Abstract
Additive manufacturing, popularly known as “3D printing”, enables us to fabricate advanced scaffolds and cell-scaffold constructs for tissue engineering. 4D printing makes dynamic scaffolds for human tissue regeneration, while bioprinting involves living cells for constructing cell-laden structures. However, 3D/4D printing and bioprinting have limitations. This article provides an up-to-date review of 3D/4D printing and bioprinting in tissue engineering. Based on 3D/4D printing, 5D printing is conceptualized and explained. In 5D printing, information as the fifth dimension in addition to 3D space and time is embedded in printed structures and can be subsequently delivered, causing change/changes of the environment of 5D printed objects. Unlike 3D/4D printing that makes passive/inactive products, 5D printing produces active or intelligent products that interact with the environments and cause their positive changes. Finally, the application of 5D printing in tissue engineering is illustrated by our recent work. 3D/4D/5D printing and bioprinting are powerful manufacturing platforms for tissue engineering.
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Introduction
In the long human history, two-dimensional (2D) printing has made immeasurable impacts on various aspects of our society, including education, economy, science, and engineering. In the 1980s, printing technologies started to have revolutionary developments, advancing from 2D printing to 3D printing. 3D printing began when Hull patented the world’s first 3D printer, stereolithography (SLA), in 1986 in the USA. Over the past 30 years, many other 3D printing technologies have been developed, including selective laser sintering (SLS), 3D powder binding (3DPB), fused deposition modeling (FDM), extrusion-based 3D printing, and inkjet printing. Nowadays “3D printing”, formally known as “additive manufacturing (AM)”, has been a term representing a group of technologies with which materials (or “inks” in AM) are deposited in a layer-by-layer manner to form 3D objects, non-porous or porous. Compared to conventional subtractive manufacturing, 3D printing has many distinctive advantages, including customized construction of complex 3D objects with high precision, much reduced material waste in product manufacture, and shortened or reduced product development cycles. Owing to these unique advantages, 3D printing has already made great achievements in the biomedical engineering field [1]. Tissue engineering, an essential part of biomedical engineering, has inevitably benefited hugely from the application of 3D printing. 3D printing has often been adopted to fabricate a variety of tissue engineering scaffolds with individualized structures and characteristics by using different types of biomaterials including polymers, ceramics, metals and their composites [2, 3]. For example, ** temperature and load holding time affected shape fixity and recovery, as well as stress recovery [9]. However, 4D printing makes products that only have changes for or in themselves but do not affect their environments, which can also limit their usefulness in tissue engineering.
Over the time in our history, printing technologies have advanced from 2D printing to 3D printing and now to 4D printing. Each of these advances was accompanied by the increase in physical dimensions, i.e., from 2D planes to 3D space, or by the extension into time, i.e., from 3D space to 4D’s “3D space plus time”. As information and its influences are essential nowadays in every parts of our lives, information can therefore be set the fifth dimension to advance 4D printing to 5D printing, entering a new phase of additive manufacturing [10]. Unlike 3D printing, which uses 3D space, and 4D printing, which extends from 3D space into time, 5D printing employs not only 3D space and time but also information. On the basis of 4D printing, 5D printing embeds and utilizes information in printed products. Information, the fifth dimension, can be species that will cause changes in the environment upon their release to achieve what 5D printing aims for. 5D printing will enable the fabrication of shape-morphing and information-embedded-and-disseminating objects, which should be highly useful for tissue engineering with the aim of achieving the best clinical outcomes.
In this mini-review and perspective article, recent advances in 3D/4D printing in tissue engineering are reviewed and discussed in terms of printing technologies, materials, and applications. On the basis of 3D/4D printing and their developments, the concept of 5D printing is explained and illustrated, and the potential and prospects of 5D printing in tissue engineering are discussed.
Tissue engineering and strategies
Tissue engineering, as a multidisciplinary field that integrates engineering, life science and clinical medicine, offers an exciting, vital and promising approach that can provide artificial biological substitutes for diseased or injured human body tissues or organs, thereby maintaining, restoring, and/or improving tissue and organ functions. In this section, the concept and development of tissue engineering and strategies for tissue engineering are briefly presented and discussed.
Concept and development
Organ transplantation is used ultimately to save the life of patients with critical tissue loss or organ failure. It has made great successes in clinical medicine by replacing dysfunctional tissues or organs in patients with normal, functional ones from donors. However, organ transplantation faces many challenges, including shortage of donors, possible disease transmission, and rejection of a foreign tissue or organ by patients’ immune system. Against this background, tissue engineering came up strongly over 30 years ago and has since been developed as a vital and promising approach that provides artificial biological substitutes for diseased or lost tissues or organs. The term “tissue engineering” was first proposed in the mid-1980s and was used to describe technologies that could be used in operations for tissues and organs during surgery [11]. In 1987, “tissue engineering” was conceptualized as an multidisciplinary field that applied the principles of engineering and life science to understand relationships of function-structure of human tissues and organs and to develop biological substitutes to restore, maintain or improve tissue functions [12]. There are generally three approaches in tissue engineering: scaffold-based, growth factor-based, or cell-based tissue engineering. In scaffold-based tissue engineering, there are three major components: cells, scaffolds, and biological signals, which are the so-called “tissue engineering triad”. These three components are necessary for mimicking the three basic elements of native human tissues, namely, cells, extracellular matrix (ECM) and bioactive biomolecules/growth factors. ECM provides a structural support for cell attachment, proliferation, differentiation and communication, while biological molecules give specifical signals/guidance for cells and newly formed tissues for their behavior and functions. Cells of the targeted tissue, or stem cells in recent years, are essential for new tissue formation.
