by com-pressed gas, piston, or screw. Previous studies have verified that cells maintained high viability and differentiation capacity after deposition using both compressed gas and piston driven system.[28,29] Screw extrusion can generate high dosing pres-sure, which is effective for printing hydrogels with high vis-cosity, while high dosing pressure is potentially harmful for the encapsulated cells. Nevertheless, this method can fabricate 3D large-scale constructs with relatively low resolution in an effec-tive manner.

3. Microstructure Construction

Tissues and organs have highly sophisticated microstruc-tures,[30] for example, the ordered lamellar structures of skin containing different types of cells and extracellular matrix (ECM),[31] the complex and fine vascular networks and the hierarchical structures of bone.[32] The sophisticated micro-structure is one of the major contributions to their specific physiological functions. Conventionally, tissue engineering aims to regenerate the physiological functions of tissues and organs through biomaterial-,[33] cell-,[34] and growth factor-based[35] approaches. However, until now, almost all of these traditional approaches were unable to precisely mimic the microstructures of tissues and organs through controlling cells deposition, cells-ECM organization in 3D. Interestingly, 3D bioprinting is able to control the hierarchical deposition of different types of cells and biomaterials in 3D manner with predesigned structures, including lamellar structures (such as skin), vascular networks, and complex structures (Table 1).

3.1. Lamellar Structures

Skin and cartilage with lamellar structures and relatively fewer types of cells are easier to be simulated and fabricated compared to other complex tissues and organs in tissue engineering. Therefore, it has great advantage to create lamellar structures via 3D bioprinting due to the layer-by-layer building fashion.

3.1.1. Skin

Since 3D bioprinting owns the ability to deposit specific type of cells and ECM in a predictable manner,[36] it is possible to fab-ricate biomimetic skin tissues with lamellar structures. Biomi-metic skin was created in the pattern of layer-by-layer printing of collagen matrix, keratinocytes (KC), and fibroblasts (FB) via inkjet-based 3D bioprinting approach. It is able to obtain two distinct cell layers of KC and FB with precisely designed thick-nesses. The printed cells maintained highly viable proliferation ability for a certain time. The as-prepared biomimetic skin exhib-ited similar morphology and biological properties to human skin.[37] In addition, the laser-assisted bioprinting approach also can generate 3D multicellular grafts: like the natural archetype of skin tissue through the layer-by-layer deposition of KC-laden and FB-laden collagens. Immunohistochemical and immuno-fluorescence staining results further confirmed that the printed grafts have typical skin-like bilayered structures with intercel-lular adhesion and cell–cell communication.[38,39]

3.1.2. Cartilage

Mature hyaline cartilage injuries caused by articular disorders or sport damages cannot self-heal, due to the characteristics

Figure 1. The three typical approaches of 3D bioprinting of cells and tissues. a,b) Laser-assisted bioprinting of lamellar structures with fibroblasts (green) and keratinocytes (red). Scale bar is 500 µm. Adapted with permission.[39] Copyright 2012, Wiley. c,d) Inkjet-based bioprinting, the CAD model and the realized branched vascular structure via inkjet bioprinting. Adapted with permission.[76] Copyright 2014, Elsevier. e,f) Extrusion-based bioprinting, the CAD model and the fabricated human ear via extrusion bioprinting. Adapted with permission.[7] Copyright 2016, Springer and [44] Copyright 2015, Wiley-VCH.

of nonvascular, nonnerves, and nonlymphatics.[40] Despite the simple features, cartilage is indeed a heterogeneous tissue with lamellar structures including four layers from top to bottom: superficial layer, middle layer, deep layer, and calcified layer. Each layer has different chondrocyte morphology and density, collagen organization, and matrix composition.[41] According to the general process of 3D bioprinting, 3D objects were fab-ricated by depositing chondrocyte-laden hydrogels (such as alginate and poly(ethylene glycol) dimethacrylate) via micro-extrusion- or inkjet-based approaches.[42–44] For improving the mechanical properties of the 3D objects, synthetic polymers (such as polycaprolactone (PCL)) were generally printed as framework. Chondrocyte-laden alginate or collagen solution was then deposited within the PCL frameworks.[45,46] Chondro-cyte-laden 3D objects with comparable mechanical properties were obtained through coprinting PCL and cell-laden hydrogels.

