The graphene used in this study was developed at Kirklareli University, Department of Mechanical Engineering. It was donated by Dr. Karabeyo&#287;lu. The graphene toxicity test was financed by the project titled &#34;Printing and Differentiation of Mesenchymal Stem Cells on 3D Bioprinters with Graphene Doped Bioinks&#34; (Application No: 1139B411802273) completed within the scope of T&#220;B&#304;TAK 2209-B-Industry-Oriented Undergraduate Thesis Support Program. The other part of the study was supported by the research fund provided by Yildiz Technical University Scientific Research Projects (TSA-2021-4713). Mesenchymal stem cells with GFP used in the time-lapse imaging stage were donated by Virostem Biotechnology. The authors thank Dar&#305;c&#305; LAB and YTU The Cell Culture and Tissue Engineering LAB team for productive discussions.

<ol>
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The authors declare that there is no conflict of interest. The project was performed in collaboration with HD Bioink, developer of the 3D bioprinting technology.

The advantages of treatments applied with engineered 3D scaffolds over conventional 2D methods are becoming more and more noticeable every day. Stem cells used alone in these therapies or along with scaffolds produced from various biomaterials with low biocompatibility and biodegradability are usually inadequate in peripheral nerve regeneration. Wharton's jelly mesenchymal stem cells (WJ-MSCs) seem to be a suitable candidate cell line, especially considering the optimization of the protocols for acquisition, their prolif...

The nervous system, which is the mechanism that bridges the internal structure of the organism and the environment, is divided into two parts: the central and peripheral nervous systems. Peripheral nerve damage is a global problem that constitutes 1.5%-5% of the patients who present to the emergency department and develops due to various traumas, leading to significant job loss1,2,3.
Today, cellular approaches to peripheral neuro-engineering are of great interest. Stem cells come first among the cells used in these approaches. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these cells are specialized but can differentiate upon appropriate stimulation in response to injury4,5. According to the stem cell hypothesis, the stem cell system is under the influence of its microenvironment, called the stem cell niche. The preservation and differentiation of stem cells are impossible without the presence of their microenvironment6, which can be reconstituted via tissue engineering using cells and scaffolds7. Tissue engineering is a multidisciplinary field that includes both engineering and biology principles. Tissue engineering provides tools for the creation of artificial tissues that can replace living tissues and can be used in the regeneration of these tissues by removing the damaged tissues and providing functional tissues8. Tissue scaffolds, one of the three cornerstones of tissue engineering, are produced using different methods from natural and synthetic materials9. Three-dimensional (3D) printing is an emerging additive manufacturing technology that is widely used to replace or restore defective tissues via its simple but versatile production of complex shapes using various methods. Bioprinting is an additive manufacturing method that enables the coexistence of cells and biomaterials, called bioinks10. Considering the interaction of nerve cells with each other, studies have shifted to conductive biomaterial candidates such as graphene. Graphene nanoplates, which have properties such as flexible electronics, supercapacitors, batteries, optics, electrochemical sensors, and energy storage, are a preferred biomaterial in the field of tissue engineering11. Graphene has been used in studies where the proliferation and regeneration of damaged tissues and organs were performed12,13.
Tissue engineering consists of three basic building blocks: scaffold, cells, and biosignal molecules. There are deficiencies in the studies on peripheral nerve damage in terms of providing these three structures completely. Various problems have been encountered in the biomaterials produced and used in the studies, such as them containing only stem cells or biosignal molecules, the lack of a bioactive molecule that will enable stem cell differentiation, the lack of biocompatibility of the biomaterial used, and the low effect on the proliferation of cells in the tissue niche, and, thus, nerve conduction not being fully realized2,13,14,15,16.&#160;This requires the optimization of nerve regeneration, reducing muscle atrophy17,18, and creating necessary homing19 with growth factors against such problems.At this point, the characterization and analysis of the neuro-activity of a surgical biomaterial prototype, to be transferred to the clinic, are very important.
Accordingly, this methods study investigates the bioink hydrogel patterning with graphene nanoplates formed by a 3D bioprinter and its effectiveness on the neurogenic differentiation of the stem cells it contains. Also, the effects of graphene on neurosphere formation and differentiation are investigated.

<table><tbody><tr><td>&lt;br/&gt; Centrifugal</td><td>Hitachi</td><td></td><td>Used in cell culture and biomaterial step</td></tr><tr><td>0.1N CaCl2</td><td>HD Bioink</td><td></td><td>Used for crosslinker</td></tr><tr><td>0.22 &amp;micro;m membrane filter</td><td>A&amp;iota;s&amp;iota;mo</td><td></td><td>Used for sterilization</td></tr><tr><td>0.45 &amp;micro;m syringe filter</td><td>A&amp;iota;s&amp;iota;mo</td><td></td><td>Used for sterilization</td></tr><tr><td>1.5mL conic tube</td><td>Eppendorfa</td><td></td><td>Used for bioink drop</td></tr><tr><td>15mL Falcon tube</td><td>Nest</td><td></td><td>Used in cell culture step</td></tr><tr><td>25 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td><td>Nest</td><td></td><td>Used for cell culture</td></tr><tr><td>3D Bioprinting</td><td>Axolotl Biosystems Bio A2 (Turkey)</td><td></td><td>Bioprinting Step</td></tr><tr><td>50 mL Falcon tube</td><td>Nest</td><td></td><td>Used in cell culture step</td></tr><tr><td>6/24/48/96 well plates (Falcon, TPP microplates)</td><td>Merck Millipore</td><td></td><td>Used in cell culture step</td></tr><tr><td>75 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td><td>Nest</td><td></td><td>Used for cell culture</td></tr><tr><td>Anti mouse IgG-FTIC-rabbit</td><td>Santa Cruz Biotechnology</td><td>J1514</td><td>Seconder antibody, used for dye</td></tr><tr><td>Anti mouse IgG-SC2781-goat</td><td>Santa Cruz Biotechnology</td><td>C3109</td><td>Seconder antibody, used for dye</td></tr><tr><td>Au coating device EM ACE600</td><td>Leica</td><td></td><td>for gold plating of biomaterial section before SEM imaging</td></tr><tr><td>Autoclave</td><td>NUVE-OT 90L</td><td></td><td>Used for the sterilization process.</td></tr><tr><td>Autoclave</td><td>NUVE-OT 90L</td><td></td><td>Used for the sterilization process.</td></tr><tr><td>Cell Cultre Cabine</td><td>Hera Safe KS</td><td></td><td>Used for the cell culture process</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium/Nutrient Mixture-F12</td><td>Sigma</td><td>RNBJ7249</td><td>Used as cell culture medium</td></tr><tr><td>FEI QUANTA 450 FEG ESEM SEM</td><td>Quanta</td><td>FEG 450</td><td>for SEM</td></tr><tr><td>Fetal Bovine Serum-FBS</td><td>Capricorn</td><td>FBS-16A</td><td>It was used by adding to the cell culture medium.</td></tr><tr><td>Freezer -80&amp;deg;C</td><td>Panasonic</td><td>MDF-U5386S-PE</td><td>We were used to store cells and the resulting exosomes</td></tr><tr><td>Gelatine-Alginate bioink powder</td><td>HD Bioink</td><td></td><td>Used for produced bioink step</td></tr><tr><td>GFP labelled-WJ-MSCs</td><td>Virostem</td><td></td><td>Used for imaging to cell-bioink interaction</td></tr><tr><td>Graphene nanoplatelets (Graphene-IGP2)</td><td>Grafen Chemical Industries Co.</td><td></td><td>Used for production 3D-G bioink</td></tr><tr><td>Immunofluorescence antibodies (N-CAD; &amp;beta;-III Tubulin)</td><td>Cell Signalling and Santa Cruz</td><td></td><td>Used for dye</td></tr><tr><td>JASCO 6600</td><td>Tetra</td><td></td><td>for FTIR</td></tr><tr><td>MTT Assay</td><td>Sigma</td><td></td><td>Viability testing</td></tr><tr><td>Penicilin/Streptomycin Solution</td><td>Capricorn</td><td>PB-S</td><td>It was added to the medium to prevent contamination in cell culture.</td></tr><tr><td>Thoma slide</td><td>Isolab</td><td></td><td>Used for counting the cell</td></tr><tr><td>Time-Lapse Imaging System</td><td>Zeiss Axio.Observer.Z1</td><td></td><td>Imaging</td></tr><tr><td>Tripsin-EDTA</td><td>Multicell</td><td></td><td>The flask was used to remove the cells covering the surface.</td></tr><tr><td>Vorteks</td><td>Biobase</td><td></td><td>For produced bioink step</td></tr><tr><td>WJ-MSCs</td><td>ATCC</td><td></td><td>Used for the cell culture process</td></tr></tbody></table>

preparation and characterization of graphene-based 3d biohybrid hydrogel bioink for peripheral neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

Graphene toxicity and 2D imaging 
Statistical analysis of the obtained MTT results was conducted with a one-way ANOVA with Tukey&#39;s test in statistical analysis software, and the graph obtained is shown in Figure 2. The graphene percentage compared to control showed a significant decrease only for the 0.001% graphene concentration (**p &#60; 0.01).. There were no significant differences between the other groups and the control (p &#62; 0.05). Therefore, the optimum g...

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Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

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2.1K Views.  Istinye University. Het stamcelsysteem staat onder invloed van zijn belangrijke componenten. Het behoud en de differentiatie van stamcellen is ondenkbaar zonder de aanwezigheid van hun micro-omgeving. Deze micro-omgeving, stamcelniche genaamd, bestaat uit cellen en steigers.Perifere neuropathie kan zijn en af en toe, ze kunnen zoals letsel aan de brachii plexus, verschillende trauma's, tumor, autonome anomalieën, immuundefinitie, en metabole ziekte. Er zijn verschillende 3D-kweekmethoden ontwikkeld om ee...

Video: Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation in situ. Unlike conventional bulk hydrogels, interconnected microscale pores in GHS facilitate degradation-independent cell infiltration as well as oxygen, nutrient, and cellular byproduct transfer. Methacryloyl-modified gelatin (GelMA), a (photo)chemically crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties, has widely been used as a cell-responsive/instructive biomaterial. Converting bulk GelMA to GHS&#160;may open a plethora of opportunities for tissue engineering and regeneration. In this article, we demonstrate the procedures of high-throughput GelMA microgel fabrication, conversion to resuspendable dry microgels (micro-aerogels), GHS formation via the chemical assembly of microgels, and granular bioink fabrication for extrusion bioprinting. We show how a sequential physicochemical treatment via cooling and photocrosslinking enables the formation of mechanically robust GHS.&#160;When light is inaccessible (e.g., during deep tissue injection), individually crosslinked GelMA HMPs may be bioorthogonally assembled via enzymatic crosslinking using transglutaminases. Finally, three-dimensional (3D) bioprinting of microporous GHS&#160;at low HMP packing density is demonstrated via the interfacial self-assembly of heterogeneously charged nanoparticles.

This article describes protocols for high-throughput gelatin methacryloyl microgel fabrication using microfluidic devices, converting microgels to resuspendable powder (micro-aerogels), the chemical assembly of microgels to form granular hydrogel scaffolds, and developing granular hydrogel bioinks with preserved microporosity for 3D bioprinting.

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

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