Scaffold-based tissue engineering
Among the three approaches in tissue engineering, scaffold-based tissue engineering has been dominantly used for regenerating human body tissues. Tissue engineering scaffolds act as artificial ECM and provide a good substrate for cell attachment and growth and facilitate neo-tissue formation [13, 14]. There are several major requirements for tissue engineering scaffolds in regenerative medicine. The top priority is the biocompatibility of scaffold material and hence the scaffold, i.e., a biocompatible scaffold that can co-exist with the host tissue without any side effect and should elicit a desirable response from the host tissue after implantation. Secondly, scaffold should be highly porous, and the pores should be interconnected, which will allow ingrowth of neo-tissues and hence facilitate tissue regeneration. Thirdly, in the conventional sense for tissue engineering, scaffolds should be biodegradable; and an ideal biodegradable scaffold is expected to have degradation kinetics comparable to the formation rate of the new tissue during its regeneration. Additionally, mechanical properties of scaffolds should be adequate for device handling and implantation. In many situations, they need to be tailored to match those of target tissues to offer an environment with suitable stress for neo-tissue formation. In addition, bioactive biomolecules may be loaded relatively easily into the scaffold matrix or onto the surface of scaffold struts for promoting tissue regeneration in vitro and in vivo [15, 16]. For example, Zhang et al. incorporated two small biomolecules (i.e., resveratrol and strontium ranelate) into 3D printed poly(ε-caprolactone)/hydrogel composite scaffolds [17]. Their results showed that the dual release of these small molecules had combinational advantages in enhancing angiogenesis and inhibiting osteoclast activities. Furthermore, advanced tissue engineering scaffolds should have an architecture that mimics the structure of ECM (pore shape, pore size, porosity, etc.) to influence positively cell behavior (attachment, spreading, etc.) and to enable deep cell penetration/migration [6, 18]. In the work by Zhang et al., 3D printing was applied to construct bone tissue engineering scaffolds with integrated hierarchical Haversian bone structure [19]. Their results showed that the Haversian bone-mimicking scaffolds contributed to the osteogenic, angiogenic, and neurogenic differentiation in vitro and speeded up the blood vessel ingrowth and new bone formation in vivo.
To obtain a scaffold that meets the aforementioned requirements, biomaterials, biomaterial processing, scaffold fabrication, and/or material and scaffold modification are critical factors and should be carefully considered, chosen and studied. Common biomaterials used for producing scaffolds include biomedical polymers, suitable biodegradable metals, bioceramics, and their composites [11, 14]. These biomaterials can be used based on specific tissue engineering applications. For example, metallic biomaterials possess excellent mechanical properties which are suitable for orthopedic applications where long-term load bearing is required [1.
When these 3D printing technologies are applied for tissue engineering, they follow a similar designing and fabrication processes, as illustrated in Fig. 1. Firstly, the medical imaging data of the diseased/injured tissue or organ of a patient are collected via a modern medical imaging technology such as computed tomography (CT) and magnetic resonance imaging (MRI). These medical imaging data are then processed using computer-aided design (CAD) software to transform them into a corresponding virtual model, which is further sliced into a series of 2D layers (with each layer corresponding to a contour of the virtual model) using softwares installed in 3D printers. These softwares are usually proprietary assets of 3D printer makers. Next, the 3D printing machine reads the sliced data and prints out a tissue engineering product using a suitable “ink” or “bioink”—inks are acellular, while bioinks contain living cells. In some situations, 3D printed tissue engineering structures need to undergo post-printing treatment(s) to obtain final products or to improve their performance (removing sacrificial materials to create hollow interconnected channels, sintering to improve mechanical properties, etc.). Finally, after culturing and maturation, 3D printed tissue engineering structures are implanted in patients to repair their dysfunctional tissues or organs.
Schematic diagram for illustrating 3D/4D printing processes in tissue engineering.
As for 4D printing technologies, in principle, any 3D printing technology that can process 4D printing inks can be used for 4D printing. 4D printing inks are the inks that involve so-called “smart materials” and 4D printed structures from these materials can change their shapes or properties under suitable stimuli during application [24]. At present, 3D printing technologies commonly used for 4D printing in tissue engineering are primarily micro-extrusion-based printing [25], FDM [26], SLA [27] and DLP [28].
With regard to bioprinting, not all 3D printing technologies for tissue engineering are suitable since living cells are involved during the bioprinting process. For example, 3D printing technologies such as FDM and SLS use high temperatures in their fabricating processes, which will lead to the total destruction of cells, and therefore cannot fabricate 3D functional living structures [26, 29]. At present, 3D printing technologies commonly used for bioprinting are micro extrusion-based printing [5]. The paramount property for biomaterials for 3D printing in tissue engineering is biocompatibility. The second most important property of these biomaterials is their printability for the 3D printing technology concerned. For 4D printing, biomaterials should also have stimuli-responsive properties, which will enable 4D printed structures to change their shape or properties when exposed to external stimuli during applications.
For 3D printing in tissue engineering, currently available biomaterials can be classified into four categories: biomedical polymers, metallic biomaterials, bioceramics, and biomedical composites, as presented in Table 2. Among them, biomedical polymers are the most commonly used biomaterials for 3D printing in tissue engineering [11]. According to their sources, polymers can be classified as natural polymers and synthetic polymers. Most of natural biomedical polymers exhibit excellent biocompatibility and biodegradability but have poor mechanical properties. Synthetic biomedical polymers show much improved mechanical properties and slower degradation in comparison with natural polymers but lack bioactive sites that are desirable for tissue engineering applications. Therefore, chemical modification or blending of polymeric biomaterials is often performed to avoid their respective shortcomings. For example, gelatin cannot maintain a stable structure at 37 °C as it is in a liquid-like state at this temperature. Blending gelatin with alginate can form hydrogel blends with improved extrudability and fast crosslinking ability and avoid this issue [34]. Currently, 3D printing can process both natural and synthetic biomedical polymers into diverse tissue engineering products for the regeneration of body tissues such as bone [13, 35, 36], articular cartilage [16, 37, 38], tendon and ligament [39, 40], and vasculature [46,47] and osteochondral tissue [112]; but most of the hydrogels exhibited very poor mechanical properties which are far below those of the native bladder. Wang et al. used coaxial electrospinning and 3D printing to fabricate bilayer scaffolds composed of a heparin-loaded electrospun layer and a 3D printed PCL layer [113]. The application of these bilayer scaffolds enhanced bladder regeneration in a rat model, as illustrated by Fig. S4.
Liver
The liver is the largest gland in the human body and regulates a variety of functions such as metabolism, bile production, and detoxification. Owing to its complex micro-architecture and multiple types of cells, the total recreation of the native liver microenvironment is highly challenging, if possible. 3D printing provides a powerful and promising platform for fabricating liver substitutes. In vitro liver microtissues or models have been made from diverse hydrogels or liver dECM using different 3D printing technologies (e.g., DLP, micro-extrusion) [114, 115]. Several studies have focused on fabricating multiscale liver lobules with multiple types of cells and interconnected vasculatures, aiming to obtain biomimetic cell-laden constructs similar to the highly complex native liver [116, 117]. For example, Janani et al. used a dual-nozzle extrusion-based bioprinting system to process two dECM-based bioinks into liver models that mimicked microarchitecture of native liver lobule [117], which had high potential for drug toxicity and screening applications. Also, liver organoids could be engineered into 3D liver constructs for disease modelling and transplantation [118]. For example, Yang et al. used a micro-extrusion based bioprinter to fabricate hepatorganoids, which were transplanted into mice with liver failure and improved the survival of these mice, as illustrated by Fig. S5 [119]. Despite the advances of 3D printing in liver tissue engineering, fabrication via 3D printing or other means of liver substitutes comparable to the native liver with extensive vasculature, lobes, lobules, hepatocytes, and sinusoids is still a daunting task and remains as one of the highest hurdles in regenerative medicine.
Heart
3D bioprinting of a functional, full-size whole heart as that in humans must be among the few greatest challenges for engineering and medicine owing to the highly vascularized, complex structures and multiple functions of the heart, particularly the eternal beating of the heart of a living person. In recent years, 3D bioprinting has been used to fabricate some relatively simple constructs for heart parts (e.g., heart valve), patches or “mini-organs”. Materials used for 3D printing of the heart are mainly hydrogels (e.g., collagen [120], alginate [34], GelMA [61]) and dECM [121]). There are various types of cells that can be used for 3D printing of heart-related constructs such as leaflet interstitial cells, smooth muscle cells, endothelial cells, cardiomyocytes, MSCs and iPSCs. In 2015, a new 3D printing technique, i.e., freeform reversible embedding of suspended hydrogels (FRESH), was developed and applied to process low-viscosity hydrogels into full-size models of the human heart [120, 122]. As shown in Fig. S6, an organ-scale human heart could be made via FRESH 3D bioprinting from low-viscosity collagen [120]. In addition, 4D printing has been used to fabricate dynamic heart constructs. For example, Wang et al. used DLP to process a 4D ink composed of a shape memory polymer and graphene into near-infrared light-sensitive cardiac constructs with highly aligned microstructure and adjustable curvature [123]. This 4D printed cardiac construct was expected to recreate the curved topology of the myocardial tissue for its seamless integration with the heart. Despite the progress in 3D printing of heart constructs, there are still many difficulties for producing heart tissue substitutes that are comparable to the natural heart, including the creation of the whole set of blood vessels of the heart, large number of cells required to rebuild a human-size heart, and long-term in vitro culture.
