Tumor-on-a-chip models combined with mini-tissues or organoids for engineering tumor tissues.

The integration of tumor-on-a-chip technology with mini-tissues or organoids has emerged as a powerful approach in cancer research and drug development. This review provides an extensive examination of the diverse biofabrication methods employed to create mini-tissues, including 3D bioprinting, spheroids, microfluidic systems, and self-assembly techniques using cell-laden hydrogels. Furthermore, it explores various approaches for fabricating organ-on-a-chip platforms. This paper highlights the synergistic potential of combining these technologies to create tumor-on-a-chip models that mimic the complex tumor microenvironment and offer unique insights into cancer biology and therapeutic responses.

3D bioprinting using a new photo-crosslinking method for muscle tissue restoration.

Three-dimensional (3D) bioprinting is a highly effective technique for fabricating cell-loaded constructs in tissue engineering. However, the versatility of fabricating precise and complex cell-loaded hydrogels is limited owing to the poor crosslinking ability of cell-containing hydrogels. Herein, we propose an optic-fiber-assisted bioprinting (OAB) process to efficiently crosslink methacrylated hydrogels. By selecting appropriate processing conditions for the photo-crosslinking technique, we fabricated biofunctional cell-laden structures including methacrylated gelatin (Gelma), collagen, and decellularized extracellular matrix. To apply the method to skeletal muscle regeneration, cell-laden Gelma constructs were processed with a functional nozzle having a topographical cue and an OAB process that could induce a uniaxial alignment of C2C12 and human adipose stem cells (hASCs). Significantly higher degrees of cell alignment and myogenic activities in the cell-laden Gelma structure were observed compared with those in the cell construct that was printed using a conventional crosslinking method. Moreover, an in vivo regenerative potential was observed in volumetric muscle defects in a mouse model. The hASC-laden construct significantly induced greater muscle regeneration than the cell construct without topographical cues. Based on the results, the newly designed bioprinting process can prove to be highly effective in fabricating biofunctional cell-laden constructs for various tissue engineering applications.

Shape-memory collagen scaffold combined with hyaluronic acid for repairing intervertebral disc.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a common cause of chronic low back pain (LBP) and a socioeconomic burden worldwide. Conservative therapies and surgical treatments provide only symptomatic pain relief without promoting intervertebral disc (IVD) regeneration. Therefore, the clinical demand for disc regenerative therapies for disc repair is high. METHODS: In this study, we used a rat tail nucleotomy model to develop mechanically stable collagen-cryogel and fibrillated collagen with shape-memory for use in minimally invasive surgery for effective treatment of IVDD. The collagen was loaded with hyaluronic acid (HA) into a rat tail nucleotomy model. RESULTS: The shape-memory collagen structures exhibited outstanding chondrogenic activities, having completely similar physical properties to those of a typical shape-memory alginate construct in terms of water absorption, compressive properties, and shape-memorability behavior. The treatment of rat tail nucleotomy model with shape-memory collagen-cryogel/HA alleviated mechanical allodynia, maintained a higher concentration of water content, and preserved the disc structure by restoring the matrix proteins. CONCLUSION: According to these results, the collagen-based structure could effectively repair and maintain the IVD matrix better than the controls, including HA only and shape-memory alginate with HA.

Esophageal wound healing by aligned smooth muscle cell-laden nanofibrous patch.

The esophagus exhibits peristalsis via contraction of circularly and longitudinally aligned smooth muscles, and esophageal replacement is required if there is a critical-sized wound. In this study, we proposed to reconstruct esophageal tissues using cell electrospinning (CE), an advanced technique for encapsulating living cells into fibers that allows control of the direction of fiber deposition. After treatment with transforming growth factor-ß, mesenchymal stem cell-derived smooth muscle cells (SMCs) were utilized for cell electrospinning or three-dimensional bioprinting to compare the effects of aligned micropatterns on cell morphology. CE resulted in SMCs with uniaxially arranged and elongated cell morphology with upregulated expression levels of SMC-specific markers, including connexin 43, smooth muscle protein 22 alpha (SM22α), desmin, and smoothelin. When SMC-laden nanofibrous patches were transplanted into a rat esophageal defect model, the SMC patch promoted regeneration of esophageal wounds with an increased number of newly formed blood vessels and enhanced the SMC-specific markers of SM22α and vimentin. Taken together, CE with its advantages, such as guidance of highly elongated, aligned cell morphology and accelerated SMC differentiation, can be an efficient strategy to reconstruct smooth muscle tissues and treat esophageal perforation.

