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.
Multimaterial additive manufacturing or three-dimensional (3D) printing of hydrogel structures provides the opportunity to engineer geometrically dependent functionalities. However, current fabrication methods are mostly limited to one type of material or only provide one type of functionality. In this paper, we report a novel method of multimaterial deposition of hydrogel structures based on an aspiration-on-demand protocol, in which the constitutive multimaterial segments of extruded filaments were first assembled in liquid state by sequential aspiration of inks into a glass capillary, followed by in situ gel formation. We printed different patterned objects with varying chemical, electrical, mechanical, and biological properties by tuning process and material related parameters, to demonstrate the abilities of this method in producing heterogeneous and multi-functional hydrogel structures. Our results show the potential of proposed method in producing heterogeneous objects with spatially controlled functionalities while preserving structural integrity at the switching interface between different segments. We anticipate that this method would introduce new opportunities in multimaterial additive manufacturing of hydrogels for diverse applications such as biosensors, flexible electronics, tissue engineering and organ printing.
In this research, a novel development of bioink from cell sheets is presented for scaffold free bioprinting applications. Poly(N-isopropylacrylamide) (PNIPAAm) coated surfaces were first prepared by using initiated chemical vapor deposition method. Cell-sheets were then grown on these thermoresponsive pNIPAAm coated surfaces and easily detached without disturbing delicate cell-extracellular matrix (ECM) and cell-cell interactions. The detached cells sheets were used to prepare cell sheet based bioink and bioprinted to form various shapes. The results showed that the prepared cell-sheet based bioink shows an increase in the structural integrity compare to cell-aggregates suggesting that the cell sheet aggregates preserved interconnected ECM proteins. The viability of cell sheet based aggregates was also compared to single cell aggregates at three different time points in a seven-day period after printing. The developed cell-sheet based bioink has several advantages in terms of shape fidelity, reproducibility and automated deposition for bioprinting applications. The results also demonstrated that the bioprinted constructs secreted collagen type I which is a strong indication of starting ECM deposition. Moreover, the immunostaining results showed that the fibronectin in cell sheets was preserved during and after the preparation process of bioink.
of the matrix. [9] Mechanical properties such as the stiffness of the 3D surrounding environment are known to affect differentiation of certain cells, [10] and are believed to be critical for neuronal maturation and neurotransmission. [9] More discrete and higher resolved structures, such as those made with additive manufacturing (AM) [11] and electrojetting technology, [12] are promising to provide reproducible conditions. Electrojetting uses electrostatic forces to fabricate monodispersed, nanomicro particles in a simple, versatile, and cost-effective method for drug delivery and tissue engineering applications. [13] Electrospinning allows processability for polymer solutions and polymer melts. [14] In addition, polymer melts can be directly written using a programmable path in a technique known as melt electrowriting (MEW). [15] The precise placement of low-micrometer diameter fibers that are stackable using additive manufacturing principles are achievable using MEW (Figure 1a,b). The advantages of MEW include solvent-free processing and avoidance of the chaotic fiber deposition often seen in electrospinning. Bioprinting approaches for cell hierarchy have been reinforced with extruded, stiffer structures [16] that reinforce the bioink and aid with in vitro handling [17] and extend a processing window for such hydrogels. [16,17] The reinforcing of matrices and bioinks with much smaller, low-micrometer scale fibers on matrices has recently been of particular interest. [18,19] Using MEW, [15] well-ordered, small diameter fibers can be distributed throughout a matrix in low volume fractions and provide significant increase in overall mechanical properties. [18][19][20] Furthermore, the mechanics of MEW fiber-reinforced hydrogels can be modulated further with sinusoidal direct-writing of the fibers. [18] Since the fiber diameter made using MEW can be readily changed with the nozzle mass flow rate and/or the collection speed, the diameter of the printed fiber can be significantly altered. [21] Therefore, MEW reinforcement of matrices (such as Matrigel shown here) have the potential to regulate the environment of cells through both mechanotransductive [22] or haptotaptic [23] cues.An experimentally designed in vitro 3D structure for electrophysiological studies requires a relevant receptor model, with the inhibitory glycine receptor (GlyR) used in this study. The GlyR is a pentameric, ligand gated ion channel, which belongs to the superfamily of Cys-loop receptors. [24] Upon binding of 2D electrophysiology is often used to determine the electrical properties of neurons. In the brain however, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, 3D electrophysiology is established within a fiber-reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Usin...
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