Other tissues and organs
In addition to the tissues and organs that have been presented and discussed above, 3D/4D printing has also been used to create scaffolds or constructs for other human body tissues/organs, such as cornea [124], gastrointestinal tract [125, 126] and kidney [127]. For example, Kong et al. used electric field direct writing to fabricate microfibrous scaffolds and then infused the scaffold with GelMA hydrogels to obtain fiber-hydrogel composites, which mimicked the stromal structure of native cornea and provided a good environment for the regeneration of corneal stroma [124]. Brassard et al. used organoid bioprinting to fabricate macro-scale tubular intestinal epithelia with in vivo-like crypts and villus domains [125]. Also, kidney organoids were used to manufacture uniformly patterned kidney tissue sheets via micro extrusion-based bioprinting, and these tissue sheets exhibited the potential for renal repair [127].
Challenges for 3D/4D printing in tissue engineering
Although additive manufacturing has made remarkable progress in tissue engineering, it still faces many challenges and some of these challenges may be insurmountable within the foreseeable future. The first challenge is to develop new biomaterials for 3D/4D printing in tissue engineering, particularly for 4D printing. A suitable biomaterial for 3D printing in tissue engineering should have good biocompatibility, printability, biodegradability, and mechanical properties [1, 5, 60]. Additionally, biomaterials for 4D printing should also have good stimulus-responsive properties which will allow 4D printed structures to change shape or properties under appropriate stimuli [24]. Another challenge is to develop new 3D/4D printing technologies with improved performance for tissue engineering applications. Current 3D printing technologies still have various, technology-specific limitations, such as printing speed, printing resolution, and multiple material printing in some printers (e.g., SLA and SLM), which has limited their further applications in producing newer and better tissue engineering products. Next, for 3D/4D bioprinting, there are still many problems for this type of new technologies in creating clinically usable biological substitutes or products. Cell issues such as cell source, cell number/density, cell viability, and cell spreading within the matrix should be carefully considered and investigated to produce clinically good cell-laden constructs for tissue regeneration. Requisite cell density and microstructural complexity must be met for producing and achieving large and functioning tissues or organs [121]. Furthermore, building up the entire blood vessel network for whole organs such as liver and heart and incorporating them into the 3D/4D printed organs are a huge challenge that must be tackled, in the short run rather than in the long run. Finally, 3D or 4D printing itself has limitations, i.e., the static structure issue in 3D printing and the environment effect issue in 4D printing, which can prevent 3D/4D printed structures from meeting the highly demanding requirements for tissue regeneration, remain. Such issues may be mitigated by moving AM forward into 5D printing.
5D printing and its application in tissue engineering
Based on the concepts and developments of 3D printing and 4D printing, Wang in his invited talk at the 2021 Materials Research Society Spring Meeting & Exhibit introduced the concept of 5D printing and presented a research work showing the application of 5D printing in tissue engineering [10]. In this section, the concept of 5D printing is presented in more detail and its application is illustrated by 5D printing in tissue engineering.
Concept and practice of 5D printing
In 3D printing, objects (non-porous or porous) are produced through the layer-by-layer precise deposition of materials in 3D space (i.e., in the traditional, physical three dimensions in space). 3D printed objects are static during their whole product lifetime (i.e., both manufacture and service time). In 4D printing, time is added as the fourth dimension, and then dynamic structures are fabricated by using 3D printing technologies and smart materials, as well as, in many cases, smart designs. 4D printed objects will undergo shape or property change(s) by responding to pre-determined external stimuli. But 3D/4D printing produces passive or inactive products which do not interact with the environment. On the basis of 3D printing and 4D printing, it should be feasible to add another dimension, the fifth dimension (which can be “information”), to 4D printing to move the additive manufacturing platform forward to 5D printing which will fabricate active or intelligent structures that interacts with the envirenment and causes possitive changes. Information nowdays plays a dominent role in our society and it is therefore natural to chose information as the fifth dimension for 5D printing. Here, information is defined in a broad sense and can be any species that will lead to the change/changes of the environment of 5D printed objects (or, the 5D printed objects themselves) upon their release to achieve what 5D printing aims at for individual applications. Therefore, 5D printing can be defined as:
5D printing produces shape-morphing and information-embedded structures, and the information, which is the 5th dimension in 5D printed structures, will be delivered in situ during applications of these structures. More importantly, with 5D printed structures, the in situ delivered information will affect the surrounding environment (or, the 5D printed structures themselves) and guide the change/changes in the environment (or, 5D printed structures). Unlike 3D/4D printing which makes passive or inactive products that can fulfill intended functions but do not change the environment, 5D printing produces active or intelligent products that interact with the environments and cause their intended, positive changes.
In 5D printing, information is involved in addition to 3D space and time for additive manufacturing. As such, 5D printed structures can not only change their own shape or properties during their applications by responding to suitable stimuli but also change the environment (or, the structures themselves) upon the release of embedded information, which will lead to wider applications of printing technologies and printed structures in diverse industries. Unlike 3D/4D printied passive or inactive structures that only provide the spceific functon/functions and hence simply serve the purspose(s), 5D printed active or intelligent structures will fulfill the functions and importantly, cause positive changes of their environments.
