Fabrication of fully aligned self-assembled cell-laden collagen filaments for tissue engineering via a hybrid bioprinting process.

Cell-laden structures play a pivotal role in various tissue engineering applications, particularly in tissue restoration. Interactions between cells within bioprinted structures are crucial for successful tissue development and regulation of stem cell fate through intricate cell-to-cell signaling pathways. In this study, we developed a new technique that combines polyethylene glycol (PEG)-infused submerged bioprinting with a stretching procedure. This approach facilitated the generation of fully aligned collagen structures consisting of myoblasts and a low concentration (2 wt%) of collagen to efficiently encourage muscle tissue regeneration. By adjusting several processing parameters, we obtained biologically safe and mechanically stable cell-laden collagen filaments with uniaxial alignment. Notably, the cell filaments exhibited markedly elevated cellular activities compared to those exhibited by conventional bioprinted filaments, even at similar cell densities. Moreover, when we implanted structures containing adipose stem cells into mice, we observed a significantly increased level of myogenesis compared to that in normally bioprinted struts. Thus, this promising approach has the potential to revolutionize tissue engineering by fostering enhanced cellular interactions and promoting improved outcomes in regenerative medicine.

Engineered 3D liver-tissue model with minispheroids formed by a bioprinting process supported with in situ electrical stimulation.

Three-dimensional (3D) bioprinting, an effective technique for building cell-laden structures providing native extracellular matrix environments, presents challenges, including inadequate cellular interactions. To address these issues, cell spheroids offer a promising solution for improving their biological functions. Particularly, minispheroids with 50-100 µm diameters exhibit enhanced cellular maturation. We propose a one-step minispheroid-forming bioprinting process incorporating electrical stimulation (E-MS-printing). By stimulating the cells, minispheroids with controlled diameters were generated by manipulating the bioink viscosity and stimulation intensity. To validate its feasibility, E-MS-printing process was applied to fabricate an engineered liver model designed to mimic the hepatic lobule unit. E-MS-printing was employed to print the hepatocyte region, followed by bioprinting the central vein using a core-shell nozzle. The resulting constructs displayed native liver-mimetic structures containing minispheroids, which facilitated improved hepatic cell maturation, functional attributes, and vessel formation. Our results demonstrate a new potential 3D liver model that can replicate native liver tissues.

Macroscale pseudo-spheroids fabricated using methacrylated collagen-coated cells.

Rationale: Cell spheroids have shown great promise as tools for creating effective three-dimensional (3D) tissue models, facilitating tissue reconstruction and organoid development, due to their high cell density and efficient cellular interactions. However, a significant challenge persists in creating large-scale tissue structures with a 3D geometrical architecture using spheroids, due to the continual condensation and reorganization of cells and their environments. Methods: The spherical cell aggregates (pseudo-cell spheroids) or macroscale cell aggregates were obtained by coating each adipose-derived stem cell (hASC) with methacrylated collagen (Col-Ma). Subsequently, the coated cells were printed into an alginate supporting bath and photocrosslinked through exposure to UV light. To assess the effectiveness of this procedure on regenerative potential, the generated cell aggregates were compared with conventional cell spheroids and bioprinted cell constructs using immunofluorescent staining and quantification of myogenic-related gene expressions. Moreover, the bioconstructs were implanted into a mouse model with volumetric muscle loss to further elucidate their regenerative and functional recovery properties. Results: The use of Col-Ma as a cell-coating material enables the rapid and physical aggregation of cells within several hours, regardless of the cell type. Furthermore, Col-Ma-coated cell aggregates can provide relatively lower hypoxic conditions than cell spheroids fabricated using the hanging drop method owing to the thin porous Col-Ma layer coated on the cells. In addition, the resulting structures maintain their geometrical architecture following cell fusion and possess the potential for efficient scale-up and 3D complex shape formation, making them more suitable for clinical applications than conventional cell spheroids. Finally, the feasibility of the Col-Ma-coated cylindrical human adipose-derived stem cells aggregates was assessed through implantation in a mouse volumetric muscle loss model, showing a significantly higher regenerative ability of muscle tissue than the normally bioprinted cell construct. Conclusion: Our newly proposed method has meaningful potential for various tissue engineering applications, supported by the improved cellular activities and efficient muscle regeneration observed in both in vitro and in vivo studies, and organ-chip models.

Efficient Myogenic Activities Achieved through Blade-Casting-Assisted Bioprinting of Aligned Myoblasts Laden in Collagen Bioink.

This study investigated mechanical stimulation combined with three-dimensional (3D) bioprinting as a new approach for introducing biophysical and biological cues for tissue regeneration. A blade-casting method in conjunction with bioprinting was employed to fabricate bioengineered skeletal muscle constructs using a bioink composed of C2C12 myoblasts and collagen type-I. Various printing process parameters were selected and optimized to achieve a highly organized cell alignment within the constructs. The resulting cell-aligned constructs demonstrated remarkable improvement in actin filament alignment and cell proliferation compared with conventionally printed cell-laden constructs. This improvement can be attributed to the synergistic effects of mechanotransduction, facilitating the cellular response to mechanical cues and the alignment of fibrillated collagen, which plays a significant role in modulating cellular functions and promoting muscle tissue regeneration. Furthermore, we assessed the impact of blade casting combined with 3D bioprinting on gene expression. The expression levels of myogenesis-related genes were substantially upregulated, with an approximately 1.6-fold increase compared to the constructs fabricated without the blade-casting technique. The results demonstrated the effectiveness of combining mechanical stimulation through blade casting with 3D bioprinting in promoting aligned cell structures, enhancing cellular functions, and driving muscle tissue regeneration.

Bioprinted hASC-laden cell constructs with mechanically stable and cell alignment cue for tenogenic differentiation.

3D bioprinting is a technology that enables the precise and controlled deposition of cells and an artificial extracellular matrix (ECM) to create functional tissue constructs. However, current 3D bioprinting methods still struggle to obtain mechanically stable and unique cell-morphological structures, such as fully aligned cells. In this study, we propose a new 3D bioprinting approach that utilizes a high concentration of bioink without cells to support mechanical properties and drag flow to fully align cells in a thin bath filled with cell-laden bioink, resulting in a hybrid cell-laden construct with a mechanical stable and fully aligned cell structure. To demonstrate the feasibility of this approach, we used it to fabricate a cell-laden construct using human adipose stem cells (hASCs) for tendon tissue engineering. To achieve appropriate processing conditions, various factors such as the bioink concentration, nozzle moving speed, and volume flow rate were considered. To enhance the biocompatibility of the cell-laden construct, we used porcine decellularized tendon ECM.In vitrocellular responses, including tenogenic differentiation of the fabricated hybrid cell structures with aligned or randomly distributed cells, were evaluated using hASCs. In addition, the mechanical properties of the hybrid cell-laden construct could be adjusted by controlling the concentration of the mechanically reinforcing strut using methacrylated tendon-decellularized extracellular matrix. Based on these results, the hybrid cell-laden structure has the potential to be a highly effective platform for the alignment of musculoskeletal tissues.

The one-step fabrication of porous hASC-laden GelMa constructs using a handheld printing system.

The fabrication of highly porous cell-loaded structures in tissue engineering applications has been a challenging issue because non-porous cell-laden struts can cause severe cell necrosis in the middle region owing to poor transport of nutrients and oxygen. In this study, we propose a versatile handheld 3D printer for the effective fabrication of porous cell-laden methacrylated gelatin (GelMa) with high porosity (≈97%) by air injection and a bubble-making system using mesh filters through which a mixture of air/GelMa bioink is passed. In particular, the pore size and foamability of the cell constructs could be manipulated using various processing parameters (rheological properties of GelMa, filter size and number, and air-bioink volume ratio). To demonstrate the feasibility of the cell construct as a tissue engineering substitute for muscle regeneration, in vitro cellular activities and in vivo regeneration ability of human adipose stem cells were assessed. The in vitro results demonstrated that the human adipose stem cells (hASCs) fabricated using the handheld 3D printer were alive and well-proliferated. Furthermore, the in vivo results showed that the hASCs-constructs directly printed from the handheld 3D printer showed significant restoration of functionality and efficient muscle regeneration in the volumetric muscle loss model of mice. Based on these results, the fabrication method of the porous cell-laden construct could be a promising tool for regenerating muscle tissues.

Three-Dimensional Artificial Skin Construct Bioprinted with a Marine-Based Biocomposite.

A variety of artificial skin scaffolds, including 3D-bioprinted constructs, have been widely studied for regenerating injured skin tissue. Here, we devised a new composite biomaterial ink using fish-skin-based decellularized extracellular matrices (dECM) from tilapia and cod fish. The composition of the biocomposite mixture was carefully selected to obtain a mechanically stable and highly bioactive artificial cell construct. In addition, the decellularized extracellular matrices were methacrylated, followed by exposure to UV light to initiate photo-cross-linking. Porcine-skin-based dECMMa (pdECMMa) and tilapia-skin-based dECMMa (tdECMMa) biomaterials were used as controls. Assessment of various biophysical parameters and in vitro cellular activities, including cytotoxicity, wound healing ability, and angiogenesis, showed that the biocomposite exhibited much higher cellular activities compared to the controls owing to the synergistic effect of the favorable biophysical properties of tdECMMa and bioactive components (collagen, glycosaminoglycans (GAGs), elastin, and free fatty acids) from the decellularized cod skin. Furthermore, the skin constructs bioprinted using the bioinks exhibited more than 90% cell viability, performed with 3 days of submerged culture and then 28 days of air-liquid culture. For all cell constructs, the expression of cytokeratin 10 (CK10) was observed on the top surface of the epidermal layer, and cytokeratin 14 (CK14) was detected in the lower section of the keratinocyte layer. However, more developed CK10 and CK14 antibodies were observed in the cell-laden biocomposite construct [tilapia-skin-based dECMMa with cod-skin-based dECM] than in the controls [porcine-skin-based dECMMa (pdECMMa) and tilapia-skin-based dECMMa (tdECMMa)]. Based on these results, we believe that the fish-skin-based biocomposite construct is a potential biomaterial ink for skin regeneration.

Hybrid cell constructs consisting of bioprinted cell-spheroids.

Bioprinted cell constructs have been investigated for regeneration of various tissues. However, poor cell-cell interactions have limited their utility. Although cell-spheroids offer an alternative for efficient cell-cell interactions, they complicate bioprinting. Here, we introduce a new cell-printing process, fabricating cell-spheroids and cell-loaded constructs together without preparation of cell-spheroids in advance. Cells in mineral oil droplets self-assembled to form cell-spheroids due to the oil-aqueous interaction, exhibiting similar biological functions to the conventionally prepared cell-spheroids. By controlling printing parameters, spheroid diameter and location could be manipulated. To demonstrate the feasibility of this process, we fabricated hybrid cell constructs, consisting of endothelial cell-spheroids and stem cells loaded decellularized extracellular matrix/ß-tricalcium phosphate struts for regenerating vascularized bone. The hybrid cell constructs exhibited strong angiogenic/osteogenic activities as a result of increased secretion of signaling molecules and synergistic crosstalk between the cells.

Fabrication of self-assembled core-sheath microfibers via formulation of alginate-based bioinks.

Core-sheath microfibrous structures are widely used in various tissue engineering applications and drug delivery systems. However, the fabrication of the various core-sheath structures using a 3D printing process supplemented with a coaxial nozzle has been challenging due to the center positioning of the core nozzle enclosed in the bigger shell nozzle. In this work, we developed a new 3D printing process using an alginate-based bioink (a mixture of photo-crosslinkable hydrogel and alginate) and its in situ crosslinking process within a single glass nozzle of the 3D printer. By manipulating the alginate weight fraction, UV intensity, flow rate, and nozzle moving speed, we could fabricate various self-assembled core-sheath structures (straight, wavy, and crimped microfibers in the core region of the structure) in which the photocrosslinked hydrogel resided in the core, and alginate was positioned in the sheath region, like a virtual coaxial nozzle.

Correction: Mineralized biomimetic collagen/alginate/silica composite scaffolds fabricated by a low-temperature bio-plotting process for hard tissue regeneration: fabrication, characterisation and in vitro cellular activities.

Correction for 'Mineralized biomimetic collagen/alginate/silica composite scaffolds fabricated by a low-temperature bio-plotting process for hard tissue regeneration: fabrication, characterisation and in vitro cellular activities' by HyeongJin Lee et al., J. Mater. Chem. B, 2014, 2, 5785-5798, https://doi.org/10.1039/c4tb00931b.

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.

Bone tissue engineering supported by bioprinted cell constructs with endothelial cell spheroids.

In bone tissue engineering, efficient formation of vascularized bone tissue is a challenging issue. Here, we introduce a new strategy for effectively using multiple cells laden in a hybrid structure, such as endothelial cell (EC) spheroids and homogeneously distributed human adipose stem cells (hASCs) for bone regeneration. Methods: To fabricate the EC spheroids, cell-mixed mineral oil was used, and microscale droplets of the cell mixture were interlayered between the bioprinted hASC-laden struts. In vitro cellular responses of spheroid-laden multiple-cell constructs have been evaluated by comparing with the cell constructs bioprinted with the mixture of hASCs and ECs. In addition, mastoid obliterated rat model has been used to observe in vivo bone formation of those cell constructs. Results: The spheroid-laden multiple-cell constructs induced outstanding angiogenesis and osteogenic activities compared to a conventionally bioprinted multiple-cell construct. The enhanced biological results were clearly due to the EC spheroids, which triggered highly cooperative crosstalk between ECs and stem cells. The co-culture of the hASC constructs with the EC spheroids exhibited enhanced osteogenic- and angiogenic-related gene expression in vitro. In addition, in a rat obliterated mastoid model, considerably greater new bone formation and more competent development of new blood vessels were observed compared to those achieved with the normally bioprinted multiple cell-loaded structure. Conclusion:In vitro and in vivo results demonstrated that the bioprinted spheroid-laden multiple-cell construct is a potential candidate for use in bone tissue engineering.

Mechanically and biologically enhanced 3D-printed HA/PLLA/dECM biocomposites for bone tissue engineering.

Poly (L-lactic acid) (PLLA)-based biocomposites have been used in tissue engineering applications because of their reasonable biocompatibility and mechanical properties. However, the imperfect bioactive and mechanical properties of the composite make it difficult to be used in the region of bone defects that require high load-bearing. Therefore, this study introduced two fabricating strategies to induce mechanically and biologically enhanced hydroxyapatite (HA)/PLLA biocomposites. By introducing an in situ plasma treatment, which was simultaneously applied during the 3D-printing process, followed by the thermal annealing process, the flexural modulus of the composite was increased by 2.1-fold compared to the normal HA/PLLA composite. Furthermore, using the combinational process, efficient coating of bioactive material [decellularized extracellular matrix (dECM) derived from porcine bones] was possible. The fabricated biocomposite scaffold was assessed for various in vitro cellular activities such as cell proliferation and osteogenic activity. Based on the mechanical and biological studies, the HA/PLLA/dECM biocomposite scaffold is one of the promising scaffolds that can be applied in bone tissue regeneration.

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.

Wound healing ability of acellular fish skin and bovine collagen grafts for split-thickness donor sites in burn patients: Characterization of acellular grafts and clinical application.