Safety Evaluation by Phenotypic and Genomic Characterization of Four Lactobacilli Strains with Probiotic Properties.

Probiotic Lactobacillus species are known to exert health benefits in hosts when administered in adequate quantities. A systematic safety assessment of the strains must be performed before the Lactobacillus strains can be designated as probiotics for human consumption. In this study, we selected Lactobacillus fermentum IDCC 3901, L. gasseri IDCC 3101, L. helveticus IDCC 3801, and L. salivarius IDCC 3551 as representative Lactobacilli probiotic strains and investigated their probiotic properties and potential risks through phenotypic and genomic characterization. Various assays including antimicrobial resistance, biogenic amine production, L-/D-lactate production, acute oral toxicity, and antipathogenic effect were performed to evaluate the safety of the four Lactobacillus strains. Genomic analysis using whole genome sequencing was performed to investigate virulence and antibiotic resistance genes in the genomes of the selected probiotic strains. The phenotypes of the strains such as enzymatic activity and carbohydrate utilization were also investigated. As a result, antibiotic resistances of the four Lactobacillus species were detected; however, neither antibiotic resistance-related genes nor virulence genes were found by genomic analysis. Moreover, the four Lactobacillus species did not exhibit hemolytic activity or ß-glucuronidase activity. The biogenic amine production and oral acute toxicity were not shown in the four Lactobacillus species, whereas they produced D-lactate with minor ratio. The four Lactobacillus species exhibited antipathogenic effect to five pathogenic microorganisms. This study provides a way to assess the potential risks of four different Lactobacillus species and validates the safety of all four strains as probiotics for human consumption.

Highly porous multiple-cell-laden collagen/hydroxyapatite scaffolds for bone tissue engineering.

Efficient vascularization within a scaffold is an essential criterion for evaluating the success of volumetric bone formation. Various strategies using angiogenic growth factors and cell-based approaches to induce effective osteogenic and angiogenic activities have been investigated. In this study, we propose a new highly porous multiple-cell-laden collagen/hydroxyapatite scaffold fabricated using a whipped bioink. After in vitro culturing of cells in the porous scaffolds for an extended culture period, osteogenic and angiogenic activities were significantly enhanced owing to the well-developed microporous cell-supporting matrix inducing efficient crosstalk between the adipose stem cells and endothelial cells compared to those of the normally bioprinted cell-constructs. Furthermore, the in vitro results were thoroughly evaluated by in vivo experiments using a posterolateral lumbar spinal fusion model of an ovariectomized mouse. Based on these results, the porous multiple-cell-laden scaffolds enhanced spine fusion in the event of osteoporosis.

A bioprinted complex tissue model for myotendinous junction with biochemical and biophysical cues.

In the musculoskeletal system, the myotendinous junction (MTJ) is optimally designed from the aspect of force transmission generated from a muscle through a tendon onto the bone to induce movement. Although the MTJ is a key complex tissue in force transmission, the realistic fabrication, and formation of complex tissues can be limited. To obtain the MTJ construct, we prepared two bioinks, muscle- and tendon-derived decellularized extracellular matrix (dECM), which can induce myogenic and tenogenic differentiation of human adipose-derived stem cells (hASCs). By using a modified bioprinting process supplemented with a nozzle consisting of a single-core channel and double-sheath channels, we can achieve three different types of MTJ units, composed of muscle, tendon, and interface zones. Our results indicated that the bioprinted dECM-based constructs induced hASCs to myogenic and tenogenic differentiation. In addition, a significantly higher MTJ-associated gene expression was detected at the MTJ interface with a cell-mixing zone than in the other interface models. Based on the results, the bioprinted MTJ model can be a potential platform for understanding the interaction between muscle and tendon cells, and even the bioprinting method can be extensively applied to obtain complex tissues.

Fabrication of bone-derived decellularized extracellular matrix/ceramic-based biocomposites and their osteo/odontogenic differentiation ability for dentin regeneration.