5D printing in tissue engineering
As discussed in the "Applications of 3D/4D printing and bioprinting in tissue engineering" section of this article, 3D/4D printing has now been extensively used in tissue engineering for making a wide range of acellular scaffolds and biological substitutes with desired architectures and properties for regenerating different body tissue and organs. With the introduction of 5D printing, tissue engineering products with much improved performance are expected. When applying 5D printing in tissue engineering, the fifth dimension, i.e., information, can be biomolecules such as growth factors (GFs). It can also be other entities (functional nanoparticles, genes, cell messages, etc.). Biomolecules are commonly used in tissue engineering to promote cell proliferation and differentiation and facilitate new tissue formation. Therefore, with the encapsulation of bioactive biomolecules, tissue engineering products can accelerate the regeneration of target tissues. With 5D printing, novel tissue engineering products with shape-morphing ability and controlled delivery of information could be developed for regenerating complex body tissues. In this section, practical application of 5D printing is illustrated through an example in the tissue engineering field, with the aim of develo** novel multi-layered tissue engineering products mimicking the native tissue for improving tissue regeneration outcome.
Scaffold-based tissue engineering provides an important route for fabricating biological substitutes for tissue maintenance, restoration and improvement. 3D printing has enabled efficient and reproducible fabrication of 3D, biocompatible, biodegradable and complex scaffolds, which can serve as an artificial ECM environment and temporary support for new tissue formation and growth. Furthermore, 4D printing allows development of dynamic tissue engineering products which can change their shapes or properties under suitable stimuli to fit the anatomical geometry or functions of the target tissues after their deployment in the body. For 5D printing, the illustrative example here is the investigation and development of novel multi-layered cell-laden constructs for generating tubular tissues (e.g., blood vessels) in the body. This type of 5D printed constructs not only have the shape-morphing ability but also provide controlled delivery of the embedded information (i.e., biomolecules in this study). Figure 9 illustrates the 5D printing process to obtain cell-laden constructs which are composed of a shape-morphing layer and a rat bone-marrow mesenchymal stem cell (rBMSC)-containing and biomolecule-delivering layer. The shape-morphing layer was 4D printed using shape memory polymer PDLLA-co-TMC. This polymer has a glass transition temperature at around 37 °C, which enables it to undergo 3D shape change upon heating from room temperature to the physiological temperature. In the current study, the printed PDLLA-co-TMC layer, which was made according to our established method [52], could transform from a 2D planar structure to a 3D curved shape by responding to heat. Briefly, as shown in Fig. 9, a 25% (w/v) PDLLA-co-TMC solution was prepared by dissolving the polymer in dichloromethane (DCM, Applied Biosystems, Ireland) and was then printed onto a glass slide coated with a thin layer of Vaseline to generate a 2D planar porous structure. The dried printed 2D structures were then reshaped into tubular structures using glass rods with different diameters at 80 °C for 90 min. Afterwards, the tubular structures with different diameters were cut and then flattened at 25 °C to take up the temporary 2D planar shape. As such, the shape-morphing layer of the constructs under construction was fabricated. The next step was to fabricate the rBMSC-containing and biomolecule-delivering layer (i.e., the information-embedded layer) using a dual-nozzle 3D printing system (3D Discovery™ Evolution, regenHU Ltd, Switzerland). In this step, two types of inks/bioinks were prepared. The first was an ink of 10% (w/v) GelMA (with a modification degree of about 50~60%) hydrogel containing two types of biomolecules: 200 ng/ml TGF-β1 (shortened as “TGF” hereafter) and 3 mg/ml ascorbic acid (AA). The second was a bioink prepared with 5% (w/v) gelatin (Gel) and 5% (w/v) GelMA (with a modification degree of about 20~30%) hydrogel blend containing rBMSCs (at 1 × 106 cell/ml). All hydrogels were sterilized by 60Co γ-ray irradiation before the addition of biomolecules or living cells. After the preparation of these two inks/bioinks, they were put into two separate syringes and used for printing alternately to form the information-embedded layer. rBMSC-laden structs were printed first using the bioink, and the ink containing biomolecules was printed into the space between two parallel rBMSC-laden structs. After printing, the information-embedded layer was exposed to a UV light at 365 nm wavelength and 365 mW power for 60 s to crosslink GelMA and Gel/GelMA hydrogels. Finally, the shape-morphing layer and information-embedded layer were combined to form bilayer scaffolds. When heated to 37 °C, the bilayer scaffolds should be able to self-bend to form curved structures as designed or self-fold to form tubular structures. With local delivery of the embedded information, i.e., the biomolecules, the bilayer scaffolds were expected to have rBMSCs induced to differentiate into smooth muscle cells (SMCs). As such, shape-morphing, rBMSC- and biomolecule-incorporated bilayer tissue engineering scaffolds had been made via 5D printing.
Schematic illustration of 5D bioprinting of bilayer cell-scaffold constructs with shape-morphing ability (provided by the first scaffold layer) and in situ delivery of information (provided by the second scaffold layer).