Due to its high polyunsaturated fatty acid content, acellular fish skin has emerged as a dermal substitute for the promotion of wound healing as it decreases scar formation while providing pain relief. However, various systematic studies on acellular fish skin, such as its biophysical analysis, in vitro activities, and clinical application, have not been sufficiently investigated. In this study, we conducted a comparative study to evaluate the wound-healing ability of acellular fish skin graft (Kerecis®) with that of the widely used bovine collagen skin graft (ProHeal®). The skin grafts were evaluated not only in terms of their biophysical properties, but also their in vitro cellular activities, using fibroblasts, keratinocytes, and human endothelial cells. The clinical study evaluated wound healing in 52 patients with acute burns who underwent skin grafting on donor sites from January 2019 to December 2020. The study was conducted with two groups; while only Kerecis® was tested in one group, Kerecis® and ProHeal® were compared in the other. In both groups, the application time of the dressing material was one to two days after split-thickness skin grafting to the donor sites. The Kerecis®-treatment group experienced faster healing than the other treatment group. In particular, the average wound healing time using the Kerecis® treatment and the ProHeal® treatment was 10.7 ± 1.5 days and 13.1 ± 1.4 days, respectively. We believe that the faster healing of the Kerecis® treatment, compared to that of the ProHeal® treatment, maybe due to the synergistic effect of the unique biophysical structure and the bioactive components of acellular fish skin.

A Microfluidic Device to Fabricate One-Step Cell Bead-Laden Hydrogel Struts for Tissue Engineering.

Cell-laden structures are widely applied for a variety of tissue engineering applications, including tissue restoration. Cell-to-cell interactions in bioprinted structures are important for successful tissue restoration, because cell-cell signaling pathways can regulate tissue development and stem cell fate. However, the low degree of cell-cell interaction in conventional cell-laden bioprinted structures is challenging for the therapeutic application of this modality. Herein, a microfluidic device with cell-laden methacrylated gelatin (GelMa) bioink and alginate as a matrix hydrogel is used to fabricate a functional hybrid structure laden with cell-aggregated microbeads. This approach effectively increases the degree of cell-to-cell interaction to a level comparable to cell spheroids. The hybrid structure is obtained using a one-step process without the exhausting procedure. It consists of cell bead fabrication and an extrusion process for the cell-bead laden structure. Different flow rates are appropriately selected to develop cell-laden struts with homogeneously distributed cell beads for each hydrogel in the process. The hybrid struts exhibit significantly higher cellular activities than those of conventional alginate/GelMa struts, which are bioprinted using similar cell densities and bioink formulations. Furthermore, hybrid struts with adipose stem cells are implanted into mice, resulting in significantly higher myogenesis in comparison to normally bioprinted struts.

Bio-printing of aligned GelMa-based cell-laden structure for muscle tissue regeneration.

Volumetric muscle loss (VML) is associated with a severe loss of muscle tissue that overwhelms the regenerative potential of skeletal muscles. Tissue engineering has shown promise for the treatment of VML injuries, as evidenced by various preclinical trials. The present study describes the fabrication of a cell-laden GelMa muscle construct using an in situ crosslinking (ISC) strategy to improve muscle functionality. To obtain optimal biophysical properties of the muscle construct, two UV exposure sources, UV exposure dose, and wall shear stress were evaluated using C2C12 myoblasts. Additionally, the ISC system showed a significantly higher degree of uniaxial alignment and myogenesis compared to the conventional crosslinking strategy (post-crosslinking). To evaluate the in vivo regenerative potential, muscle constructs laden with human adipose stem cells were used. The VML defect group implanted with the bio-printed muscle construct showed significant restoration of functionality and muscular volume. The data presented in this study suggest that stem cell-based therapies combined with the modified bioprinting process could potentially be effective against VML injuries.

Bioprinted Collagen-Based Cell-Laden Scaffold With Growth Factors for Tympanic Membrane Regeneration in Chronic Perforation Model.

