Highly Aligned Nanofibrous Scaffold Derived from Decellularized Human Fibroblasts

Native tissues are endowed with a highly organized nanofibrous extracellular matrix (ECM) that directs cellular distribution and function. The objective of this study is to create a purely natural, uniform, and highly aligned nano­fibrous ECM scaffold for potential tissue engineering applications. Synthetic nanogratings (130 nm in depth) are used to direct the growth of human dermal fibroblasts for up to 8 weeks, resulting in a uniform 70 μm‐thick fibroblast cell sheet with highly aligned cells and ECM nanofibers. A natural ECM scaffold with uniformly aligned nanofibers of 78 ± 9 nm in diameter is generated after removing the cellular components from the fibroblast sheet. The elastic modulus of the scaffold is well maintained after the decellularization process because of the preservation of elastin fibers. Reseeding human mesenchymal stem cells (hMSCs) shows the excellent capacity of the scaffold in directing and supporting cell alignment and proliferation along the underlying fibers. The scaffold's biocompatibility is further examined by an in vitro inflammation assay with seeded macrophages. The aligned ECM scaffold induces a significantly lower immune response compared to its unaligned counterpart, as detected by the pro‐inflammatory cytokines secreted from macrophages. The aligned nanofibrous ECM scaffold holds great potential in engineering organized tissues.     

Programmed Shape‐Morphing Scaffolds Enabling Facile 3D Endothelialization

Rapid formation of a confluent endothelial monolayer is the key to the success of small‐diameter vascular grafts, which is significantly important for treating dangerous and even sometimes deadly vascular disorders. However, the difficulty to homogenously locate endothelial cells onto the lumen of small‐diameter tubular scaffolds makes 3D endothelialization greatly challenging. Here, novel shape‐morphing scaffolds enabling programmed deformation from planar shapes to small‐diameter tubular shapes are designed and developed by combining biocompatible shape memory polymer and electrospun nanofibrous membrane. Endothelial cells can be conveniently seeded and attached on the 2D surface of the scaffolds and subsequently self‐rolled into 3D organization at physiological temperature. Endothelial cell responses and functions are varied on the shape‐morphing scaffolds with different nanofibrous electrospun membranes as the inner layer, arisen from the inducement of scaffolds with different morphological, physical, and biochemical characteristics. Owing to excellent properties of the nanofibrous membrane fabricated by the coelectrospinning of poly‐ε‐caprolactone (PCL) and gelatin methacrylate (GelMA), the shape‐morphing scaffolds with a nanofibrous PCL/GelMA inner layer support desirable homogeneous endothelial cell attachment as well as the rapid formation of biomimetic cell–scaffold interaction and cell–cell interaction under the 3D cell culture condition, therefore offering a visible approach for facile 3D endothelialization.     

Microfluidic Spinning of Cell‐Responsive Grooved Microfibers

Engineering living tissues that simulate their natural counterparts is a dynamic area of research. Among the various models of biological tissues being developed, fiber‐shaped cellular architectures, which can be used as artificial blood vessels or muscle fibers, have drawn particular attention. However, the fabrication of continuous microfiber substrates for culturing cells is still limited to a restricted number of polymers (e.g., alginate) having easy processability but poor cell–material interaction properties. Moreover, the typical smooth surface of a synthetic fiber does not replicate the micro‐ and nanofeatures observed in vivo, which guide and regulate cell behavior. In this study, a method to fabricate photocrosslinkable cell‐responsive methacrylamide‐modified gelatin (GelMA) fibers with exquisite microstructured surfaces by using a microfluidic device is developed. These hydrogel fibers with microgrooved surfaces efficiently promote cell encapsulation and adhesion. GelMA fibers significantly promote the viability of cells encapsulated in/or grown on the fibers compared with similar grooved alginate fibers used as controls. Importantly, the grooves engraved on the GelMA fibers induce cell alignment. Furthermore, the GelMA fibers exhibit excellent processability and could be wound into various shapes. These microstructured GelMA fibers have great potential as templates for the creation of fiber‐shaped tissues or tissue microstructures.     