To confirm our design concepts and the realization of these concepts for demonstrating 5D printing in tissue engineering, additional tissue engineering scaffolds were made. Subsequently, various experiments were performed. PDLLA-co-TMC based single-layer scaffolds and bilayer scaffolds were fabricated for studying their morphology, structure and shape-morphing ability. Firstly, single-layer tubular PDLLA-co-TMC scaffolds with different diameters were made. As shown in Fig. 10(a), porous temporary 2D planar PDLLA-co-TMC scaffolds of different sizes were 4D printed. These temporary structures were planar at 25 °C when viewed from the top and side. After being heated to 37 °C via immersion in 37 °C water, they could automatically fold into tubular structures of different diameters. The shape-morphing process was quick and was completed within one minute, similar to what we had shown in a previous study [81]. Afterwards, bilayer scaffolds were fabricated where the first layer was a shape-morphing layer and the second layer was an information-embedded layer without cells, as shown in Fig. 10(b). The information-embedded layer was printed using GelMA-based hydrogels. It could be seen that the bilayer scaffolds remained in the temporary planar state at 25 °C when viewed from the top and side. They could also quickly complete the self-tubing process upon heating to above 37 °C, indicating that the addition of the second information-embedded layer did not affect the self-tubing ability of 4D printed PDLLA-co-TMC scaffolds, which was consistent with our previous study [52]. The structure and morphology of both types of scaffolds in the permanent tubular shape were examined using a field emission scanning electron microscope (Hitachi S3400N VP SEM, Japan), and SEM images are shown in Fig. 10(c). From the top view, it could be seen that both types of scaffolds exhibited the designed, regular macropores, demonstrating good structural features of the printed scaffolds. In addition, the GelMA-based hydrogel layer was seen through the regular macropores of the outer PDLLA-co-TMC layer in the bilayer scaffolds. From the side view, it could be seen that both types of scaffolds exhibited circular structures and the information-embedded layer of bilayer scaffolds was tightly attached to the PDLLA-co-TMC layer, suggesting successful fabrication of shape-morphing and information-embedded bilayer scaffolds via 5D printing.
4D/5D printed porous scaffolds. (a) Photographs showing top and side views of single-layer PDLLA-co-TMC scaffolds in the temporary planar state at 25 °C and in the permanent tubular shape at 37 °C with different diameters; (b) photographs showing top and side views of PDLLA-co-TMC/Gel-GelMA bilayer scaffolds in the temporary planar state at 25 °C and in the permanent tubular shape at 37 °C with different diameters; (c) SEM images providing top and side views of printed scaffolds in the permanent tubular shape (Scale bar: 5 mm).
PDLLA-co-TMC is an amorphous shape memory polymer. Depending on the PDLLA to TMC ratio in the polymer, it exhibits different glass transition temperatures and mechanical properties. In this 5D printing demonstration study, PDLLA-co-TMC with a PDLLA to TMC ratio of 9:1 was used. At this ratio, PDLLA-co-TMC has a glass transition temperature (Tg) slightly higher than human body temperature of 37 °C. This characteristic of this PDLLA-co-TMC polymer allows our 5D printed scaffolds to stay in the temporary planar state stably at 25 °C and then transform to the tubular shape upon heating to the body temperature according to our programmed design. In the current study, the self-tubing process of printed scaffolds had resulted from the temperature gradient within the PDLLA-co-TMC scaffold layer caused by the resha** process at 80 °C. Such temperature gradient, from the surface to the interior, of PDLLA-co-TMC scaffolds, could affect the degree of molecular orientation in PDLLA-co-TMC, leading to the self-folding ability of PDLLA-co-TMC scaffolds or PDLLA-co-TMC scaffold layer in complex scaffold/constructs. Finally, the ability of self-folding into tubular structures of bilayer scaffolds was well noted even though the PDLLA-co-TMC shape-morphing layer was joined by an information-embedded layer in the bilayer scaffolds. This observation indicated well the desired shape-morphing property of 5D printed bilayer scaffolds.
To confirm the role/effects of the fifth dimension, i.e., information, in 5D printed structures, experiments were conducted on 5D printing of cell-laden and biomolecule-delivery constructs and on the effects of in situ delivered information (i.e., biomolecules) on rBMSC behaviour. Firstly, bioprinting of the rBMSC-containing Gel/GelMA bioink was performed to investigate and optimize cell state immediately after bioprinting and after in vitro culture of bioprinted structures for 1 and 3 days. The viability of rBMSCs incorporated in Gel/GelMA hydrogel scaffolds was studied using a LIVE/DEAD assay and the results are shown in Fig. 11. Using this assay, living cells were stained by calcein AM and would show green fluorescence under a fluorescence microscope, while dead cells were stained by ethidium homodimer EthD-1 and would show red fluorescence. From the day 0 result (i.e., immediately after bioprinting), it could be observed that most cells survived the bioprinting process even though some dead cells could be seen. The cell death was caused mainly by the exposure to the UV light and by the occurrence of radical polymerization of GelMA, as was explained by other researchers according to their similar investigations [128]. Furthermore, cells in the hydrogel at this time point stayed in the rounded shape, which was commonly observed in bioprinting studies of cell-laden hydrogels [34, 66, 101, 102]. After 1-day in vitro culture, interestingly, it was noted that most cells had been released from the hydrogel and that nearly no dead cells could be seen. It could be speculated that rBMSCs came out of the Gel/GelMA hydrogel owing to biodegradation of the hydrogel. The bioink was composed of rBMSCs, gelatin and lowly modified GelMA together with a low concentration of photoinitiator (2-hydroxy-2-methylpropiophenone, ~ 0.1% (v/v)). Such hydrogels after UV crosslinking of about 60 s (UV light intensity: 360 mW) could degrade fairly quickly when cultured at 37 °C. In addition, from the 10 × magnification images, the released rBMSCs could be seen to attach to the substrate and became spreading, which were very good for the subsequent cell differentiation under the influence of locally delivered biomolecules, i.e., the information. After 3-day in vitro culture, the released and adhering rBMSCs grew well and exhibited good proliferation, suggesting the good bioprinting part of 5D printing in the current study, as well as good fabrication of rBMSC-laden structs in the information-embedded layer of 5D bioprinted constructs.
