Name: preparation and characterization of graphene-based 3d biohybrid hydrogel bioink for peripheral neuroengineering
Uploaded: 2022-05-16T14:00:00
Description: Watch this Scientific Journal Video about Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering at JoVE.com

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.

1. Culturing of Wharton&#39;s jelly mesenchymal stem cells
<ol>
	<li>Take the Wharton&#39;s jelly mesenchymal stem cells (WJ-MSCs, from ATCC) out of a &#8722;80 &#176;C freezer. Culture WJ-MSCs in DMEM-F12 medium containing 10% fetal calf serum (FBS), 1% Pen-Strep, and 1% L-glutamine&#160;in a sterile laminar flow at room temperature, as described in Yurie et al.20.</li>
	<li>Cryopreserve some of the cells at 1 x 106 cells/mL with freezing medium containing 35% FBS, 5...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+peripheral+nerve+injury:+A+wartime+review.">Traumatic peripheral nerve injury: A wartime review.</a> Journal of Craniofacial Surgery. 21 (4), 998-1001 (2010).</li><li>Mushtaq, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+peripheral+nerve+injury+in+trauma+in+emergency+settings.">Frequency of peripheral nerve injury in trauma in emergency settings.</a> Cureus. 13 (3), 14195 (2021).</li><li>Allahverdiyev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Principles of Somatic and Stem Cell Culture Systems, 1st Edition. , (2018).</li><li>Allahverdiyev, A. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+clinical+applications+of+Wharton's+jelly-derived+mesenchymal+stromal+cells.">Characteristics and clinical applications of Wharton's jelly-derived mesenchymal stromal cells.</a> Current Research in Translational Medicine. 68 (1), 5-16 (2020).</li><li>Yoo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+peripheral+nerve+regeneration+through+elastic+nerve+guidance+conduits+prepared+using+a+porous+PLCL+membrane+with+a+3D+printed+collagen+hydrogel.">Augmented peripheral nerve regeneration through elastic nerve guidance conduits prepared using a porous PLCL membrane with a 3D printed collagen hydrogel.</a> Biomaterials Science. 22, 1-12 (2020).</li><li>Jansen, K., Meek, M. F., vander Werff, J. F. A., van Wachem, P. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td> 
			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 &#181;m membrane filter</td>
			<td>A&#953;s&#953;mo</td>
			<td></td>
			<td>Used for sterilization</td>
		</tr>
		<tr>
			<td>0.45 &#181;m syringe filter</td>
			<td>A&#953;s&#953;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&#39;s Modified Eagle&#39;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&#176;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; &#946;-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...

Watch this Scientific Journal Video about Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering at JoVE.com

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 ...

