Using decellularized extracellular matrix (dECM) hydrogels as bioinks has been an important step forward for bioprinting of functional tissue constructs, considering their rich microenvironment and their high degree of biomimicry. However, directly using dECM hydrogels as bioinks may not be suitable for bioprinting processes because of the loss of shape fidelity and geometrical precision of bioprinted structure due to their slow gelation kinetics. In this article, the development and direct bioprinting of dECM hydrogel bioink from bovine Achilles tendon were presented. The developed bioink is used for a microcapillary-based bioprinting process without any support structure and/or any additional cross-linker components. The reported decellularization and solubilization methods yield dECM pre-gels which turn into stable hydrogels in a short time at physiological conditions. The gelation kinetics and mechanical strength of bioinks with different concentrations and digestion times are characterized. A support structure-free 3D bioprinting of the developed bioink is shown by aspirating dECM bioinks and then in situ gelation and extrusion through a fine microcapillary nozzle. The viability assays indicate that the developed dECM bioink has no cytotoxic effect on encapsulated NIH 3T3 cells and the cells show lineage-specific morphology in the early days of culture as well.

Melt electrospun fibers, in general, have larger diameters than normally achieved with solution electrospinning. This study uses a modified nozzle to direct‐write melt electrospun medical‐grade poly(ε‐caprolactone) onto a collector resulting in fibers with the smallest average diameter being 275 ± 86 nm under certain processing conditions. Within a flat‐tipped nozzle is a small acupuncture needle positioned so that reduces the flow rate to ≈0.1 µL h−1 and has the sharp tip protruding beyond the nozzle, into the Taylor cone. The investigations indicate that 1‐mm needle protrusion coupled with a heating temperature of 120 °C produce the most consistent, small diameter nanofibers. Using different protrusion distances for the acupuncture needle results in an unstable jet that deposited poor quality fibers that, in turn, affects the next adjacent path. The material quality is notably affected by the direct‐writing speed, which became unstable above 10 mm min−1. Coupled with a dual head printer, first melt electrospinning, then melt electrowriting could be performed in a single, automated process for the first time. Overall, the approach used here resulted in some of the smallest melt electrospun fibers reported to date and the smallest diameter fibers from a medical‐grade degradable polymer using a melt processing technology.

Melt electrowriting (MEW) is a direct‐writing technology for small diameter fibers; however, due to electrostatic attraction, the technique is restricted in how close these microfibers can be positioned on the collector. Here, the minimum interfiber distance between parallel poly(ε‐caprolactone) MEW microfibers is determined for different fiber diameters and number of layers on noncoated and star‐shaped poly(ethylene oxide‐stat‐propylene oxide) (sP(EO‐stat‐PO))‐coated glass coverslips. The effect of the fiber diameter, the number of fiber layers, and shape of turning loops affect precision and the minimum interfiber distance. Single fibers with diameter of 5, 10, and 15 µm have a minimum interfiber distance without fiber bridging of 33 ± 2.7, 54 ± 2.2, and 62 ± 2.7 µm, respectively. Increasing the number of layers to ten increases this minimum interfiber distance approximately twofold to 60 ± 3.5, 97 ± 4.5, and 102 ± 2.7 µm for the increasing fiber diameters. The sP(EO‐stat‐PO) slightly increases the minimum interfiber distance for the 15 µm diameter group only, with spacing for the 5 and 10 µm fibers unaffected by the coating. Identifying and determining the fabrication limits for MEW is highly instructional for users working and designing scaffolds with this technology.

(1) Background: Intervertebral disc (IVD) repair represents a major challenge. Using functionalised biomaterials such as silk combined with enforced hydrogels might be a promising approach for disc repair. We aimed to test an IVD repair approach by combining a genipin-enhanced fibrin hydrogel with an engineered silk scaffold under complex load, after inducing an injury in a bovine whole organ IVD culture; (2) Methods: Bovine coccygeal IVDs were isolated from ~1-year-old animals within four hours post-mortem. Then, an injury in the annulus fibrosus was induced by a 2 mm biopsy punch. The repair approach consisted of genipin-enhanced fibrin hydrogel that was used to fill up the cavity. To seal the injury, a Good Manufacturing Practise (GMP)-compliant engineered silk fleece-membrane composite was applied and secured by the cross-linked hydrogel. Then, IVDs were exposed to one of three loading conditions: no load, static load and complex load in a two-degree-of-freedom bioreactor for 14 days. Followed by assessing DNA and matrix content, qPCR and histology, the injured discs were compared to an uninjured control IVD that underwent the same loading profiles. In addition, the genipin-enhanced fibrin hydrogel was further investigated with respect to cytotoxicity on human stem cells, annulus fibrosus, and nucleus pulposus cells; (3) Results: The repair was successful as no herniation could be detected for any of the three loading conditions. Disc height was not recovered by the repair DNA and matrix contents were comparable to a healthy, untreated control disc. Genipin resulted being cytotoxic in the in vitro test but did not show adverse effects when used for the organ culture model; (4) Conclusions: The current study indicated that the combination of the two biomaterials, i.e., genipin-enhanced fibrin hydrogel and an engineered silk scaffold, was a promising approach for IVD repair. Furthermore, genipin-enhanced fibrin hydrogel was not suitable for cell cultures; however, it was highly applicable as a filler material.

Impairments in neuronal circuits underly multiple neurodevelopmental and neurodegenerative disorders. 3D cell culture models enhance the complexity of in vitro systems and provide a microenvironment closer to the native situation than with 2D cultures. Such novel model systems will allow the assessment of neuronal network formation and their dysfunction under disease conditions. Here, mouse cortical neurons are cultured from embryonic day E17 within in a fiber‐reinforced matrix. A soft Matrigel with a shear modulus of 31 ± 5.6 Pa is reinforced with scaffolds created by melt electrowriting, improving its mechanical properties and facilitating the handling. Cortical neurons display enhance cell viability and the neuronal network maturation in 3D, estimated by staining of dendrites and synapses over 21 days in vitro, is faster in 3D compared to 2D cultures. Using functional readouts with electrophysiological recordings, different firing patterns of action potentials are observed, which are absent in the presence of the sodium channel blocker, tetrodotoxin. Voltage‐gated sodium currents display a current–voltage relationship with a maximum peak current at −25 mV. With its high customizability in terms of scaffold reinforcement and soft matrix formulation, this approach represents a new tool to study neuronal networks in 3D under normal and, potentially, disease conditions.