Recent advances in bioprinting techniques: approaches, applications and future prospects

Inkjet-based bioprinting is a type of bioprinting technology based on the conventional inkjet printing process with desktop inkjet printers. It is a noncontact printing process that deposits precise picoliter droplets of “bioink” onto a hydrogel substrate or culture dish under computer control. The common methods can be further classified into thermal and piezoelectric actuator methods based on the droplet actuation mechanism [20]. In thermal technology, ink droplets are generated by heating so that an inflated bubble forces the ink out of the narrow nozzle and onto the substrates (Fig. 2a). The localized temperature can reach hundreds of degrees in only a few microseconds to generate pulse pressure [21]. This technology is inexpensive and has been used broadly [22, 23]. However, the droplets prepared using the thermal technology are mixed, unordered and unequal in size. Because of frequent nozzle blockages, smooth printing is difficult. Shear and thermal stress also affect the viability of the cellular and protein inks [24]. In piezoelectric technology, drops are generated by the transient pressure from piezoelectric actuator (Fig. 2b). In contrast to thermal technology, the piezoelectric method does not use heat and does not cause orifice clogging, allowing droplets to remain directional with regular and equal size [25, 26]. However, piezoelectric technology can cause damage to the cell membrane and cell lysis if used too frequently [27]. Greater than 90 % viability has been reported for piezoelectrically deposited mammalian cells, including human osteoblasts, fibroblasts, and bovine chondrocytes [26].

Scientists have made great progress in patterning molecules, cells and organs by inkjet printing. Molecules such as DNA have been successfully printed [28], facilitating studies of cancer pathogenesis and treatment. In addition, thermal inkjet printing has been demonstrated to be biocompatible with Chinese hamster ovary (CHO) cells and rat embryonic motoneurons [29]. Less than 8 % of CHO cells were lysed in the printing process, indicating that mammalian cells can be successfully printed by inkjet bioprinting and retain their functions, with good prospects for creating living tissue structures or organs. Further developments in bioprinting technology have resulted in advancements in the printing of functional blood vessels and heart valves. In 2015, Jana and Lerman studied the bioprinting of cardiac valves to solve clinical transplantation shortages. Cardiac constructs are complex and important, particularly the four valves of the heart. Although heart valves have been successfully printed, the functional requirements of elasticity and physiological conditions remain to be fulfilled [24]. Similarly, in 2014, Duan B examined printing of blood vessels and observed drawbacks similar to those observed for bioprinted heart valves. Hydrogels, the main biomaterials used in inkjet bioprinting, are too soft to withstand normal physiological conditions [30, 31]. Thus, to successfully print organs that maintain good biological function in vivo, new biological materials that are more suitable for the human body must be developed to match the mechanical and biological properties of native organs.

Pressure-assisted bioprinting (PAB) is based on extrusion to create desired 3D patterns and constructs. The biomaterials used for printing are usually solutions, pastes, or dispersions [32] that are extruded by coordinating the motion of pneumatic pressure or plunger- or screw-based pressure in the form of a continuous filament through a microscale nozzle orifice or a microneedle onto a stationary substrate. After layer-by-layer application, complete 3D patterns and constructs are eventually formed (Fig. 2c).

The advantages of PAB technology include room temperature processing, direct incorporation of cells and homogenous distribution of cells. PAB has been applied to the printing of cell and organs with confirmed retention of activity. Bioprinted cells include mouse pre-osteoblasts, human mesenchymal stem cells, endothelial cells, and osteogenic progenitors. Bioprinted cells have been used to repair ovine calvarial defects [33]. The feasibility of multicellular bioprinted constructs incorporating goat multipotent stromal cells (MPSCs) and endothelial progenitor cells with retention of heterogeneous cell organization in the subcutaneous tissue of immunodeficient mice and production extracellular matrix has been demonstrated. The multipotent stromal cells in the multicellular bioprinted constructs differentiated into bone structures, and the endothelial progenitor cells differentiated into blood vessels. These results support the ability of multicellular bioprinted grafts to retain activity in vivo [34, 35].

Laser-assisted bioprinting (LAB) uses a laser as the energy source to deposit biomaterials onto a substrate. This technique usually consists of three parts: a pulsed laser source, a ribbon coated with liquid biological materials that are deposited on the metal film, and a receiving substrate [24]. The lasers irradiate the ribbon, causing the liquid biological materials to evaporate and reach the receiving substrate in droplet form. The receiving substrate contains a biopolymer or cell culture medium to maintain cellular adhesion and sustained growth after transfer of cells from the ribbon (Fig. 2d). LAB mainly uses nanosecond lasers with UV or near-UV wavelengths as energy sources to print hydrogels, cells, proteins and ceramic materials [36, 37]. The resolution varies from pico- to micro-scale features and is affected by many factors: the thickness of the biological materials on the film, their rheological properties, the energy of the laser pulse, the wettability of the substrate, and the printing speed and organization of the structure [38, 39].