Cell viability of rBMSCs at different culture time after bioprinting as shown by merged fluorescence images with live cells being stained in green and dead cells being stained in red.
To investigate the role/influence of the 5th dimension (i.e., embedded information, with its in situ delivery) on the incorporated living cells in 5D printed constructs, three types of the information layer containing different biomolecules, i.e., information, were fabricated by printing 10% (w/v) GelMA hydrogels containing AA, TGF, or TGF + AA (shortened as “TA” hereafter). GelMA hydrogel without biomolecules was also printed and set as the control. The printed information layers were UV-crosslinked for 60 s to stabilize the structure. In inks for the information layer, pure GelMA hydrogel with modified substitution of about 50%~60% was used so that the printed structures could stay stable and maintain a controlled and sustained delivery of the information (i.e., biomolecules). The bioprinted information layer scaffolds were cultured under the normal condition at 37 °C and after 5-day culture, the expression of F-actin and SMC-specific proteins (i.e., α-SMA and CALP) was studied to reveal the effects of locally delivered information (i.e., biomolecules) on rBMSCs, and the results are shown in Fig. 12. Abundant F-actin (red colored) together with cell nucleus (blue colored) was seen for cells in all three types of scaffolds. However, rBMSCs cultured in information-free scaffolds (the first column on the left in Fig. 12) showed relatively smaller and narrower cytoskeleton and lower cell density in comparison with those in information-embedded scaffolds (the AA, TGF and TA columns in Fig. 12). These results have demonstrated that the released embedded-information could improve the proliferation and cytoskeleton development of rBMSCs. Furthermore, as required by the definition of 5D printing and hence important for the cell-laden constructs manufactured, it was also shown that while no SMC-specific protein molecules were seen in the control group, cells in scaffolds embedded with AA or TGF displayed expressions of SMC-specific markers, i.e., α-smooth muscle actin (α-SMA) and h1-calponin (CALP). These results suggested that AA or TGF biomolecules alone were able to induce the differentiation of rBMSCs towards SMCs, which was in agreement with studies conducted by others [129, 130]. In addition, it could be seen that rBMSCs in scaffolds containing both TGF and AA exhibited significantly higher expression levels of α-SMA and CALP than scaffolds containing TGF or AA alone. This result indicated that TGF and AA had a synergetic effect on the differentiation of rBMSCs into SMCs, which was also observed in Narita et al.’s study [15]. Therefore, it is shown that the local delivery of information (TGF and AA), i.e., the 5th dimension in 5D printed structures, could affect the local environment for rBMSCs and cause the differentiation of rBMSCs towards SMCs, fullfilling what 5D printing aimed at in the current study. In the context of tissue engineering, these results have shown the importance and significant effects of incoporating apporriate biomolecules (including growth factors) in advanced MSC-laden constructs for guilding/controlling the cell hevhaior.
Expression of F-actin and also two SMC-specific proteins (α-SMA and CALP, owing to induced differentiation of rBMSCs) as shown by immunofluorescent staining of different biomolecules after 5-day in vitro culture. (AA, TGF and TA refer to ascorbic acid, TGF-β1 and TGF + AA, respectively.).
In human history, printing technologies, from 2D to 3D to 4D printing, play very important roles in various aspects of social development, including information assembly and dissemination, education, religion, manufacturing, etc. Over the past 30 years, 3D printing, since its first appearance in 1986, has made astonishing achievements in different areas and industries, including product design and development, consumer goods, transportation, healthcare, environmental protection, science, and education. Tissue engineering has greatly benefited from 3D printing, which has significantly expanded our ability to fabricate complex and advanced tissue engineering products with great precision, controlled architectures, and enhanced properties for regenerating different body tissues and organs. Applications of 3D printing for different tissues and organs (skin, vasculature, bone, articular cartilage, etc.) are now commonly seen and frequently reported. Over the past 10 years, 4D printing, since its introduction in 2013, has gained increasing attention in different areas such as soft robotics, smart sensors, and biomedical devices. Also, 4D printing in tissue engineering has been increasingly explored for, for example, fabricating self-tubing structures for blood vessel repair and for making shape-morphing bone scaffolds to match the defect shape and size after scaffold deployment. The progress in 3D and 4D printing has now enabled additive manufacturing to move forward into 5D printing which uses embedded information as the fifth dimension, in addition to 3D space and time. Just like 3D and 4D printing, 5D printing with the added information dimension will have great influences in different fields, such as biomedical engineering, of which tissue engineering is an essential part. For tissue engineering, in this article, we have shown a demonstration study for 5D printing which fabricated new bilayer scaffolds with both shape-morphing ability and controlled delivery of information (i.e., incorporated biomolecules). The 5D printed scaffolds could automatically fold up by responding to heating to the body temperature and the embedded and control-delivered information could change the environment of locally released living MSCs and induce the MSCs to differentiate into SMCs, which will be highly useful for the regeneration of tubular tissues such as vasculature and gastrointestinal tract. This demonstration study has also indicated the great potential of 5D printing in fabricating advanced scaffolds or constructs for regenerating complex human body tissues and organs.