Furthermore, this approach also can be used to fabricate an ear by co-bioprinting PCL, chondrocyte-laden and adipocyte-laden hydrogels.[47] A bionic ear was created via 3D bioprinting of chondrocyte-seeded bioink, along with an intertwined con-ducting polymer consisting of infused silver nanoparticles.[48] The chondrocyte cells maintained high viability during in vitro culture. Meanwhile, the inductive coil antenna enables the readout of inductively coupled signals from cochlea-shaped electrodes. However, it remains a challenge to fully mimic the lamellar structure of cartilage with different chondrocyte mor-phology and density, collagen organization, and matrix compo-sition via 3D bioprinting.

3.2. Vascular Structures

Vascular networks play important role in supplying cells/tis-sues with essential oxygen and nutrients. Many strategies

have been developed to build vascular networks in tissue engi-neering including cell-based,[49] growth factor-based,[50] scaffold-based,[51,52] and AV loop model[53] methods. More details about these approaches can be found in previous reviews.[54,55] In this section, we will focus on the creation of vascular structures by 3D bioprinting such as i) direct fabrication of vascular channel structures via core–shell flow and coaxial nozzle method, ii) creation of the connected channel structures via sacrificial(fugitive) materials, and iii) bioprinting cell drops/filaments forming vascular structures.

3.2.1. Core–Shell Flow and Nozzle Method (Coaxial 3D Printing)

One-step preparation of cell-laden hydrogel fibers with vascular-like structures has been achieved through core–shell flow and nozzle method. In this method, 2 wt% cell-laden alginate solution was injected through middle pipette, and crosslink solution (CaCl2) was injected simultaneously from core to sheath flow. Once the algi-nate solution and crosslinking solution were contacted in the tip of the pipette, alginate was crosslinked immediately to maintain the hollow structure.[56–58] Specifically, cell-laden alginate objects with designed hollow channels and 3D structures can be fabricated by using core–shell flow and 3D bioprinting (Figure 2a).[59–61] In addi-tion, bio-blood-vessels were constructed by coaxial 3D bioprinting of cell-laden alginate/decellularized extracellular matrix and fugi-tive CPF-127 hydrogels.[62] However, the as-prepared hollow fibers and 3D channels, due to their thin wall, were fragile and difficult to be handled. By extruding alginate pastes with high concentration (e.g., 16.7 wt%) through core–shell printing nozzles, 3D channel structures with good structural integrity were achieved thanks to the enhanced stiffness of concentrated alginate pastes during 3D printing (Figure 2b).[63] However, the high concentration of algi-nate had adverse effect on the viability of embedded cells.

Table 1. Examples of microstructure construction via 3D bioprinting.

Microstructure Bioinks Cells Construction strategy Important findings Ref.

Lamellar structures

(skin)

Collagen I Fibroblasts,

keratinocytes

Laser-assisted

3D bioprinting

Typical bilayered structures mimicking skin

with favorable intercellular adhesion

and cell–cell communication

[39]

Lamellar structures

(cartilage)

Fibrin–collagen,

PCL

Chondrocyte Hybrid printing different

materials in layers

Five-layer tissue construct of 1 mm

thickness formed cartilage-like tissues

both in vitro and in vivo

[45,46]

Hollow tubes

structures (vascular)

Alginate Fibroblasts, endothelial

cells, stem cells

Extrusion-based, core–shell

(coaxial) 3D bioprinting

3D objects with channel structures

mimicking vascular structures,

while nonconnected channels between

layers and hollow fibers

[59–61]

Hollow tubes

structures (vascular)

Hydrogel, carbohydrate glass,

pluronic F-127, Ad-HA, CD-HA

Fibroblasts,

endothelial cells

Extrusion-based, fugitive lattice

bioprinting (sacrificial ink)

Large size constructs with fully

connected vascular networks

[65,66]

Hollow tubes

structures (vascular)

Crosslinked hyaluronic

acid alginate, agarose

Fibroblasts, human

MG-63 cells, stem cells

Cells droplets or filaments

assembly bioprinting

Branched structures were achieved;

structural support from molding

template, gelation from substrate and

buoyant force from medium

[80–82,84]

Complex solid

structures (bone,

skeletal muscle, liver)

Composite hydrogel,

PCL, GelMA

Human AFSCs,

chondrocytes,

myoblasts, hiPSCs

Co-bioprinting different

type of cells and materials

Complex tissue and solid organ

constructs with controlled cells location,

mechanical stability, and microchannels

mimicking vascular networks;

array of liver lobule structure

[20,91]

3D bioprinting of channel structures using core–shell flow or coaxial nozzles is an efficient and convenient method. How-ever, it is difficult to create branched tube-like structures using this method.