The goal of this study was to fabricate bioactive cell-laden biocomposites supplemented with bone-derived decellularized extracellular matrix (dECM) with calcium phosphate ceramic, and to assess the effect of the biocomponents on the osteogenic and odontogenic differentiation of human dental pulp stem cells (hDPSCs). By evaluating the rheological properties and selecting printing parameters, mechanically stable cell-laden 3D biocomposites with high initial cell-viability (>90%) and reasonable printability (≈0.9) were manufactured. The cytotoxicity of the biocomposites was evaluated via MTT assay and nuclei/F-actin fluorescent analyses, while the osteo/odontogenic differentiation of the hDPSCs was assessed using histological and immunofluorescent analyses and various gene expressions. Alkaline phosphate activity and alizarin red staining results indicate that the dECM-based biocomposites exhibit significantly higher osteogenic activities, including calcification, compared to the collagen-based biocomposites. Furthermore, immunofluorescence images and gene expressions demonstrated upregulation of dentin matrix acidic phosphoprotein-1 and dentin sialophosphoprotein in the dECM-based biocomposites, indicating acceleration of the odontogenic differentiation of hDPSCs in the printed biocomposites. The hDPSC-laden biocomposite was implanted into the subcutaneous region of mice, and the biocomposite afforded clear induction of osteo/odontogenic ectopic hard tissue formation 8 weeks post-transplantation. From these results, we suggest that the hDPSC-laden biocomposite is a promising biomaterial for dental tissue engineering.

Bioprinted hASC-laden collagen/HA constructs with meringue-like macro/micropores.

Extrusion-based bioprinting is one of the most effective methods for fabricating cell-laden mesh structures. However, insufficient cellular activities within the printed cylindrical cell-matrix blocks, inducing low cell-to-cell interactions due to the disturbance of the matrix hydrogel, remain to be addressed. Hence, various sacrificial materials or void-forming methods have been used; however, most of them cannot solve the problem completely or require complicated fabricating procedures. Herein, we suggest a bioprinted cell-laden collagen/hydroxyapatite (HA) construct comprising meringue-like porous cell-laden structures to enhance osteogenic activity. A porous bioink is generated using a culinary process, i.e., the whipping method, and the whipping conditions, such as the material concentration, time, and speed, are selected appropriately. The constructs fabricated using the meringue-like bioink with MG63 cells and human adipose stem cells exhibit outstanding metabolic and osteogenic activities owing to the synergistic effects of the efficient cell-to-cell interactions and HA stimulation released from the porous structure. The in vitro cellular responses indicate that the meringue-like collagen bioink for achieving an extremely porous cell-laden construct can be a highly promising cell-laden material for various tissue regeneration applications.

Highly bioactive cell-laden hydrogel constructs bioprinted using an emulsion bioink for tissue engineering applications.

The insufficient pore structure of cell-laden hydrogel scaffolds has limited their application in various tissue regeneration applications owing to low cell-to-cell/matrix interactions and low transfer of nutrients and metabolic wastes. Herein, we designed a highly porous cell-laden hydrogel scaffold fabricated using an emulsion bioink consisting of methacrylated collagen (CMA), mineral oil (MO), and human adipose stem cells (hASCs) to induce efficient cell infiltration and cellular activities. By selecting the most appropriate concentration of CMA and MO, the emulsion bioink can be successfully formulated with proper yield stress and printability. The cell-laden scaffold exhibited significantly greater cell growth and cytoskeletal reorganization than the normally printed cell-laden CMA scaffold. Furthermore, two bioactive components (kartogenin and bone morphogenetic protein-2) were physically encapsulated in the oil droplets of the cell construct, and the molecules in the cell constructs enhanced chondrogenic or osteogenic differentiation of hASCs in the printed structure. Based on these results, the cell-printed structure using an emulsion bioink can not only provide a good cellular microenvironment but also be a new potential method to accelerate stem cell differentiation by combining bioactive molecules and cell-laden scaffolds.

Highly elastic 3D-printed gelatin/HA/placental-extract scaffolds for bone tissue engineering.