With the recent development of bioprinting technology, various attempts have been made to replace bioprinting technologies and regenerative medicine are more directed towards transplantation/reconstructive surgeries only with the implantation of scaffolds. The purpose of this study is to determine whether the growth factors, human umbilical cord serum (hUCS) and bFGF (basic fibroblast growth factor), have a synergistic effect on eardrum regeneration, when used with a cell-printed scaffold in a chronic tympanic membrane perforation (TMP) model. In this study, in vitro cellular activities for bioprinted cell-laden collagen scaffolds using human adipose stem cells (hASCs) and supplemented with 10 [Formula: see text]/mL hUCS and 10 ng/mL bFGF were performed. The mixture of the growth factors in the cell-laden structures effectively affects various in vitro cellular responses including the proliferation of hASCs and the migration of keratinocytes due to the synergistic effect of the growth factors and hASCs. For the in vivo evaluation, a rat TMP model was used, and the TMP regeneration was assessed by otoscopic examination, hearing threshold measurement, and histologic examination. Although the cell-laden structure containing hUCS was more enhancing effect compared to the structure with bFGF, more synergistic effect in the structure using hUCS/bFGF was observed. Based on the results, we believe that the cell-laden structure incorporating hUCS and bFGF can induce significant regeneration of chronic tympanic membrane perforation.

Engineered Myoblast-Laden Collagen Filaments Fabricated Using a Submerged Bioprinting Process to Obtain Efficient Myogenic Activities.

The skeletal muscle tissue comprises a hierarchical fibrous structure with fully aligned myofibers. To obtain a unique aligned engineering construct for regenerating muscle tissue, we adopted a submerged bioprinting process. Here, 3 wt % collagen and 6 wt % alginate solutions were used as a matrix cell-encapsulating bioink and supporting solution in the printing bath, respectively. By manipulating the processing parameters (various alginate weight fractions in the bath, nozzle moving speed, and hydrostatic pressure), cell-laden filaments (∼50 µm in diameter) were successfully fabricated. They presented a high degree of alignment of the fibrillated collagen and meaningful initial viability (∼90%) of the C2C12 myoblasts. In vitro cellular responses indicated that fully aligned F-actin filaments of myoblasts were developed, resulting in a high degree of alignment/formation of myotubes, compared to that in the controls (>100 µm diameter of cell-laden filaments). Furthermore, the expression levels of various myogenic genes (Myod1, Myh2, and Myog) were measured using a reverse transcription polymerase chain reaction on day 21 of the cell culture, and the results showed that the cell-laden filaments with a small diameter had considerably greater gene expression levels (2.2-8-fold) than those with a relatively large diameter. Thus, the printing process described herein can provide a new potential biofabricating platform to obtain cell-laden engineering constructs for various tissues.

Electrohydrodynamic-direct-printed cell-laden microfibrous structure using alginate-based bioink for effective myotube formation.

In this study, a fully aligned microfibrous structure fabricated using fibrin-assisted alginate bioink and electrohydrodynamic direct-printing was proposed for skeletal muscle tissue engineering. To safely construct the aligned alginate/fibrin microfibrous structure laden with myoblasts or endothelial cells, various printing conditions, such as an applied electric field, distance between the nozzle and target, and nozzle moving speed, were selected appropriately. Furthermore, to accelerate the formation of myotubes more efficiently, the alginate/fibrin bioink with vascular endothelial cells was co-printed into a spatially patterned structure within a myoblast-laden structure. The myoblast-laden structure co-cultured with endothelial cells presented fully aligned myotube formation and significantly greater myogenic differentiation compared to the myoblast-laden structure without the endothelial cells owing to the more abundant secretion of angiogenic cytokines. Also, when adipose stem cell- and endothelial cell-laden fibrous structure was implanted in a mouse volumetric muscle loss model, accelerated volumetric muscle repair was observed compared to the defect model. Based on the results, this study demonstrates an alginate-based bioink and new bio-fabricating method to obtain microfibrous cell-laden alginate/fibrin structures with mechanically stable and topographical cues. The proposed method can provide a myoblast/endothelial cell-laden fibrous alginate structure to efficiently induce engineering of skeletal muscle tissue, which could be used in muscle-on-a-chip or recovering structures of volumetric muscle defects.