5D printing is still at its very early stage of development and faces several major challenges. Firstly, it has been conceptualized only recently and is not yet widely known among researchers. Secondly, the information used in 5D printing to cause changes in the environment is currently very limited and requires further developments in the research community. For 5D printing in tissue engineering, apart from growth factors that were used in the demonstration study and presented in this article, other cell signals/messages (i.e., some proteins or molecules produced by cells) as potential, usable information should be investigated. These cell messages can be incorporated into 5D printed structures as the fifth dimension in AM and will be sent to the target tissues after deployment of the 5D printed structures, followed by changing the environment and triggering the changes of cells. Thirdly, currently available polymers suitable for 5D printing (Table S8) are very limited and hence new or novel biomaterials for 5D printing should be researched extensively and developed. Fourthly, there are still very few studies on 5D printing, and hence more research is needed to explore and realize its potential. Finally, the application of 5D printing in tissue engineering is not often reported and in this regard, more research is needed to see the benefits and potential. Nevertheless, as a new development of additive manufacturing, 5D printing can find many promising applications in translational clinical research and practices. 5D bioprinted cell-laden structures with embedded information and dynamic features will better recapitulate native tissues and organs, which can be used to gain deeper understanding of cell signaling pathways in basic research. Also, 5D printed information-embedded tissue engineering scaffolds will provide improved performance and hence can speed up tissue regeneration, leading to improved clinical outcomes. Furthermore, 5D printing can be used to produce personalized products with customized information and geometry for individual patients, leading to further developments in precision medicine.
Concluding remarks
This mini-review and prospective article has provided a concise and up-to-date review of recent advances in 3D/4D printing and bioprinting in tissue engineering. Current major 3D/4D printing technologies usable for tissue engineering and also biomaterials processable by 3D printing technologies for tissue engineering applications are presented and discussed. Applications of 3D/4D printing and bioprinting in tissue engineering for obtaining different static and dynamic scaffolds or cell-scaffold constructs for regenerating body tissues and organs, from relatively simple skin to very complex heart, are provided and analyzed. Finally, the concept of 5D printing is presented and explained, and using tissue engineering, the practice of 5D printing is demonstrated. The information embedded in 5D printed tissue engineering scaffolds and cell-scaffold constructs can be biomolecules such as GFs. The demonstration study has showed successful fabrication via 5D printing of shape-morphing, MSC-containing and biomolecule-delivering bilayer tissue engineering scaffolds. Such bilayer scaffolds can not only change from 2D planar shape to 3D tubular structures at the human body temperature but also deliver the embedded information to induce the incorporated rBMSCs to proliferate and differentiate for regenerating the target tissue, demonstrating the power of 5D printing for tackling challenges in regenerating complex body tissues. Moving from 3D/4D printing to 5D printing, AM goes forward from making passive/inactive products to producing active/intelligent structures that can cause intended, positive changes in the environment. 5D printing opens a new arena for the development of additive manufacturing and in the tissue engineering area, leading to the fabrication of novel tissue engineering scaffolds with high clinical performance for the regeneration of different human tissues and organs.
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Acknowledgments
M. Wang thanks Hong Kong’s Research Grants Council (RGC) for the financial support for his research on additive manufacturing and tissue engineering through various research grants, including recent Grants 17200519, 17202921, 17201622 and N_HKU749/22, and also The University of Hong Kong (HKU) through research grants in its Seed Fund for Basic Research Scheme. He thanks a donor in Hong Kong for her generous donation to support his research in biomaterials and tissue engineering. J. Lai thanks HKU for awarding him a PhD scholarship for conducting research at HKU. Assistance provided by members of M. Wang’s research group at HKU and by technical staff in HKU’s Department of Mechanical Engineering, Faculty of Medicine, Faculty of Dentistry and Electron Microscopy Unit is acknowledged.
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JL and MW: design of the review; JL: research and drafting of the manuscript; MW: concept development of 5D printing, research supervision, writing, reviewing and editing.
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A shape memory cycle for 4D printed objects (Figure S1). Examples of 3D/4D printed tissue engineering scaffolds (Figures S2–S6). Summary of recent work on 3D/4D printing for different tissue engineering applications (Table S1–S7). Typical polymers for 3D/4D/5D printing in tissue engineering (Table S8). Supplementary file1 (DOCX 7479 kb)
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Lai, J., Wang, M. Developments of additive manufacturing and 5D printing in tissue engineering. Journal of Materials Research 38, 4692–4725 (2023). https://doi.org/10.1557/s43578-023-01193-5
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DOI: https://doi.org/10.1557/s43578-023-01193-5