Bioengineering scaffolds have been improved to achieve efficient regeneration of various damaged tissues. In this study, we attempted to fabricate mechanically and biologically activated 3D printed scaffold in which porous gelatin/hydroxyapatite (G/H) as a matrix material provided outstanding mechanical properties with recoverable behavior, and human placental extracts (hPE) embedded in the scaffold were used as bioactive components. Methods: Various cell types (human adipose-derived stem cells; hASCs, pre-osteoblast; MC3T3-E1, human endothelial cell line; EA.hy926, and human dermal fibroblast; hDFs) were used to assess the effect of the hPE on cellular responses. High weight fraction (~ 70 wt%) of hydroxyapatite (HA) in a gelatin solution supplemented with glycerol was used for the G/H scaffold fabrication, and the scaffolds were immersed in hPE for the embedding (G/H/hPE scaffold). The osteogenic abilities of the scaffolds were investigated in cultured cells (hASCs) assaying for ALP activity and expression of osteogenic genes. For the in vivo test, the G/H and G/H/hPE scaffolds were implanted in the rat mastoid obliteration model. Results: The G/H/hPE scaffold presented unique elastic recoverable properties, which are important for efficient usage of implantable scaffolds. The effects of G/H and G/H/hPE scaffold on various in vitro cell-activities including non-toxicity, biocompatibility, and cell proliferation were investigated. The in vitro results indicated that proliferation (G/H = 351.1 ± 13.3%, G/H/hPE = 430.9 ± 8.7% at day 14) and expression of osteogenic markers (ALP: 3.4-fold, Runx2: 3.9-fold, BMP2: 1.7-fold, OPN: 2.4-fold, and OCN: 4.8-fold at day 21) of hASCs grown in the G/H/hPE scaffold were significantly enhanced compared with that in cells grown in the G/H scaffold. In addition, bone formation was also observed in an in vivo model using rat mastoid obliteration. Conclusion:In vitro and in vivo results suggested that the G/H/hPE scaffold is a potential candidate for use in bone tissue engineering.

Production of Vitamin K by Wild-Type and Engineered Microorganisms.

Vitamin K is a fat-soluble vitamin that mainly exists as phylloquinone or menaquinone in nature. Vitamin K plays an important role in blood clotting and bone health in humans. For use as a nutraceutical, vitamin K is produced by natural extraction, chemical synthesis, and microbial fermentation. Natural extraction and chemical synthesis methods for vitamin K production have limitations, such as low yield of products and environmental concerns. Microbial fermentation is a more sustainable process for industrial production of natural vitamin K than two other methods. Recent advanced genetic technology facilitates industrial production of vitamin K by increasing the yield and productivity of microbial host strains. This review covers (i) general information about vitamin K and microbial host, (ii) current titers of vitamin K produced by wild-type microorganisms, and (iii) vitamin K production by engineered microorganisms, including the details of strain engineering strategies. Finally, current limitations and future directions for microbial production of vitamin K are also discussed.

Self-aligned myofibers in 3D bioprinted extracellular matrix-based construct accelerate skeletal muscle function restoration.

To achieve rapid skeletal muscle function restoration, many attempts have been made to bioengineer functional muscle constructs by employing physical, biochemical, or biological cues. Here, we develop a self-aligned skeletal muscle construct by printing a photo-crosslinkable skeletal muscle extracellular matrix-derived bioink together with poly(vinyl alcohol) that contains human muscle progenitor cells. To induce the self-alignment of human muscle progenitor cells, in situ uniaxially aligned micro-topographical structure in the printed constructs is created by a fibrillation/leaching of poly(vinyl alcohol) after the printing process. The in vitro results demonstrate that the synergistic effect of tissue-specific biochemical signals (obtained from the skeletal muscle extracellular matrix-derived bioink) and topographical cues [obtained from the poly(vinyl alcohol) fibrillation] improves the myogenic differentiation of the printed human muscle progenitor cells with cellular alignment. Moreover, this self-aligned muscle construct shows the accelerated integration with neural networks and vascular ingrowth in vivo, resulting in rapid restoration of muscle function. We demonstrate that combined biochemical and topographic cues on the 3D bioprinted skeletal muscle constructs can effectively reconstruct the extensive muscle defect injuries.

Lotus-Root-Like Microchanneled Collagen Scaffold.

In the human body, there are numerous microtubular tissue structures, such as muscles, vessels, nerves, and tendons. Tissue engineering scaffolds have been regarded as a high-potential candidate for providing such aligned instructive niches to facilitate cell-recruitment and differentiation, and eventually, successful tissue regeneration. Moreover, scaffolds derived from the extracellular matrix (ECM) can provide excellent biocompatibility. However, the fabrication of such microtubular hierarchical scaffolds using ECM has proven to be difficult, and thus, innovative fabrication approaches are required. Herein, we have developed a biofabrication system involving a sequential removal of supporting materials (polycaprolactone (PCL) and poly(vinyl alcohol) (PVA)) to fabricate a uniaxially aligned microtubular collagen scaffold, a lotus-like structure. To generate the unique morphological structures of the scaffold, we manipulated various material-related and processing factors, such as the molecular weight of PVA and the weight fraction of collagen coating. Physical and biological activities of the aligned hierarchical microtubular collagen scaffolds were compared with those of the controls (conventional collagen struts and microtubular collagen scaffolds void of a uniaxial topographical cue). In conclusion, the instructive niche on the aligned hierarchical microtubular collagen structure induced high degrees of myoblast alignment and efficient myogenic differentiation.

Bone-derived dECM/alginate bioink for fabricating a 3D cell-laden mesh structure for bone tissue engineering.

Alginate bioink has been widely employed to fabricate 3D cell-laden structures because of its low toxicity, appropriate biocompatibility, and easy/fast cross-linking ability. However, the low bioactivity of the hydrogel is a main shortcoming, so that physical or chemical modification with bioactive components is a promising strategy to efficiently increase the biological activity of alginate hydrogel. The present study proposes a new method to obtain bioactive alginate-based bioink by supplementing it with methacrylated (Ma)-decellularized extracellular matrix (dECM) derived from bone tissues. We demonstrate that the appropriate processing conditions and concentration of Ma-dECM in the bioink offer not only reasonable printability for fabricating 3D cell-laden structures, but also meaningful cell viability of the printed cell-laden construct. Moreover, the biologically improved microenvironment of alginate-based cell-laden structures formed using our method demonstrated a substantial effect on the osteogenic differentiation of the human adipose derived stem cells that were laden in the bioink.

New Bioink Derived from Neonatal Chicken Bone Marrow Cells and Its 3D-Bioprinted Niche for Osteogenic Stimulators.

This study examined whether neonatal chicken bone marrow cells (cBMCs) could support the osteogenesis of human stromal cells in a three-dimensional (3D) extracellular bioprinting niche. The majority (>95%) of 4-day-old cBMCs subcultured 5 times were positive for osteochondrogenesis-related genes (Col I, Col II, Col X, aggrecan, Sox9, osterix, Bmp2, osteocalcin, Runx2, and osteopontin) and their related proteins (Sox9, collagen type I, and collagen type II). LC-MS/MS analysis demonstrated that cBMC-conditioned medium (c-medium) contained proteins related to bone regeneration, such as periostin and members of the TGF-ß family. Next, a significant increase in osteogenesis was detected in three human adipose tissue-derived stromal cell (hASC) lines, after exposure to c-medium concentrates in 2D culture (p < 0.05). To evaluate biological function in a 3D environment, we employed the cBMC-derived bioactive components as a cell-supporting biomaterial in collagen bioink, which was printed to construct a 3D hASC-laden scaffold for observing osteogenesis. Complete osteogenesis was detected in vitro. Moreover, after transplantation of the hASC-laden structure into rats, prominent bone formation was observed compared with that in control rats receiving scaffold-free hASC transplantation. These results demonstrated that substance(s) secreted by chick bone marrow cells clearly activated the osteogenesis of hASCs in 2D- or 3D-niches.

Angiogenic factors secreted from human ASC spheroids entrapped in an alginate-based hierarchical structure via combined 3D printing/electrospinning system.

Human adipose-derived stem cell spheroids have been widely used in the treatment or regeneration of damaged skin tissues, and their success is believed to be due in part to angiogenic factors released from the spheroids. To achieve the sustained release of bioactive components from implanted spheroids within a defective area, the use of a biocompatible scaffolding biomaterial is required. In this study, we developed an alginate-based scaffolding structure, which was processed using three-dimensional printing and electrospinning for use as a spheroid-entrapping structure. A micro-sized alginate strut and electrospun alginate nanofibers functioned not only to firmly entrap the spheroids, but also to enable the stable release of various angiogenic and wound healing-related factors. We also demonstrated the function of these factors using a tube-forming assay and found that conditioned media from the spheroid-scaffold group improved capillary-like structure formation in human umbilical vein endothelial cells compared to the single cell-scaffold group. Our results suggest that this spheroid-entrapping alginate hybrid structure could represent a new platform for stem cell therapy using spheroid transplantation.

An intestinal model with a finger-like villus structure fabricated using a bioprinting process and collagen/SIS-based cell-laden bioink.