3.2.2. Method of Using Sacrificial Materials

To address the challenge of creating branched tube-like struc-tures, a method of using sacrificial materials is proposed to build channel networks with bifurcated structures by micromolding in a chip using gelatin as sacrificial materials.[64] The sacrificial materials are widely used in 3D printing for temporary support of the overhanging parts in 3D objects. After the complex 3D objects were printed, the sacrificial materials were removed. Therefore, the employed sacrificial materials should be printable and can be easily removed. A carbohydrate-glass was selected as a sacrificial material in this process because it is printable and easily washed out. Hydrogels containing living cells were casted into the printed carbohydrate-glass based 3D lattice. After crosslinking of the hydrogels, the carbohydrate-glass was dis-solved in medium creating a fully connected channel network (Figure 3a).[65] Additionally, other cell-friendly sacrificial mate-rials were applied in this strategy such as Pluronic F-127 and gelatin.[66,67] Specifically, human umbilical vein endothelial cells were able to adhere to the inner wall of channels when they were connected to a perfusion system (Figure 3b).[68–71] Gener-ally, the method of using sacrificial materials is composed of three steps: 3D printing of fugitive ink lattice, cast of cell-laden hydrogels, and removal of the fugitive ink.

Direct printing of fugitive ink into cell-laden hydrogels is more efficient and flexible to create channel networks,

compared to the method of using sacrificial materials.[72] Recently, a self-healing hydrogel based on adamantane/β-cyclodextrin modi-fied hyaluronic acid (Ad-HA or CD-HA) was prepared as the receiving material for crea-tion of channel structures.[73] Once the nozzle was inserted into the receiving hydrogel, the hydrogel was deformed and rapidly healed around the injected ink leading to the non-mixing of the printed ink and the receiving hydrogel. Finally, the channel structures were highly retained after the ink was removed. Therefore, it is possible to create very com-plex channel structures (such as the biomi-metic vascular-like structures in 3D) through this strategy (Figure 3c). However, to fulfill this strategy, fugitive inks and cell-laden hydrogels should meet the requirements of good cytocompatibility and the maintenance of the printed structures without mutual penetration.

Sacrificial materials-based 3D bioprinting is a promising approach to fabricate large size constructs with biomimetic vascular-like net-works.[69,70,74] However, several issues should be carefully addressed. For example, sacri-ficial materials must meet various require-

ments including excellent printability, facile wash-out, suitable stiffness to carry their own weight, stable interfaces with target hydrogels, and cytocompatibility. Meanwhile, it seems that this approach has limited capability to deposit multiple types of cells in organized positions and designed microstructures.

3.2.3. Cell-Laden Droplets or Filaments Assembly

Microsized droplets or filaments containing living cells have been used as cellular building blocks to reconstruct tissues with designed microstructures via microfluidic technique and 3D bioprinting.[75–78] The printed cell-laden microdrops or rods must maintain 3D vascular-like structures rather than spreading out diffusely. However, cell-laden drops or rods are generally liquid gels exhibiting diffusion behavior on the receiver after bioprinting. In order to overcome this problem, three typical strategies have been developed including i) molding template, ii) gelation from substrate, and iii) buoyant force supporting from medium. For example,Norotte et al.[79] designed an approach to print agarose rods as molding templates and simultaneously co-bioprint uniform multicellular droplets layer-by-layer (Figure 4a). The molding templates not only controlled the microstructures of 3D objects including channel diameter, wall thickness, and branch archi-tecture, but also provided mechanical support to retain the 3D channel structures.[80] After the molding templates were removed, 3D channel structures were obtained.