The stem cell system is under the influence of its important components. The preservation and differentiation of stem cell is unthinkable without the presence of their microenvironment. This microenvironment, called stem-cell niche, consist of cell and scaffolds.Peripheral neuropathy can be and occasionally, they may such as injury to the brachii plexus, various trauma, tumor, autonomic anomalies, immune definition, and metabolic disease. Various 3D culture methods have been developed to provide a better and more natural niche for the stem cells. Spheroid formation and 3D bioprinting are relatively new and promising methods for 3D cultures.3D bioprinting can also be used in neuroengineering studies. Consequently, within this study, graphene-based and alginate/gelatin-based bioinks were developed and studied for their regenerative features. To begin, culture Wharton's jelly mesenchymal stem cells or WJMSCs in DMEM F12 medium containing 10%fetal calf serum or FBS, 1%pen/strep, and 1%L-glutamine under a sterile laminar flow at room temperature.When the cultured cells are 80%confluent in the flask, pour the medium and wash the cells with five milliliters of PBS. Then add five milliliters of 0.25%trypsin and 2.21 millimolar sodium EDTA and incubate at 37 degrees Celsius. After five minutes, add 10 milliliters of DMEM F12 medium containing 10%FBS to the cells.Suspend the cells, collect the medium and transfer it to a centrifuge tube. Next, centrifuge the cells and discard the supernatant before reseeding the cells in a new flask with a fresh nutrient medium containing 10%FBS. To prepare the bioink of the control group without graphene, weigh 4.5 milligrams of alginate and 1.5 milligrams of gelatin and transfer them to a centrifuge tube.Then add 50 milliliters of DMEM F12 medium containing 10%FBS to the tube. Again, weigh 4.5 milligrams of alginate and 1.5 milligrams of gelatin and transfer them to a centrifuge tube. Then add 50 microliters of 0.1%graphene to the tube and make the volume 50 milliliters with DMEM F12 medium containing 10%FBS.Mix the bioinks by pipetting and vortexing before autoclaving at 121 degrees Celsius under 1.5. atmospheric pressure for 20 minutes. After autoclaving, centrifuge the solution to remove formed bubbles and place the bioinks at 37 degrees Celsius until the cells are prepared.For the cell bioink interaction, create the bioink groups. Group one includes 3D-B and 3D-G printed with bioink for bioprinting. Group two includes 3D-BS and 3D-GS bioinks on which spheroids were formed after bioprinting.For group one, count the cells to one by 10 to the seventh cells in 0.5 milliliters of medium. Then add 4.5 milliliters of bioink. Transfer the mixture to the cartridges in the sterile cabinet using syringes.Install the cartridges in the corresponding extruder section of the bioprinter. For the second group, take five milliliters of bioink from each of the bioink groups and transfer them to sterile cartridges with the help of an injector. Use the bioprinter with two coaxial print heads and the pneumatic-driven extrusion technology.Set the XYZ resolution per micro step to 1.25 micrometers, extrusion width to 400 micrometers, and the extrusion height to 200 micrometers. Use a 20 by 20 by 5 millimeter grid to create 3D models. Create 3D models using open source web-based CAD programs.For the 3D bioprinting process, set the average pressure of the printer to 7.5 psi. Then set the cartridge and bed temperature to 37 degrees Celsius and speed to 60%Place the system in the home position during the writing phase. Position the axes automatically and select and set the extruder before starting the bioprinting process.After the printing, remove the sample and place it under a laminar flow cabinet. Next, spray the bioinks with 0.1 normal calcium chloride solution or add one milliliter solution with a pipette at room temperature and wait for 10 to 20 seconds. Then wash the printed patterns twice with PBS containing calcium and magnesium.Add two milliliters of DMEM F12 medium with 10%FBS on top of each cell containing bioink group and incubate the plates at 37 degrees Celsius with 5%carbon dioxide for 30 minutes. Next, add two milliliters of suspension medium containing one by 10 to the sixth cells to each group and incubate the plates. After 24 hours, observe and photograph all batches of bioink for spheroid formation under an inverted microscope for WJMSCs differentiation to neuron-like cells except for the control group.Add two milliliters of neurogenic differentiation medium per well and refresh every two days. Follow for seven days to observe neural differentiation. Next, using timelapse imaging, examine the effects of graphene on stem cells and monitor cell interactions within the bioink.The effect of graphene concentration on cell proliferation is demonstrated here. Compared to the control, significant decrease was observed for the 0.001%graphene concentration. There were no significant differences between the other groups and the control.The cell graphene interactions showed that graphene was tolerated in the 2D system and was taken by the cells through endocytosis. Timelapse imaging showed that cells that survived in the 3D graphene medium maintained their vitalities through GFP brightness until the end of the incubation. SEM images and FIIR analysis of the 3D-B and 3D-G bioink groups are shown here.Bioink cell interactions were demonstrated both on the surface and internally. The cells were morphologically round and attached to the material. In 3D neuronal differentiation, it was considered that the borders of the spheroid cells in both groups were transparent and lively, and the spheroids in the graphene group were relatively larger and trapped the graphene inside the the cell.Immunostaining of 2D and 3D cells is shown here. The green images represent neuron-like structures. The 2D positive control sample expressed fewer neuron-like structure markers than the 3D samples.We discovered that graphene-based bioinks were more successful in terms of differentiation of stem cells into neuron-like cells. We propose that graphene-based bioinks would be excellent tools for treatment of peripheral nerve disorders in further studies. Today, the stem cell system can be created by tissue engineering with natural and synthetic biomaterials The creation of artificial tissues that can replace living tissues which can be used in regeneration of these tissues, removing the damage, and providing function is provided by tissue engineering.

preparation-characterization-graphene-based-3d-biohybrid-hydrogel

Research

JoVE Journal

Bioengineering

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

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 ...

Quantitative Characterization of Bioink Properties for Continuous DLP Printing

Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing parameters of digital light processing (DLP). However, the acquisition of working curves wastes materials and requires high formability of materials, which are not suitable for biomaterials. In addition, the reduction of cell activity due to multiple exposures and the failure of structural formation due to repeated positioning are both unavoidable problems in conventional DLP bioprinting. This work introduces a new method of obtaining the working curve and the improvement process of continuous DLP printing technology based on such a working curve. This method of obtaining the working curve is based on the absorbance and photorheological properties of the biomaterials, which do not depend on the formability of the biomaterials. The continuous DLP printing process, obtained from improving the printing process by analyzing the working curve, increases the printing efficiency more than tenfold and greatly improves the activity and functionality of cells, which is beneficial to the development of tissue engineering.

Author Spotlight: Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing  

This study uses temperature and material composition to control the yield stress properties of yield stress fluids. The solid-like state of the ink can protect the printing structure, and the liquid-like state can continuously fill the printing position, realizing the digital light processing 3D printing of extremely soft bioinks.

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing ...