Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death.

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.

3D Bioprinting of Collagen-based Microfluidics for Engineering Fully-biologic Tissue Systems.

Microfluidic and organ-on-a-chip devices have improved the physiologic and translational relevance of in vitro systems in applications ranging from disease modeling to drug discovery and pharmacology. However, current manufacturing approaches have limitations in terms of materials used, non-native mechanical properties, patterning of extracellular matrix (ECM) and cells in 3D, and remodeling by cells into more complex tissues. We present a method to 3D bioprint ECM and cells into microfluidic collagen-based high-resolution internally perfusable scaffolds (CHIPS) that address these limitations, expand design complexity, and simplify fabrication. Additionally, CHIPS enable size-dependent diffusion of molecules out of perfusable channels into the surrounding device to support cell migration and remodeling, formation of capillary-like networks, and integration of secretory cell types to form a glucose-responsive, insulin-secreting pancreatic-like microphysiological system.

Primary Glial Cell and Glioblastoma Morphology in Cocultures Depends on Scaffold Design and Hydrogel Composition.

3D cell cultures better replicate the in vivo environment compared to 2D models. Glioblastoma multiforme, a malignant brain tumor, highly profits from its cellular environment. Here, the U87 glioblastoma cell line in the presence/absence of primary astrocytes is studied. Thiolated hyaluronic acid (HA-SH) hydrogel reinforced with microfiber scaffolds is compared to Matrigel. Hyaluronic acid is a major extracellular matrix (ECM) component in the brain. Poly(ɛ-caprolactone) (PCL) scaffolds are written by meltelectrowriting in a box and triangular shaped design with pore sizes of 200 µm. Scaffolds are composed of 10-layers of PCL microfibers. It is found that scaffold design has an impact on cellular morphology in the absence of hydrogel. Moreover, the used hydrogels have profound influences on cellular morphology resulting in spheroid formation in HA-SH for both the tumor-derived cell line and astrocytes, while cell viability is high. Although cocultures of U87 and astrocytes exhibit cell-cell interactions, polynucleated spheroid formation is still present for U87 cells in HA-SH. Locally restricted ECM production or inability to secrete ECM proteins may underlie the observed cell morphologies. Thus, the 3D reinforced PCL-HA-SH composite with glioma-like cells and astrocytes constitutes a reproducible system to further investigate the impact of hydrogel modifications on cellular behavior and development.

Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation.

3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30-500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca2+ imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases.

Brain and Breast Cancer Cells with PTEN Loss of Function Reveal Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Utilizing 3D Scaffolds and Aligned Microfiber Tracts.

BACKGROUND: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. METHODS: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. RESULTS: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. CONCLUSIONS: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.

Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix-A Model System to Study Disease Mechanisms.

3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease.

Melt Electrospinning of Nanofibers from Medical-Grade Poly(ε-Caprolactone) with a Modified Nozzle.

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.

The importance of plasmin for the healing of the anterior cruciate ligament.

AIMS: The anterior cruciate ligament (ACL) is known to have a poor wound healing capacity, whereas other ligaments outside of the knee joint capsule such as the medial collateral ligament (MCL) apparently heal more easily. Plasmin has been identified as a major component in the synovial fluid that varies among patients. The aim of this study was to test whether plasmin, a component of synovial fluid, could be a main factor responsible for the poor wound healing capacity of the ACL. METHODS: The effects of increasing concentrations of plasmin (0, 0.1, 1, 10, and 50 µg/ml) onto the wound closing speed (WCS) of primary ACL-derived ligamentocytes (ACL-LCs) were tested using wound scratch assay and time-lapse phase-contrast microscopy. Additionally, relative expression changes (quantitative PCR (qPCR)) of major LC-relevant genes and catabolic genes were investigated. The positive controls were 10% fetal calf serum (FCS) and platelet-derived growth factor (PDGF). RESULTS: WCS did not differ significantly among no plasmin versus each of the tested concentrations (six donors). The positive controls with PDGF and with FCS differed significantly from the negative controls. However, we found a trend demonstrating that higher plasmin concentrations up-regulate the expression of matrix metalloproteinase 13 (MMP13), 3 (MMP3), and tenomodulin (TNMD). CONCLUSION: The clinical relevance of this study is the possibility that it is not solely the plasmin, but also additional factors in the synovial fluid of the knee, that may be responsible for the poor healing capacity of the ACL.Cite this article: Bone Joint Res 2020;9(9):543-553.