The typical example of fabricating branched vascular net-works using the strategy of gelation from substrate is 3D bio-printing of cell-laden alginate bioinks. When alginate droplets were printed onto the receiver containing CaCl2, the upper

Figure 2. a) Direct creation of hollow channel structures via coaxial bioprinting of cell-laden alginate and CaCl2 solution, adapted with permission.[61] Copyright 2015, Elsevier. b) Direct creation of hollow channel structures via core–shell nozzle-assisted printing of highly concen-trated alginate bioinks, adapted with permission.[63] Copyright 2013, Wiley-VCH.

alginate drops were crosslinked by the diffusion of calcium ions from bottom alginate layers, which promises the stable branched vascular networks in 3D (Figure 4b).[81] However, the gelation timing and degree are of importance. Fast and strong gelation resulted in the separation of drops, while slow and weak gelation resulted in the collapse of structures. In addition, alginate droplets or filaments were also directly printed into CaCl2 solutions to build the vascular-like structures by control-ling the movement of the receiver. The CaCl2 solution not only acted as crosslinker for the gelation of alginate, but also pro-vided buoyant force to support the overhang structures. Tubular structures with a height of 10 mm were obtained by depositing 210 layers of cells-laden alginate droplets into CaCl2 solution (Figure 4c).[82] The operating conditions such as the distance between the nozzle tip and liquid level should be carefully con-trolled to avoid the clogging of the nozzle and printing error.[83] However, only a few materials such as alginate can be used in this approach. Another interesting method is to print cell-laden hydrogels into a hydrophobic high-density fluid.[84] The hydro-phobicity and high density of the receiver solution could con-trol the deposition of hydrogel droplets and the buoyant force provided support for the overhang structures. The human-scale objects with branch vascular-like networks were obtained through this method (Figure 4d).

3.3. Complex Structures

3.3.1. Musculoskeletal Tissues

Human tissues/organs such as liver and bone have com-plex microstructures containing multiple types of cells and ECM. It is, absolutely, a great challenge to accurately repli-cate the microstructures of human tissues/organs. 3D bio-printing of biomimetic tissues/organs is still in its infancy.[85] The inkjet-based and microextrusion-based approaches have been developed to deposit different types of cells and mate-rials by using multiple printing nozzles. For example, Mer-ceron et al.[86] designed a microextrusion-based 3D bioprinting system consisting of four syringes to engineer a muscle-tendon unit. Four syringes were loaded with C2C12 myoblasts-laden bioinks, NIH/3T3 fibroblasts-laden bioinks, polyurethane (PU), and PCL, respectively. Materials and cell-laden bioinks were coprinted to yield 3D constructs with biomimetic structures of native tissues via layer-by-layer process. The PU and PCL mate-rials possessed high mechanical properties for structural sup-port and the printed cells maintained high viability, their initial tissue development, and differentiation ability. The strategy of coprinting materials and cells is also popular for the fabrication of hard tissues such as bone.[87–89] Recently, human-scale tissue

Figure 3. Creation of vascular-like structures by the approach of using sacrificial materials. a) Carbohydrate-glass was selected as sacrificial elements to be printed as 3D lattice. Then, hydrogels containing living cells were casted into the printed lattice. After dissolving the carbohydrate-glass lattice, connected channel networks were created. Adapted with permission.[65] Copyright 2012, Nature Publishing Group. b) 3D cell-laden vascularized tis-sues that exceed 1 cm in thickness were created by using cell-friendly Pluronic F-127 as sacrificial material. Adapted with permission.[71] Copyright 2016, National Academy of Sciences. c) Schematic illustration of direct printing of a supramolecular ink (sacrificial material) (red) into another supra-molecular ink (receiving hydrogel) (green) was presented. After crosslinking of the receiving hydrogel and removal of the sacrificial ink, vascular-like networks were created. Adapted with permission.[73] Copyright 2015, Wiley-VCH.

constructs with structural integrity were developed using a tissue–organ printer. Multiple types of cells, PCL, and sacrificial materials were coprinted into complex tissue and solid organ constructs with controlled cells location, mechanical stability, and microchannels mimicking vascular-like networks.[20]

3.3.2. Soft Tissues

The strategy of coprinting of PCL and cells was not only applied for the fabrication of hard tissues, but also for the preparation of soft tissues. Pati et al.[90] reported that human adipose tissue-derived mesenchymal stem cells (hASCs)-laden decellularized adipose tissue matrix bioink and PCL were coprinted for soft tissue engineering applications.

The approach of co-bioprinting also can be used to print mate-rials with different concentrations. Recently, a two-step 3D bio-printing approach was developed to create patient-individual hepatic model with biomimetic microarchitecture of native liver. In this case, 5% wt/vol gelatin methacryloyl (GelMA) solution containing human induced pluripotent stem cells (hiPSCs)-derived hepatic cells was printed into a spatial pattern for the first layer. Then, 2.5% GelMA and 1% Glycidal methacrylate-hyaluronic acid solution containing endothelial and mesenchymal cells was printed for the second complementary layer. The printed 3D model recapitulating

native liver lobule structure showed phenotypic and functional enhancements and higher liver-gene expression levels.[91]

4. Conclusion and Perspective

3D bioprinting is a promising technology to fabricate artifi-cial tissues/organs for tissue engineering and regeneration medicine applications. Microstructures including lamellar and vascular-like structures in resolution of tens of micrometers have been realized via laser-, inkjet-, and microextrusion-based bioprinting. Especially for the fabrication of vascular-like net-works, several approaches have been developed including core–shell flow and nozzles, sacrificial materials, and cell droplets or filaments assembly. However, the relationship between printing resolution and printing speed should be carefully addressed. It would be exciting if 3D bioprinting can precisely deposit every single cell in temporally and spatially controlled manner.[92] However, this would compromise the printing efficiency and would be a big challenge for the fabrication of human-scale tissues and organs. Conversely, 3D bioprinting of cell clusters with cell–cell communication would be a promising strategy for the fabrication of large-size tissues and organs.[93]

To address the issue of the conflict between printing resolu-tion and printing speed, next-generation 3D bioprinting device

Figure 4. Fabrication of vascular-like structures via bioprinting of cell droplets or filaments. a) Strategy of creating branched vascular-like structures via printing agarose rods as molding template and simultaneously co-bioprinting uniform multicellular droplets layer-by-layer. Adapted with permission.[79] Copyright 2009, Elsevier. b) Creation of branched vascular structures using the strategy of gelation from substrate: alginate droplets were printed onto the substrate containing CaCl2, and then the alginate drops were crosslinked by Ca2+ ions diffused from the substrate. Scale bar is 200 µm. Adaptedwith permission.[81] Copyright 2012, Wiley-VCH. c) Alginate drops or filaments were directly printed into CaCl2 solutions to create branched structures. Scale bar is 3 mm. Adapted with permission.[82] Copyright 2015, Wiley. d) Creation of tubes by printing hydrogels into a hydrophobic high-density fluid. Adapted with permission.[84] Copyright 2012, IOP Publishing.

and strategy should be developed. For example, a novel 3D printing method named continuous liquid interface production with feature resolution below 100 µm and significantly high printing speed was reported.[94] This method is a kind of “face” printing approach, instead of droplet or line printing used in laser-, inkjet-, and microextrusion-based approaches. Therefore, it can significantly increase the printing speed without com-promising printing resolution. This novel method and printing strategy might be interesting for creating cell-laden biomimetic microstructures for 3D bioprinting of tissues in next step. In addition, 4D printing, the programmed action of the shape change in time dependence based on 3D printed objects, has now developed fast.[95,96] It can create complex 3D microstruc-tures in programmed action and does not require sacrificial materials for mechanical support. Interestingly, external stim-ulation can be introduced in 3D printing device to reorganize the microstructures in micro and nano scales.[97] For example, magnetic force was used to control the orientation of aniso-tropic particles in polymer ink during 3D printing.[98] It would be exciting to control the reorganization of printed cells by using this method. Furthermore, 3D bioprinting is also applied to create artificial tissues by combining other techniques, for example a hybrid 3D bioprinting approach using microscaf-folds and extrusion is reported to couple the advantages of solid scaffolds with 3D bioprinting.[99]

The other issue is the change of the created microstructures during post-bioprinting. 3D bioprinted objects are biologically dynamic, and the microstructures of the objects were influ-enced by several factors including cell proliferation, migration, and differentiation, the degradation of hydrogels and ECM reconstruction. Deposition of cells and materials in 3D con-structs are controllable by 3D bioprinting, while the prolifera-tion, migration, and differentiation of the deposited cells and the degradation of bioinks are generally unable to be accurately controlled. Therefore, the dynamic change of bioprinted micro-structures should be carefully evaluated.

AcknowledgementThe Natural Science Foundation of China (Grant No. 81741107), Basic Research Program of Shenzhen (Grant No. JCYJ20160308090821437), The Medical Scientific Research Foundation of Guangdong Province (Grant No. A2016253), and Scientific Research Foundation for Newly Introduced Teachers of Shenzhen University (Grant No. 2016078) were granted.

Conflict of InterestThe authors declare no conflict of interest.

Keywords3D bioprinting, artificial tissues/organs, microstructures, tissue engineering

Received: January 23, 2018Revised: March 12, 2018

Published online:

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