The surface of the small intestine has a finger-like microscale villus structure, which provides a large surface area to realize efficient digestion and absorption. However, the fabrication of a villus structure using a cell-laden bioink containing a decellularized small intestine submucosa, SIS, which can induce significant cellular activities, has not been attempted owing to the limited mechanical stiffness, which sustains the complex projective finger-like 3D structure. In this work, we developed a human intestinal villi model with an innovative bioprinting process using a collagen/SIS cell-laden bioink. Methods: A Caco-2-laden microscale villus structure (geometry of the villus: height = 831.1 ± 36.2 µm and diameter = 190.9 ± 3.9 µm) using a bioink consisting of collagen type-I and SIS was generated using a vertically moving 3D bioprinting process. By manipulating various compositions of dECM and a crosslinking agent in the bioink and the processing factors (printing speed, printing time, and pneumatic pressure), the villus structure was achieved. Results: The epithelial cell-laden collagen/SIS villi showed significant cell proliferation (1.2-fold) and demonstrated meaningful results for the various cellular activities, such as the expression of tight-junction proteins (ZO-1 and E-cadherin), ALP and ANPEP activities, MUC17 expression, and the permeability coefficient and the glucose uptake ability, compared with the pure 3D collagen villus structure. Conclusion: In vitro cellular activities demonstrated that the proposed cell-laden collagen/dECM villus structure generates a more meaningful epithelium layer mimicking the intestinal structure, compared with the pure cell-laden collagen villus structure having a similar villus geometry. Based on the results, we believe that this dECM-based 3D villus model will be helpful in obtaining a more realistic physiological small-intestine model.

Efficient myotube formation in 3D bioprinted tissue construct by biochemical and topographical cues.

Biochemical and biophysical cues directly affect cell morphology, adhesion, proliferation, and phenotype, as well as differentiation; thus, they have been commonly utilized for designing and developing biomaterial systems for tissue engineering applications. To bioengineer skeletal muscle tissues, the efficient and stable formation of aligned fibrous multinucleated myotubes is essential. To achieve this goal, we employed a decellularized extracellular matrix (dECM) as a biochemical component and a modified three-dimensional (3D) cell-printing process to produce an in situ uniaxially aligned/micro-topographical structure. The dECM was derived from the decellularization of porcine skeletal muscles and chemically modified by methacrylate process to enhance mechanical stability. By using this ECM-based material and the 3D printing capability, we were able to produce a cell-laden dECM-based structure with unique topographical cues. The myoblasts (C2C12 cell line) laden in the printed structure were aligned and differentiated with a high degree of myotube formation, owing to the synergistic effect of the skeletal muscle-specific biochemical and topographical cues. In particular, the increase of the gene-expression levels of the dECM structure with topographical cues was approximately 1.5-1.8-fold compared with those of a gelatin methacrylate (GelMA)-based structure with the same topographical cues and a dECM-based structure without topographical cues. According to these in vitro cellular responses, the 3D printed dECM-based structures with topographical cues have the potential for bioengineering functional skeletal muscle tissues, and this strategy can be extended for many musculoskeletal tissues, such as tendons and ligaments and utilized for developing in vitro tissue-on-a-chip models in drug screening and development.

A Myoblast-Laden Collagen Bioink with Fully Aligned Au Nanowires for Muscle-Tissue Regeneration.

Contact guidance can promote cell alignment and is thus widely employed in tissue regeneration. In particular, skeletal muscle consists of long fibrous bundles of multinucleated myotubes formed by the fusion and differentiation of the satellite cells of myoblasts. Herein, a functional bioink and cell-printing process supplemented with an electric field are proposed for obtaining highly aligned myoblasts in a collagen-based bioink. To achieve the goal, we mixed Au nanowires (GNWs) with the collagen-based bioink to provide aligned topographical cues to the laden cells. Because the aligned GNWs could clearly provide topographical cues to the cells, we adjusted various processing parameters (flow rate, nozzle speed, and processing temperature) and applied an external electric field to optimally align the GNWs. By selecting an appropriate condition, the GNWs in the printed C2C12-laden structure were well aligned in the printing direction, and they eventually induced a high degree of myoblast alignment and efficient myotube formation. Through the several in vitro cellular activities and in vivo works revealing the myogenesis of the cell-laden structure, we conclude that the collagen/GNW-based cell-laden structure fabricated using the proposed method is a new prospective platform for the effective formation of muscle tissues.