A Hybrid Peptide Amphiphile Fiber PEG Hydrogel Matrix for 3D Cell Culture

Since the traditional 2D surface for cell growth has been shown to be increasingly insufficient in contemporary cell biology, more and more research is performed on 3D matrices that can better represent the natural extracellular matrix (ECM) in many aspects. To create such a complex nonuniform 3D matrix, four‐armed polyethylene glycol with azides and (1R,8S,9S)‐bicyclo[6.1.0]non‐4‐yn‐9‐yl groups is functionalized to form the hydrogel basis. Together with these, a matrix metalloproteinase cleavable peptide sequence as a functional motif is also built in to add degradability to the hydrogel. In addition, self‐assembled peptide amphiphile (PA) fibers containing a cellular binding peptide sequence (RGDS) are encapsulated in the hydrogel to mimic the natural fibrous structure of the ECM and to stimulate cell adhesion. Rheology studies confirm that the polymer dissolved in the PA fiber solution forms a stable hydrogel with acceptable mechanical properties (G′ = 3.8 kPa). In addition, it is shown that this hydrogel network is degradable under the action of a metalloproteinase enzyme. Finally, the hybrid hydrogel is used to culture and it is demonstrated that both HeLa cells and human mesenchymal stem cells show adherence, good viability, and a well‐spread shape inside the hybrid hydrogel after 5 days of incubation when all components are present.     

Digital Plasmonic Patterning for Localized Tuning of Hydrogel Stiffness

The mechanical properties of the extracellular matrix (ECM) can dictate cell fate in biological systems. In tissue engineering, varying the stiffness of hydrogels—water‐swollen polymeric networks that act as ECM substrates—has previously been demonstrated to control cell migration, proliferation, and differentiation. Here, “digital plasmonic patterning” (DPP) is developed to mechanically alter a hydrogel encapsulated with gold nanorods using a near‐infrared laser, according to a digital (computer‐generated) pattern. DPP can provide orders of magnitude changes in stiffness, and can be tuned by laser intensity and speed of writing. In vitro cellular experiments using A7R5 smooth muscle cells confirm cell migration and alignment according to these patterns, making DPP a useful technique for mechanically patterning hydrogels for various biomedical applications.     

Sulfated Hydrogel Matrices Direct Mitogenicity and Maintenance of Chondrocyte Phenotype through Activation of FGF Signaling

Deciphering the roles of chemical and physical features of the extracellular matrix (ECM) is vital for developing biomimetic materials with desired cellular responses in regenerative medicine. Here, it is demonstrated that sulfation of biopolymers, mimicking the proteoglycans in native tissues, induces mitogenicity, chondrogenic phenotype, and suppresses catabolic activity of chondrocytes, a cell type that resides in a highly sulfated tissue. Through tunable modification of alginate it is shown that increased sulfation of the microenvironment promotes fibroblast growth factor (FGF) signaling‐mediated proliferation of chondrocytes in a 3D matrix independent of stiffness, swelling, and porosity. Furthermore, for the first time it is shown that a biomimetic hydrogel acts as a 3D signaling matrix to mediate a heparan sulfate/heparin‐like interaction between FGF and its receptor leading to signaling cascades inducing cell proliferation, cartilage matrix production, and suppression of dedifferentiation markers. Collectively, this study reveals important insights on mimicking the ECM to guide self‐renewal of cells via manipulation of distinct signaling mechanisms.     

Chinese‐Noodle‐Inspired Muscle Myofiber Fabrication

Much effort has been made to engineer artificial fiber‐shaped cellular constructs that can be potentially used as muscle fibers or blood vessels. However, existing microfiber‐based approaches for culturing cells are still limited to 2D systems, compatible with a restricted number of polymers (e.g., alginate) and always lacking in situ mechanical stimulation. Here, a simple, facile, and high‐throughput technique is reported to fabricate 3D cell‐laden hydrogel microfibers (named hydrogel noodles), inspired by the fabrication approach for Chinese Hele noodle. A magnetically actuated and noncontact method to apply tensile stretch on hydrogel noodles has also been developed. With this method, it is found that cellular strain‐threshold and saturation behaviors in hydrogel noodles differ substantially from their 2D analogs, including proliferation, spreading, and alignment. Moreover, it is shown that these cell‐laden microfibers can induce muscle myofiber formation by tensile stretching alone. This easily adaptable platform holds great potential for the creation of functional tissue constructs and probing mechanobiology in three dimensions.     

Collective Organization Behaviors of Multi-Cell Systems Induced by Engineered ECM-Cell Mechanical Coupling

Cells in vivo are surrounded by fibrous extracellular matrix (ECM), which can mediate the propagation of active cellular forces through stressed fiber bundles and regulate various biological processes. However, the mechanisms for multi-cellular organization and collective dynamics induced by cell-ECM mechanical couplings, which are crucial for the development of novel ECM-based biomaterial for cell manipulation and biomechanical applications, remain poorly understood. Herein, the authors design an in vitro quasi-3D experimental system and demonstrate a transition between spreading and aggregating in collective organizational behaviors of discrete multi-cellular systems, induced by engineered ECM-cell mechanical coupling, with the observed phenomena and underlying mechanisms differing fundamentally from those of cell monolayers. During the process of collective cell organization, the collagen substrate undergoes reconstruction into a dense fiber network structure, which is correlated with local cellular density and consistent with observed enhanced cells' motility; and the weakening of fiber bundle formation within the hydrogel reduces cells’ movement. Moreover, cells can respond to the curvature and shape of the original cell population and form different aggregation patterns. These results elucidate important physical factors involved in collective cell organization and provide important references for potential applications of biomaterials in new therapies and tissue engineering.     

Anisotropic Nanoscale Presentation of Cell Adhesion Ligand Enhances the Recruitment of Diverse Integrins in Adhesion Structures and Mechanosensing‐Dependent Differentiation of Stem Cells

The nanoscale anisotropic patterns of bioactive ligands in the extracellular matrix regulate cell adhesion behaviors. However, the mechanisms of such regulation remain unclear. Here, RGD‐bearing gold nanorods (AuNRs) are conjugated with different aspect ratios (ARs, from 1 to 7) on cell culture substrates to decouple the effect of nanoscale anisotropic presentation of cell adhesive RGD peptides on cell adhesion. Compared with AuNRs with small ARs, AuNRs with large ARs significantly promote cell spreading, the alignment of the basal cytoskeletal structure, and nanopodia attachment. Furthermore, both ‐β3 and ‐β1 class integrins are recruited to AuNRs with large ARs, thereby promoting the development of focal adhesion toward fibrillar adhesion, whereas the recruitment of diverse integrins and the development of cell adhesion structures are hindered by small ARs AuNRs. The anisotropic presentation of ligands by large AR AuNRs better activates mechanotransduction signaling molecules. These findings are confirmed both in vitro and in vivo. Hence the enhanced mechanotransduction promotes osteogenic differentiation in stem cells. These findings demonstrate the potential use of well‐controlled synthetic nanoplatforms to unravel the fundamental mechanisms of cell adhesion and associated signaling at the molecular level and to provide valuable guidance for the rational design of biomaterials with tailored bioactive functions.     

Characterization of a Novel Fiber Composite Material for Mechanotransduction Research of Fibrous Connective Tissues

Mechanotransduction is the fundamental process by which cells detect and respond to their mechanical environment, and is critical for tissue homeostasis. Understanding mechanotransduction mechanisms will provide insights into disease processes and injuries, and may support novel tissue engineering research. Although there has been extensive research in mechanotransduction, many pathways remain unclear, due to the complexity of the signaling mechanisms and loading environments involved. This study describes the development of a novel hydrogel‐based fiber composite material for investigating mechanotransduction in fibrous tissues. By encapsulating poly(2‐hydroxyethyl methacrylate) rods in a bulk poly(ethylene glycol) matrix, it aims to create a micromechanical environment more representative of that seen in vivo. Results demonstrated that collagen‐coated rods enable localized cell attachment, and cells are successfully cultured for one week within the composite. Mechanical analysis of the composite indicates that gross mechanical properties and local strain environments could be manipulated by altering the fabrication process. Allowing diffusion between the rods and surrounding matrix creates an interpenetrating network whereby the relationships between shear and tension are altered. Increasing diffusion enhances the shear bond strength between rods and matrix and the levels of local tension along the rods. Preliminary investigation into fibroblast mechanotransduction illustrates that the fiber composite upregulates collagen I expression, the main protein in fibrous tissues, in response to cyclic tensile strains when compared to less complex 2D and 3D environments. In summary, the ability to create and manipulate a strain environment surrounding the fibers, where combined tensile and shear forces uniquely impact cell functions, is demonstrated.     

Engineered Extracellular Matrices as Biomaterials of Tunable Composition and Function

Engineered and decellularized extracellular matrices (ECM) are receiving increasing interest in regenerative medicine as materials capable to induce cell growth/differentiation and tissue repair by physiological presentation of embedded cues. However, ECM production/decellularization processes and control over their composition remain primary challenges. This study reports engineering of ECM materials with customized properties, based on genetic manipulation of immortalized and death‐inducible human mesenchymal stromal cells (hMSC), cultured within 3D porous scaffolds under perfusion flow. The strategy allows for robust ECM deposition and subsequent decellularization by deliberate cell‐apoptosis induction. As compared to standard production and freeze/thaw treatment, this grants superior preservation of ECM, leading to enhanced bone formation upon implantation in calvarial defects. Tunability of ECM composition and function is exemplified by modification of the cell line to overexpress vascular endothelial growth factor alpha (VEGF), which results in selective ECM enrichment and superior vasculature recruitment in an ectopic implantation model. hMSC lines culture under perfusion‐flow is pivotal to achieve uniform scaffold decoration with ECM and to streamline the different engineering/decellularization phases in a single environmental chamber. The findings outline the paradigm of combining suitable cell lines and bioreactor systems for generating ECM‐based off‐the‐shelf materials, with custom set of signals designed to activate endogenous regenerative processes.     

Injectable Peptide Decorated Functional Nanofibrous Hollow Microspheres to Direct Stem Cell Differentiation and Tissue Regeneration

Injectable microspheres are attractive stem cell carriers for minimally invasive procedures. For tissue regeneration, the microspheres need to present the critical cues to properly direct stem cell differentiation. In natural extracellular matrix (ECM), growth factors (GFs) and collagen nanofibers provide critical chemical and physical cues. However, there have been no reported technologies that integrate synthetic nanofibers and GFs into injectable microspheres. In this study, functional nanofibrous hollow microspheres (FNF‐HMS), which can covalently bind GF‐mimicking peptides, are synthesized. Two different GF‐mimicking peptides, Transforming Growth Factor‐β1 mimicking peptide Cytomodulin (CM) and Bone Morphogenetic Protein‐2 mimicking peptide P24, are separately conjugated onto the FNF‐HMS to induce distinct differentiation pathways of rabbit bone marrow‐derived mesenchymal stem cells (BMSCs). While no existing biomaterials are reported to successfully deliver CM to induce chondrogenesis, the developed FNF‐HMS are shown to effectively present CM to BMSCs and successfully induced their chondrogenesis for ­cartilage formation in both in vitro and in vivo studies. In addition, P24 is conjugated onto the newly developed FNF‐HMS and is capable of retaining its bioactivity and inducing ectopic bone formation in nude mice. These results demonstrate that the novel FNF‐HMS can effectively deliver GF‐mimicking peptides to modulate stem cell fate and tissue regeneration.     

Biomimetic Scaffolds for Tissue Engineering

Biomimetic scaffolds mimic important features of the extracellular matrix (ECM) architecture and can be finely controlled at the nano‐ or microscale for tissue engineering. Rational design of biomimetic scaffolds is based on consideration of the ECM as a natural scaffold; the ECM provides cells with a variety of physical, chemical, and biological cues that affect cell growth and function. There are a number of approaches available to create 3D biomimetic scaffolds with control over their physical and mechanical properties, cell adhesion, and the temporal and spatial release of growth factors. Here, an overview of some biological features of the natural ECM is presented and a variety of original engineering methods that are currently used to produce synthetic polymer‐based scaffolds in pre‐fabricated form before implantation, to modify their surfaces with biochemical ligands, to incorporate growth factors, and to control their nano‐ and microscale geometry to create biomimetic scaffolds are discussed. Finally, in contrast to pre‐fabricated scaffolds composed of synthetic polymers, injectable biomimetic scaffolds based on either genetically engineered‐ or chemically synthesized‐peptides of which sequences are derived from the natural ECM are discussed. The presence of defined peptide sequences can trigger in situ hydrogelation via molecular self‐assembly and chemical crosslinking. A basic understanding of the entire spectrum of biomimetic scaffolds provides insight into how they can potentially be used in diverse tissue engineering, regenerative medicine, and drug delivery applications.     

Biomimetic Structures: Biological Implications of Dipeptide‐Substituted Polyphosphazene–Polyester Blend Nanofiber Matrices for Load‐Bearing Bone Regeneration

Successful bone regeneration benefits from three‐dimensional (3D) bioresorbable scaffolds that mimic the hierarchical architecture and mechanical characteristics of native tissue extracellular matrix (ECM). A scaffold platform that integrates unique material chemistry with nanotopography while mimicking the 3D hierarchical bone architecture and bone mechanics is reported. A biocompatible dipeptide polyphosphazene‐polyester blend is electrospun to produce fibers in the diameter range of 50–500 nm to emulate dimensions of collagen fibrils present in the natural bone ECM. Various electrospinning and process parameters are optimized to produce blend nanofibers with good uniformity, appropriate mechanical strength, and suitable porosity. Biomimetic 3D scaffolds are created by orienting blend nanofiber matrices in a concentric manner with an open central cavity to replicate bone marrow cavity, as well as the lamellar structure of bone. This biomimicry results in scaffold stress–strain curve similar to that of native bone with a compressive modulus in the mid‐range of values for human trabecular bone. Blend nanofiber matrices support adhesion and proliferation of osteoblasts and show an elevated phenotype expression compared to polyester nanofibers. Furthermore, the 3D structure encourages osteoblast infiltration and ECM secretion, bridging the gaps of scaffold concentric walls during in vitro culture. The results also highlight the importance of in situ ECM secretion by cells in maintaining scaffold mechanical properties following scaffold degradation with time. This study for the first time demonstrates the feasibility of developing a mechanically competent nanofiber matrix via a biomimetic strategy and the advantages of polyphosphazene blends in promoting osteoblast phenotype progression for bone regeneration.     

Nanostructured Biomaterials for Regeneration

Biomaterials play a pivotal role in regenerative medicine, which aims to regenerate and replace lost/dysfunctional tissues or organs. Biomaterials (scaffolds) serve as temporary 3D substrates to guide neo tissue formation and organization. It is often beneficial for a scaffolding material to mimic the characteristics of extracellular matrix (ECM) at the nanometer scale and to induce certain natural developmental or/and wound healing processes for tissue regeneration applications. This article reviews the fabrication and modification technologies for nanofibrous, nanocomposite, and nanostructured drug‐delivering scaffolds. ECM‐mimicking nanostructured biomaterials have been shown to actively regulate cellular responses including attachment, proliferation, differentiation, and matrix deposition. Nanoscaled drug delivery systems can be successfully incorporated into a porous 3D scaffold to enhance the tissue regeneration capacity. In conclusion, nanostructured biomateials are a very exciting and rapidly expanding research area, and are providing new enabling technologies for regenerative medicine.     

Wet‐Spun Biodegradable Fibers on Conducting Platforms: Novel Architectures for Muscle Regeneration

Novel biosynthetic platforms supporting ex vivo growth of partially differentiated muscle cells in an aligned linear orientation that is consistent with the structural requirements of muscle tissue are described. These platforms consist of biodegradable polymer fibers spatially aligned on a conducting polymer substrate. Long multinucleated myotubes are formed from differentiation of adherent myoblasts, which align longitudinally to the fiber axis to form linear cell‐seeded biosynthetic fiber constructs. The biodegradable polymer fibers bearing undifferentiated myoblasts can be detached from the substrate following culture. The ability to remove the muscle cell‐seeded polymer fibers when required provides the means to use the biodegradable fibers as linear muscle‐seeded scaffold components suitable for in vivo implantation into muscle. These fibers are shown to promote differentiation of muscle cells in a highly organized linear unbranched format in vitro and thereby potentially facilitate more stable integration into recipient tissue, providing structural support and mechanical protection for the donor cells. In addition, the conducting substrate on which the fibers are placed provides the potential to develop electrical stimulation paradigms for optimizing the ex vivo growth and synchronization of muscle cells on the biodegradable fibers prior to implantation into diseased or damaged muscle tissue.     

Deciphering,Designing, and Realizing Self‐Folding Biomimetic Microstructures Using a Mass‐Spring Model and Inkjet‐Printed,Self‐Folding Hydrogels

Flat, organic microstructures that can self‐fold into 3D microstructures are promising for tissue regeneration, for being capable of distributing living cells in 3D while forming highly complex, biomimetic architectures to assist cells in performing regeneration. However, the design of self‐folding microstructures is difficult due to a lack of understanding of the underlying formation mechanisms. This study helps bridge this gap by deciphering the dynamics of the self‐folding using a mass‐spring model. This numerical study reveals that self‐folding procedure is multi‐modal, which can become random and unpredictable by involving the interplays between internal stresses, external stimulation, imperfection, and self‐hindrance of the folding body. To verify the numerical results, bilayered, hydrogel‐based micropatterns capable of self‐folding are fabricated using inkjet‐printing and tested. The experimental and numerical results are consistent with each other. The above knowledge is applied to designing and fabricating self‐folding microstructures for tissue‐engineering, which successfully creates 3D, cell‐scaled, and biomimetic microstructures, such as microtubes, branched microtubes, and hollow spheres. Embedded in self‐folded microtubes, human mesenchymal stem cells proliferate and form linear cell‐organization mimicking the cell morphology in muscles and tendons. The above knowledge and study platforms can greatly contribute to the research on self‐folding microstructures and applications to tissue regeneration.     