Researchers have demonstrated the feasibility of using laser-based technology to print cells, for example, human dermal fibroblasts, mouse C2C12 myoblasts, bovine pulmonary artery endothelial cells (BPAECs), breast cancer (MCF-7) cells and rat neural stem cells [39‐41]. In 2013, Michael et al. [42] successfully created Graftskin skin substitutes by utilizing LAB technology, a landmark event in the field of laser-assisted bioprinting. Fibroblasts and keratinocytes were used as the cell sources to fabricate the skin constructs, which were subsequently transplanted into the skin folds of nude mice to perform an in vivo evaluation. Eleven days later, the graft adhered well to the tissue around the skin wound. The keratinocytes proliferated and differentiated well and grew to the skin stratum corneum and basal layer. Compared with other bioprinting technologies, LAB has unique advantages, including a nozzle-free, non-contact process, the printing of cells with high activity and high resolution, and the control of ink droplets and precise delivery characteristics. Ink bubble dynamics, shear stress and laser pulse energy all play important roles in the bioprinting process [43‐45].

Stereolithography (STL) technology is a solid freeform, nozzle-free technology that was developed in the late 1980s [46]. A liquid, photo-sensitive polymer formulation is solidified upon illumination. STL uses digital micromirror arrays to control the light intensity to polymerize light-sensitive polymer materials. This technique is mainly applied to fabricate structures from curable acrylics and epoxies. The number of photocrosslinkable polymers is increasing, and multiple resins can be used for one structure [32]. Digital light projection controls the printing process in this top-down system. Compared with other solid freeform techniques, STL has the highest fabrication accuracy, and an increasing number of materials can be used in this process. Furthermore, STL can print light-sensitive hydrogels layer-by-layer, and the total printing time depends only on the thickness of the structure [47].

However, in addition to these advantages, there are numerous restrictions, such as the lack of proper biocompatible and biodegradable polymers, harmful effects from residual toxic photocuring reagents, the inability to completely remove the supporting structure and the inability to form horizontal gradients in the constructs [46]. UV-sensitive photoinitiators were once used, although UV is harmful to cellular DNA and causes skin cancer. In 2015, Wang [47] investigated the use of customized visible light in a STL system, including a commercial beam projector and bioinks, a mixture of PEGDA, methacrylated gelatin (GelMA), and eosin Y-based photoinitiator. They first described the detailed protocol of the visible light-based STL system and revealed the necessity of an infrared ray (IR)-filtering water filter to the system. Their experimental results with NIH 3T3 cells demonstrated that this system with customized visible light could support the bioprinting of visible light-curable hydrogels with 50-μm resolution and high cell viability (∼85 %) for at least 5 days.

In general, biomaterials can be categorized into a large variety of hydrogel, metallic, ceramic, polymeric and composite materials. The physical characteristics of biomaterials determines the optimal printing type. For example, low-viscosity materials are more attractive for bioprinting because cells can grow well in the low-pressure environment [48]. Other material properties, such as pore size and interconnectivity, also influence the encapsulated cells [49].

Cell printing is the key element for the printing of tissue and organs. However, the choice of bioink materials is limited by the stringent printing conditions. The stiffness, functional groups, and surface morphology of biomaterials have a significant impact on cellular behavior. Cells are usually encapsulated in biodegradable hydrogels that mimic a tissue-like environment for building bioprinted ink [62]. The characteristics of hydrogels can protect the inner cells from the shear force generated in the printing process and maintain their bio-functions, such as the self-renewal ability and multi-lineage differentiation potency of stem cells. The cytocompatibility of laser-assisted cell printing technology with cells post-deposition was recently demonstrated, and this technique has been widely used for its high resolution and accuracy in single-cell deposition.

The extracellular microenvironment or niche provides various stimuli, such as physical, chemical and biological factors, to direct cell adhesion, proliferation and differentiation. Obvious effects on cell behavior have been confirmed by directly designing the surface morphology of scaffolds [72].

Biological molecules, including proteins and nucleic acids, have been successfully deposited with bioprinting technologies such as inkjet printing [14]. An advantage of inkjet printing is the ability to control the concentration gradient of internal ingredients using different bioink densities [73].

Growth factors such as BMP2, epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) have also been measured by molecular patterning. To print 3D artificial tissues, studies of 2D molecular arrays may provide clues about the function of growth factors in their niche.

As the mode of transport for nutrition and metabolic waste, functional blood vessels play important roles in cardiovascular diseases [80] and the construction of artificial organs, particularly these with a rich blood supply. Advances in developmental biology and iconography have enabled significant progress in printing vasculature in vitro. However, due to the unique functions and specific structures of the vasculature in different tissues, creating a vascular system remains a critical, unmet challenge.

Bioprinting enables the fabrication of network structures using hydrogels or other materials as bioink. Bertassoni et al. [81] successfully bioprinted a vascular network with GelMA, which improved metabolic transportation, cellular viability and the formation of endothelial monolayers. Kolesky et al. [82] successfully co-printed multiple materials with cells and vasculature using thermally reversible gelation to fabricate complex tissues.

