A novel bioink and a dispensing technique for 3D tissue‐engineering applications are presented. The technique incorporates a coaxial extrusion needle using a low‐viscosity cell‐laden bioink to produce highly defined 3D biostructures. The extrusion system is then coupled to a microfluidic device to control the bioink arrangement deposition, demonstrating the versatility of the bioprinting technique. This low‐viscosity cell‐responsive bioink promotes cell migration and alignment within each fiber organizing the encapsulated cells.

The myocardium behaves like a sophisticated orchestra that expresses its true potential only if each member performs the correct task harmonically. Recapitulating its complexity within engineered 3D functional constructs with tailored biological and mechanical properties, is one of the current scientific priorities in the field of regenerative medicine and tissue engineering. In this study, driven by the necessity of fabricating advanced model of cardiac tissue, we present an innovative approach consisting of heterogeneous, multi-cellular constructs composed of Human Umbilical Vein Endothelial Cells (HUVECs) and induced pluripotent cell-derived cardiomyocytes (iPSC-CMs). Cells were encapsulated within hydrogel strands containing alginate and PEG-Fibrinogen (PF) and extruded through a custom microfluidic printing head (MPH) that allows to precisely tailor their 3D spatial deposition, guaranteeing a high printing fidelity and resolution. We obtained a 3D cardiac tissue compose of iPSC-derived CMs with a high orientation index imposed by the different defined geometries and blood vessel-like shapes generated by HUVECs which, as demonstrated by in vivo grafting, better support the integration of the engineered cardiac tissue with host’s vasculature.

We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3-5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo. Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application.

In this work we demonstrate how to print 3D biomimetic hydrogel scaffolds for cartilage tissue engineering with high cell density (>10(7) cells ml(-1)), high cell viability (85 ÷ 90%) and high printing resolution (≈100 μm) through a two coaxial-needles system. The scaffolds were composed of modified biopolymers present in the extracellular matrix (ECM) of cartilage, namely gelatin methacrylamide (GelMA), chondroitin sulfate amino ethyl methacrylate (CS-AEMA) and hyaluronic acid methacrylate (HAMA). The polymers were used to prepare three photocurable bioinks with increasing degree of biomimicry: (i) GelMA, (ii) GelMA + CS-AEMA and (iii) GelMA + CS-AEMA + HAMA. Alginate was added to the bioinks as templating agent to form stable fibers during 3D printing. In all cases, bioink solutions were loaded with bone marrow-derived human mesenchymal stem cells (BM-MSCs). After printing, the samples were cultured in expansion (negative control) and chondrogenic media to evaluate the possible differentiating effect exerted by the biomimetic matrix or the synergistic effect of the matrix and chondrogenic supplements. After 7, 14, and 21 days, gene expression of the chondrogenic markers (COL2A1 and aggrecan), marker of osteogenesis (COL1A1) and marker of hypertrophy (COL10A1) were evaluated qualitatively by means of fluorescence immunocytochemistry and quantitatively by means of RT-qPCR. The observed enhanced viability and chondrogenic differentiation of BM-MSCs, as well as high robustness and accuracy of the employed deposition method, make the presented approach a valid candidate for advanced engineering of cartilage tissue.

Skeletal muscle tissue engineering (SMTE) aims at repairing defective skeletal muscles. Until now, numerous developments are made in SMTE; however, it is still challenging to recapitulate the complexity of muscles with current methods of fabrication. Here, after a brief description of the anatomy of skeletal muscle and a short state‐of‐the‐art on developments made in SMTE with “conventional methods,” the use of 3D bioprinting as a new tool for SMTE is in focus. The current bioprinting methods are discussed, and an overview of the bioink formulations and properties used in 3D bioprinting is provided. Finally, different advances made in SMTE by 3D bioprinting are highlighted, and future needs and a short perspective are provided.

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

We demonstrate how to generate highly ordered porous matrices from dextran-methacrylate (DEX-MA) using microfluidics. We use a flow focusing device to inject an aqueous solution of DEX-MA and surfactant to break the flow of an organic solvent (cyclohexane) into monodisperse droplets at a high volume fraction (above 74% v/v) to form an ordered high internal phase emulsion (HIPE). We collect the crystalline HIPE structure and freeze it by gelling. The resulting polyHIPEs are characterized by an interconnected and ordered morphology. The size of pores and interconnects ranges between hundreds and tens of micrometers, respectively. The technique that we describe allows for precise tuning of all the structural parameters of the matrices, including their porosity, the size of the pores and the lumen of interconnects between the pores. The resulting ordered and precisely tailored HIPE gels represent a new class of scaffolds for applications in tissue engineering

Fiber‐based approaches hold great promise for tendon tissue engineering enabling the possibility of manufacturing aligned hydrogel filaments that can guide collagen fiber orientation, thereby providing a biomimetic micro‐environment for cell attachment, orientation, migration, and proliferation. In this study, a 3D system composed of cell‐laden, highly aligned hydrogel yarns is designed and obtained via wet spinning in order to reproduce the morphology and structure of tendon fascicles. A bioink composed of alginate and gelatin methacryloyl (GelMA) is optimized for spinning and loaded with human bone morrow mesenchymal stem cells (hBM‐MSCs). The produced scaffolds are subjected to mechanical stretching to recapitulate the strains occurring in native tendon tissue. Stem cell differentiation is promoted by addition of bone morphogenetic protein 12 (BMP‐12) in the culture medium. The aligned orientation of the fibers combined with mechanical stimulation results in highly preferential longitudinal cell orientation and demonstrates enhanced collagen type I and III expression. Additionally, the combination of biochemical and mechanical stimulations promotes the expression of specific tenogenic markers, signatures of efficient cell differentiation towards tendon. The obtained results suggest that the proposed 3D cell‐laden aligned system can be used for engineering of scaffolds for tendon regeneration.