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.

Anisotropically Aligned Cell-Laden Nanofibrous Bundle Fabricated via Cell Electrospinning to Regenerate Skeletal Muscle Tissue.

For muscle regeneration, a uniaxially arranged micropattern is important to mimic the structure of the natural extracellular matrix. Recently, cell electrospinning (CE) has been tested to fabricate cell-laden fibrous structures by embedding cells directly into micro/nanofibers. Although homogenous cell distribution and a reasonable cell viability of the cell-laden fibrous structure fabricated using the CE process are achieved, unique topographical cues formed by an aligned fibrous structure have not been applied. In this study, a CE process to achieve not only homogeneous cell distribution with a high cell viability, but also highly aligned cells, which are guided by aligned alginate fibers is employed. To attain the aligned cell-laden fibrous structure, various processing conditions are examined. The selected condition is applied using C2C12 myoblast cells to ensure the biocompatibility and guidance of cell elongation and alignment. As a control, a cell-printed scaffold using a 3D bioprinter is used to compare the efficiency of cell alignment and differentiation of myoblasts. Highly arranged, multinucleated cell morphology is confirmed in the CE scaffold, which successively facilitates myogenic differentiation. It is believed that this study will be a new platform for obtaining cell alignment and will significantly benefit the efforts on muscle regeneration.

An Innovative Collagen-Based Cell-Printing Method for Obtaining Human Adipose Stem Cell-Laden Structures Consisting of Core-Sheath Structures for Tissue Engineering.

Three-dimensional (3D) cell printing processes have been used widely in various tissue engineering applications due to the efficient embedding of living cells in appropriately designed micro- or macro-structures. However, there are several issues to overcome, such as the limited choice of bioinks and tailor-made fabricating strategies. Here, we suggest a new, innovative cell-printing process, supplemented with a core-sheath nozzle and an aerosol cross-linking method, to obtain multilayered cell-laden mesh structure and a newly considered collagen-based cell-laden bioink. To obtain a mechanically and biologically enhanced cell-laden structure, we used collagen-bioink in the core region, and also used pure alginate in the sheath region to protect the cells in the collagen during the printing and cross-linking process and support the 3D cell-laden mesh structure. To achieve the most appropriate conditions for fabricating cell-embedded cylindrical core-sheath struts, various processing conditions, including weight fractions of the cross-linking agent and pneumatic pressure in the core region, were tested. The fabricated 3D MG63-laden mesh structure showed significantly higher cell viability (92 ± 3%) compared with that (83 ± 4%) of the control, obtained using a general alginate-based cell-printing process. To expand the feasibility to stem cell-embedded structures, we fabricated a cell-laden mesh structure consisting of core (cell-laden collagen)/sheath (pure alginate) using human adipose stem cells (hASCs). Using the selected processing conditions, we could achieve a stable 3D hASC-laden mesh structure. The fabricated cell-laden 3D core-sheath structure exhibited outstanding cell viability (91%) compared to that (83%) of an alginate-based hASC-laden mesh structure (control), and more efficient hepatogenic differentiations (albumin: ∼ 1.7-fold, TDO-2: ∼ 7.6-fold) were observed versus the control. The selection of collagen-bioink and the new printing strategy could lead to an efficient way to achieve 3D cell-laden mesh structures that mimic the anatomical architecture of a patient's defective region.

Sulfuretin induces osteoblast differentiation through activation of TGF-ß signaling.

The identification and examination of potential determinants controlling the progression of cell fate toward osteoblasts can be intriguing subjects. In this study, the effects of sulfuretin, a major compound isolated from Rhus verniciflua Stokes, on osteoblast differentiation were investigated. Treatments of sulfuretin induced alkaline phosphatase (ALP) activity in mesenchymal C3H10T1/2 cells and mineralization in preosteoblast MC3T3-E1 cells. Pro-osteogenic effects of sulfuretin were consistently observed in freshly isolated primary bone marrow cells. In mechanical studies, sulfuretin specifically induced expression of TGF-ß target genes, such as SMAD7 and PAI-1, but not other signaling pathway-related genes. Similar to the results of gene expression analysis, reporter assays further demonstrated TGF-ß-specific induction by sulfuretin. Furthermore, disruption of TGF-ß signaling using treatment with TGF-ß-specific inhibitor, SB-431542, and introduction of SMAD2/3 small interfering RNA impaired the effects of sulfuretin in inducing ALP activity and expression of ALP mRNA. Together, these data indicate that the pro-osteogenic effects of sulfuretin are mediated through activation of TGF-ß signaling, further supporting the potential of sulfuretin in the prevention of bone-related diseases such as bone fracture and osteoporosis.

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.

Mechanically improved electrospun PCL biocomposites reinforced with a collagen coating process: preparation, physical properties, and cellular activity.

In this work, we fabricated highly aligned electrospun poly(ε-caprolactone)(PCL)/collagen biocomposites, which were consisted of multi-layered structure. The aligned directions of the composites were controlled with two rotating collectors, and various weight fractions (1, 2, 3 wt%) of collagen were embedded between the mat of PCL microfibers to improve the mechanical property and biological activities of osteoblast-like cells (MG63). The PCL/collagen biocomposite showed nine times of increment in mechanical strength of random PCL/collagen composite. An increase in collagen content in the biocomposites displayed significant increase of mechanical properties, hydrophilic property, water-absorption ability, and even cell viability of osteoblast-like cells (MG63).

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.

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.

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.

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.

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.

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.

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.

Polycaprolactone scaffolds fabricated with an advanced electrohydrodynamic direct-printing method for bone tissue regeneration.

Electrohydrodynamic (EHD) direct writing has been used in diverse microelectromechanical systems and various supplemental methods for biotechnology and electronics. In this work, we expanded the use of EHD-induced direct writing to fabricate 3D biomedical scaffolds designed as porous structures for bone tissue engineering. To prepare the scaffolds, we modified a grounded target used in conventional EHD direct printing using a poly(ethylene oxide) solution bath, elastically cushioning the plotted struts to prevent crumbling. The fabricated scaffolds were assessed for not only physical properties including surface roughness and water uptake ability but also biological capabilities by culturing osteoblast-like cells (MG63) for the EHD-plotted polycaprolactone (PCL) scaffold. The EHD-scaffolds showed significantly roughened surface and enhanced water-absorption ability (400% increase) compared with the pure rapid-prototyped PCL. The results of cell viability, alkaline phosphatase activity, and mineralization analyses showed significantly enhanced biological properties of the scaffold (20 times the cell viability and 6 times the mineralization) compared with the scaffolds fabricated using RP technology. Because of the results, the modified EHD direct-writing process can be a promising method for fabricating 3D biomedical scaffolds in tissue engineering.

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.

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.