In the printing of cardiac valves, tissue-engineered valve scaffolds are generally focused on the rebuilding of the aortic valve [24]. Researchers have performed extensive studies of printing aortic valve structures with hydrogels [30, 31, 83]. Cell-laden, valve-shaped structures were printed with a high cell survival rate (greater than 90 %). However, bioink materials are deficient in flexibility and elasticity, and their mechanical characteristics still do not meet clinical needs.

Bone and cartilage regeneration is the most mature field utilizing printing technology because the composition of hard tissues is uncomplicated and is mainly composed of inorganic elements. A variety of biomaterials have been produced to construct bone and cartilage scaffolds by many manufacturing approaches, including gas foaming [84, 85], salt leaching [86, 87] and freeze drying [88, 89]. However, the structural and mechanical properties of artificial scaffolds can be more accurately controlled by 3D bioprinting than by other technologies.

A cement powder system was recently fabricated containing HA and TCP as the ideal composition for human bone replacement to repair large defects [90]. The dimensional accuracy of the bioprinted scaffolds was greater than 96.5 %. In Gao’s work [91], hMSCs and nanoparticles of bioactive glass (BG) and HA were co-printed to control the spatial placement of cells. hMSCs encapsulated in this compound exhibited high cell viability (86.62 ± 6.02 %) and compressive modulus (358.91 ± 48.05 kPa) after 21 days in culture. In Park et al. work [92], HA and Col-1 hydrogels printed as tissue-mimetic structures were investigated independently to elucidate their effects on the behavior of chondrocytes and osteoblasts. Chondrocytes on HA hydrogels and osteoblasts on Col-1 hydrogels maintained better proliferative capacity and cell function than chondrocytes on Col-1 hydrogels and osteoblasts on HA hydrogels.

Bioprinting has been a popular technology for the creation of cartilage tissue engineering scaffolds from a variety of materials, ranging from ceramics to nanomaterials. Markstedt et al. [77] developed a printable bioink using a combination of nanofibrillated cellulose and alginate with human chondrocytes as living soft tissue. The mixture exhibited excellent shear-thinning behavior and cell viability of 86 % after 7 days of 3D culture. The high plasticity of the ink allowed shapes resembling cartilage tissues, such as an ear and a meniscus, to be printed successfully.

Although printable scaffolds have been extensively applied in bone and cartilage tissue engineering, bioinks with suitable physical properties are still needed.

Skin protects the body from attack by foreign substances and maintains the integrity of the body. Many chronic diseases and burn wounds cause irreversible damage to skin, and thus bioprinting technology is particularly critical in the preparation of skin substitutes for transplantation to repair damaged skin.

The LAB technique has been applied to the printing of bio-skin. Michael et al. [42] successfully created skin substitutes using LAB technology and transplanted them to skin wounds of nude mice. Eleven days later, the graft was able to adhere well to the tissue around the skin wound, and the cells in the graft were able to proliferate and differentiate. In 2014, Lee et al. used keratinocytes and fibroblasts as materials to bioprint skin tissue. The resulting skin tissue had representative morphology and biology. The skin bioprinted by 3D technology was able to maintain its shape and had high flexibility, reproducibility and culture throughput. In addition, cells that cause skin diseases can be added to the biomaterials, and skin tissue printed with pathogenic cells can be used to study the pathophysiology of skin diseases [93].

Compared to other organs, the liver has strong regeneration ability. Patients who require liver transplantation can receive lobes of liver from a healthy donor [94] or can also wait for their own liver tissue to regenerate. However, healthy donors are in short supply, and the regeneration period for self-liver tissue is long. Therefore, bioprinting of liver tissue by tissue engineering is particularly important.

Many researchers have studied liver bioprinting. Primary hepatocytes and stem cell-derived hepatocytes have been used as the bioink to bioprint liver tissue [95]. Although the cells lose a certain amount of activity and functionality in the printing process, liver tissue containing both cell types can be sustained for a period of time. In contrast to traditional printing technologies, 3D printing technology can provide the exact size and shape of the liver suitable for the needs of patients with liver resection [96]. Recently, a new technique has been used to maintain cell activity and functionality for longer times [97]. The technique utilizes bioprinting technology to form structures called “canaliculi” that are similar to the liver hepatic cord. The primary hepatocytes and the “canaliculus” structures are cultured together in the collagen matrix. Then, a biomimetic ECM system evaluates the activity and functionality of the primary hepatocytes on different ECM-based hydrogels [98]. After the activity and functionality of the primary hepatocytes have been confirmed, the cells can be maintained for 4 weeks. Further increases in cellular activity would facilitate expanded applications of bioprinted liver tissue. Chang et al. [99] also demonstrated that multilayered tissues can be used as in vitro 3D liver models. This multilayered tissue contained rat and human hepatocytes, and the multilayered cellular architecture could be used as a liver analog to help detect drug toxicities and for other medical and biological testing. Thus, 3D bioprinted livers can be used not only for liver resection in patients and other liver surgeries but also for simulated liver experiments in vitro.

The use of biological printing for the liver would have profound impact. Although some achievements have been made, hurdles must still be overcome, including cost and time, and the mechanical properties of printed liver tissue must be highly consistent with that of the native liver.