The purpose of 3D bioprinting technology is to design and create functional 3D tissues or organs in situ for in vivo applications. 3D cell-printing, or additive biomanufacturing, allows the selection of biomaterials and cells (bioink), and the fabrication of cell-laden structures in high resolution. 3D cell-printed structures have also been used for applications such as research models, drug delivery and discovery, and toxicology. Recently, numerous attempts have been made to fabricate tissues and organs by using various 3D printing techniques. However, challenges such as vascularization are yet to be solved. This article reviews the most commonly used 3D cell-printing techniques with their advantages and drawbacks. Furthermore, up-to-date achievements of 3D bioprinting in in vivo applications are introduced, and prospects for the future of 3D cell-printing technology are discussed.

Electrospinning has gained great interest in the field of regenerative medicine, due to its fabrication of a native extracellular matrix-mimicking environment. The micro/nanofibers generated through this process provide cell-friendly surroundings which promote cellular activities. Despite these benefits of electrospinning, a process was introduced to overcome the limitations of electrospinning. Cell-electrospinning is based on the basic process of electrospinning for producing viable cells encapsulated in the micro/nanofibers. In this review, the process of cell-electrospinning and the materials used in this process will be discussed. This review will also discuss the applications of cell-electrospun structures in tissue engineering. Finally, the advantages, limitations, and future perspectives will be discussed.

The most important requirements of biomedical substitutes used in muscle tissue regeneration are appropriate topographical cues and bioactive components for the induction of myogenic differentiation/maturation. Here, we developed an electric field-assisted 3D cell-printing process to fabricate cell-laden fibers with a cell-alignment cue. Methods : We used gelatin methacryloyl (GelMA) laden with C2C12 cells. The cells in the GelMA fiber were exposed to electrical stimulation, which induced cell alignment. Various cellular activities, such as cell viability, cell guidance, and proliferation/myogenic differentiation of the microfibrous cells in GelMA, were investigated in response to parameters (applied electric fields, viscosity of the bioink, and encapsulated cell density). In addition, a cell-laden fibrous bundle mimicking the structure of the perimysium was designed using gelatin hydrogel in conjunction with a 4D bioprinting technique. Results : Cell-laden microfibers were fabricated using optimized process parameters (electric field intensity = 0.8 kV cm -1 , applying time = 12 s, and cell number = 15 × 10 6 cells mL -1 ). The cell alignment induced by the electric field promoted significantly greater myotube formation, formation of highly ordered myotubes, and enhanced maturation, compared to the normally printed cell-laden structure. The shape change mechanism that involved the swelling properties and folding abilities of gelatin was successfully evaluated, and we bundled the GelMA microfibers using a 4D-conceptualized gelatin film. Conclusion : The C2C12-laden GelMA structure demonstrated effective myotube formation/maturation in response to stimulation with an electric field. Based on these results, we propose that our cell-laden fibrous bundles can be employed as in vitro drug testing models for obtaining insights into the various myogenic responses.

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its innate properties, or in response to external stimuli. Specifically, the SME can be seen in hybrid structures composed of a smart material and static material due to their inhomogeneity and different properties. [9,10] On the other hand, various stimuli, such as light, temperature, and humidity, can cause SMEs when a smart material converts the stimulus/energy into dynamic movement. With a detailed understanding of the properties of smart materials and their responses to stimuli, 4D printing can be utilized to precisely fabricate a "programmed" dimension that transforms and/or recovers its shape in response to stimuli. Many such smart materials have been developed, thus confirming the feasibility of 4D printing (Figure 1a).In this review, the concept of 4D bioprinting and smart materials will be defined by 4D mechanisms of SMEs and stimulus-responsive mechanisms. Then, 4D bioprinting will be categorized according to the types and cases of biomedical smart materials and applications. The current limitations and future aspects will also be discussed. Four-dimensional Mechanisms: Shape-Morphing EffectsFour-dimensional printing is essentially a 3D printing technique combined with one more dimension, that is, the SME by pre-set stimuli. The SME in 4D printing occurs after printing of the 3D structure, which is printer-independent but still predictable as the effect is programmed beforehand. [11] The types of SME-inducing stimuli will be discussed in the following section. In this section, SMEs will be classified according to the shape recovery ability of the 4D-printed structure. The SME of 4D structures with no shape recovery ability can be classified as one-way SME, while SMEs with shape recovery ability is classified as two-or multi-way SME. One-Way Shape MorphingOne-way SME refers to a 4D structure that is designed to change its structure once and is not able to recover its original shape by itself. This irreversibility of one-way SME is shown in Figure 1b. [12] One-way SME is distinguished from normal deformations, such as degradation, contraction, and swelling, as the deformation can be designed and predicted, that is, artificially programmed. One-way SME is a relatively simple mechanism compared to two-or multi-way SME. BioprintingThe development of the three-dimensional (3D) printer has resulted in significant advances in a number of fields, including rapid prototyping and biomedical devices. For 3D structures, the inclusion of dynamic responses to stimuli is added to develop the concept of four-dimensional (4D) printing. Typically, 4D printing is useful for biofabrication by reproducing a stimulus-responsive dynamic environment corresponding to physiological activities. Such a dynamic environment can be precisely designed with an understanding of shape-morphing effects (SMEs), which enables mimicking the functionality or intricate geometry of tissues. Here, 4D bioprinting is investigated for clinical use, for example, in drug delivery systems, tissue engineering, and surgery in vivo. This review presents ...