3D-bioprinting is a promising technique to mimic the complex anatomy of natural tissues, as it comprises a precise and gentle way of placing bioinks containing cells and hydrogel. Although hydrogels expose an ideal growth environment due to their extracellular matrix (ECM)-like properties, high water amount and tissue like microstructure, they lack mechanical strength and possess a diffusion limit of a couple of hundred micrometers. Integration of electrospun fibers could hereby benefit in multiple ways, for instance by controlling mechanical characteristics, cell orientation, direction of diffusion and anisotropic swelling behavior. The aim of this study was to create an advanced ECM-biomimicking scaffold material for tissue engineering, which offers enhanced diffusion properties. PCL bulk membranes were successfully electrospun and fragmented using a cryo cutting technique. Subsequently, these short single fibers (500 µm), swelling properties (20%–60% of control), and higher mechanical strength, while its viscosity (5–30 mPas*s) and gelation kinetics (28 °C) remained almost unaffected. The diffusion tests indicate a high level of size selectivity, which can be utilized for targeted biomolecule transport in the future. Finally, applying 3D-bioprinting technology (drop-on-demand vs. microextrusion) a print setting dependent post-dispensing orientation of the fibers could be induced, which ultimately paves way for the fabrication of metamaterials with anisotropic material properties. As expected, the fiber-filled bioink was found to be non-cytotoxic in cell culture trials using HUVECs and HepG2 (>80% viability). In summary, microfiber integration holds great promise for 3D-bioprinting of tissue percursors with advanced metamaterial properties and thus offers high applicability in various fields of research, such as in-vitro tissue models, tissue engineered implants or cultivated meat.

Hydrogels emerged as versatile biomaterials for tissue engineering due to their extra cellular matrix similarity and mechanical and biochemical properties. Still, hydrogels expose limited stiffness, anisotropy and nutrient diffusion. By reinforcing hydrogels with synthetic and natural fibers, these drawbacks can be effectively addressed, thereby enabling the modeling of advanced biomimetic tissue. This review discusses recent progress in the fabrication of fiber-integrated hydrogels and brings together developments from biomaterials, biofabrication, mechanobiology, and organ-model engineering. Fiber-addition impact on viscoelastic, time-dependent und nonlinear material properties, on multiscale and hierarchical constructs and on mechanical and biological readouts are analyzed. Specifically, the integration of both synthetic and natural fibers into hydrogel matrices is highlighted which significantly broaden their structural and biochemical versatility. These fiber-added hydrogels display improved properties including enhanced stiffness (up to 10-fold increase), anisotropy (>80 % alignment) and nutrient diffusion (4-fold increase). Moreover, the incorporation of fibers directly impacts cellular behavior by promoting adhesion, migration, proliferation and differentiation. Finally, bone, muscle and nerve tissue are exemplary presented in more detail to highlight the broad potential of these composite materials. In conclusion, fiber-embedded hydrogels represent a decisive step toward enhanced 4D-metamaterials.

Electrical stimulation enhances functionality and accelerates maturation in biofabricated tissues, which are particularly important for muscle tissue engineering applications. Accordingly, there is demand for 3D-printable electrically conductive cytocompatible scaffolds that enable patient-specific geometries and localized electrical stimulation, as well as incorporate further maturation-promoting geometrical cues. Filament-based scaffolds from fused filament fabrication could overcome current limitations in geometric freedom, size and partially cytotoxic additives. In this study, biodegradable polylactic acid (PLA)-based conductive filaments incorporating graphene nanoplatelets (GNPs) or carbon nanofibers (CNFs) were developed via melt-mixing extrusion to possibly enable the electrical functionalization of muscle scaffolds. A two-stage process combining twin-screw and single-screw extrusion was preferred to allow for higher filler incorporation. Filament morphology, printability, electrical conductivity, and cytocompatibility were systematically evaluated. Homogeneous filaments containing up to 16 wt.% GNPs or 3.6 wt.% CNFs were successfully produced and processed by fused filament fabrication into scaffold geometries supporting myoblast orientation. Electrical conductivity was measured above 16 wt.% GNPs, with up to 2.7 µS/m, with printed constructs capable of connecting a circuit. GNP-based filaments were cytocompatible, supporting myoblast attachment and elongated morphology. An adjustable electrical stimulation setup demonstrated improved muscle maturation and contractile responses of C2C12 myoblasts, highlighting biodegradable conductive filaments’ potential for electrically active muscle tissue scaffolds.

Biofabrication of hierarchical tissues with features ranging various size ranges and controllable anisotropy remains a challenge in 3D-bioprinting. To overcome this hurdle, the application of multi-functional microfibers acting as cell-instructive bioink additive, recently gained particular attention. In this work, we investigate a microfluidic spinning process for the fabrication of collagen microfibers with adjustable diameters ranging from 5 to 50 μm. The thread was collected on a rotating winder and fragmented into microfibers of defined length (60–300 μm). By integrating microfiber fragments into an agarose-hyaluronan hydrogel, fine-tuning of its viscosity range (10–1000 mPa∗s), and thus the precise control of the extruded strands’ diameter (0.3–1.4 mm) was achieved. While remaining strong shear-thinning behavior (n-value 0.6), E-modulus and yield stress were decreased in fiber-filled hydrogel, hinting at an interaction of agarose polymer chains with microfibers. Remarkably, the orientation of collagen microfibers could be directed either parallel or orthogonal to the printing path. This allows the biofabrication of hydrogel structures with adjustable domains of defined anisotropy. Finally, the fibers showed excellent biofunctionality both in 2D and 3D. Besides a high degree of alignment of individual cells along the microfiber axis (>80 % of cells), hMSCs built a dense, branched network in 3D. Moreover, PC12 and C2C12 were successfully differentiated in 2D and 3D. Specifically, neurite length was higher on smaller fiber diameters, even spanning non-adjacent clusters. Elongated, multi-nuclei myotubes were formed, indicating C2C12 differentiation. In summary, the work demonstrates the great potential of 3D-bioprinting in cross-scale organization of fragmented collagen microfibers.