Melt Electrowritten In Vitro Radial Device to Study Cell Growth and Migration.

The development of in vitro assays for 3D microenvironments is essential for understanding cell migration processes. A 3D-printed in vitro competitive radial device is developed to identify preferred Matrigel concentration for glioblastoma migration. Melt electrowriting (MEW) is used to fabricate the structural device with defined and intricate radial structures that are filled with Matrigel. Controlling the printing path is necessary to account for the distance lag in the molten jet, the applied electric field, and the continuous direct-writing nature of MEW. Circular printing below a diameter threshold results in substantial inward tilting of the MEW fiber wall. An eight-chamber radial device with a diameter of 9.4 mm is printed. Four different concentrations of Matrigel are dispensed into the radial chambers. Glioblastoma cells are seeded into the center and grow into all chambers within 8 days. The cell spreading area demonstrates that 6 and 8 mg mL-1 of Matrigel are preferred over 2 and 4 mg mL-1 . Furthermore, topographical cues via the MEW fiber wall are observed to promote migration even further away from the cell seeding depot. Previous studies implement MEW to fabricate cell invasive scaffolds whereas here it is applied to 3D-print in vitro tools to study cell migration.

Cortical Neurons form a Functional Neuronal Network in a 3D Printed Reinforced Matrix.

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.

3D Electrophysiological Measurements on Cells Embedded within Fiber-Reinforced Matrigel.

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. Using melt electrowriting (MEW) of scaffolds to reinforce Matrigel, 3D electrophysiology is performed on a glycine receptor-transfected Ltk-11 mouse fibroblast cell line. The glycine receptor is an inhibitory ion channel associated when mutated with impaired neuromotor behavior. The average thickness of the MEW scaffold is 141.4 ± 5.7 µm, using 9.7 ± 0.2 µm diameter fibers, and square pore spacings of 100, 200, and 400 µm. For the first time, the electrophysiological characterization of glycine receptor-transfected cells is demonstrated with respect to agonist efficacy and potency in a 3D matrix. With the MEW scaffold reinforcement not interfering with the electrophysiological measurement, this approach can now be further adapted and developed for different kinds of neuronal cultures to study and understand pathological mechanisms under disease conditions.

Development of Bioink from Decellularized Tendon Extracellular Matrix for 3D Bioprinting.

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.

Genipin-Enhanced Fibrin Hydrogel and Novel Silk for Intervertebral Disc Repair in a Loaded Bovine Organ Culture Model.

(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.

Three-dimensional direct cell bioprinting for tissue engineering.

Bioprinting is a relatively new technology where living cells with or without biomaterials are printed layer-by-layer in order to create three-dimensional (3D) living structures. In this article, novel bioprinting methodologies are developed to fabricate 3D biological structures directly from computer models using live multicellular aggregates. Multicellular aggregates made out of at least two cell types from fibroblast, endothelial and smooth muscle cells are prepared and optimized. A novel bioprinting approach is proposed in order to continuously extrude cylindrical multicellular aggregates through the bioprinter's glass microcapillaries. The multicellular aggregates are first aspirated into a capillary and then compressed to form a continuous cylindrical multicellular bioink. To overcome surface tension-driven droplet formation, the required compression ratio is calculated. Based on the developed bioprinting strategies, multicellular aggregates and their support structures are bioprinted to form 3D tissue constructs with predefined shapes. The effect of the bioprinting process was examined for fusion, cell viability at different compression ratios, and f-actin cytoskeletal organization. The results show that the bioprinted 3D constructs fuse rapidly and have high cell viability after printing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2530-2544, 2017.

Multifunctional 3D printing of heterogeneous hydrogel structures.

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.