Screening of Nanocomposite Scaffolds Arrays Using Superhydrophobic‐Wettable Micropatterns

Platforms containing multiple arrays for high‐throughput screening are demanded in the development of biomaterial libraries. Here, an array platform for the combinatorial analysis of cellular interactions and 3D porous biomaterials is described. Using a novel method based on computer‐aided manufacturing, wettable regions are printed on superhydrophobic surfaces, generating isolated spots. This freestanding benchtop array is used as a tool to deposit naturally derived polymers, chitosan and hyaluronic acid, with bioactive glass nanoparticles (BGNPs) to obtain a scaffold matrix. The effect of fibronectin adsorption on the scaffolds is also tested. The biomimetic nanocomposite scaffolds are shown to be osteoconductive, non‐cytotoxic, promote cell adhesion, and regulate osteogenic commitment. The method proves to be suitable for screening of biomaterials in 3D cell cultures as it can recreate a multitude of combinations on a single platform and identify the optimal composition that drives to desired cell responses. The platforms are fully compatible with commercially routine cell culture labware and established characterization methods, allowing for a standard control and easy adaptability to the cell culture environment. This study shows the value of 3D structured array platforms to decode the combinatorial interactions at play in cell microenvironments.     

3D Molecularly Functionalized Cell‐Free Biomimetic Scaffolds for Osteochondral Regeneration

Clinically, cartilage damage is frequently accompanied with subchondral bone injuries caused by disease or trauma. However, the construction of biomimetic scaffolds to support both cartilage and subchondral bone regeneration remains a great challenge. Herein, a novel strategy is adopted to realize the simultaneous repair of osteochondral defects by employing a self‐assembling peptide hydrogel (SAPH) FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine) to coat onto 3D‐printed polycaprolactone (PCL) scaffolds. Results show that the SAPH‐coated PCL scaffolds exhibit highly improved hydrophilicity and biomimetic extracellular matrix (ECM) structures compared to PCL scaffolds. In vitro experiments demonstrate that the SAPH‐coated PCL scaffolds promote the proliferation and osteogenic differentiation of rabbit bone mesenchymal stem cells (rBMSCs) and maintain the chondrocyte phenotypes. Furthermore, 3% SAPH‐coated PCL scaffolds significantly induce simultaneous regeneration of cartilage and subchondral bone after 8‐ and 12‐week implantation in vivo, respectively. Mechanistically, by virtue of the enhanced deposition of ECM in SAPH‐coated PCL scaffolds, SAPH with increased stiffness facilitates and remodels the microenvironment around osteochondral defects, which may favor simultaneous dual tissue regeneration. These findings indicate that the 3% SAPH provides efficient and reliable modification on PCL scaffolds and SAPH‐coated PCL scaffolds appear to be a promising biomaterial for osteochondral defect repair.     

Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs

Bioprinting is the most convenient microfabrication method to create biomimetic three‐dimensional (3D) cardiac tissue constructs, that can be used to regenerate damaged tissue and provide platforms for drug screening. However, existing bioinks, which are usually composed of polymeric biomaterials, are poorly conductive and delay efficient electrical coupling between adjacent cardiac cells. To solve this problem, a gold nanorod (GNR)‐incorporated gelatin methacryloyl (GelMA)‐based bioink is developed for printing 3D functional cardiac tissue constructs. The GNR concentration is adjusted to create a proper microenvironment for the spreading and organization of cardiac cells. At optimized concentrations of GNR, the nanocomposite bioink has a low viscosity, similar to pristine inks, which allows for the easy integration of cells at high densities. As a result, rapid deposition of cell‐laden fibers at a high resolution is possible, while reducing shear stress on the encapsulated cells. In the printed GNR constructs, cardiac cells show improved cell adhesion and organization when compared to the constructs without GNRs. Furthermore, the incorporated GNRs bridge the electrically resistant pore walls of polymers, improve the cell‐to‐cell coupling, and promote synchronized contraction of the bioprinted constructs. Given its advantageous properties, this gold nanocomposite bioink may find wide application in cardiac tissue engineering.