This study is financially supported by Knut and Alice Wallenberg Foundation (Wallenberg Wood&#160;Science&#160;Center), Swedish Research Council,VR (Bioheal, DNR 2016-05709 and DNR 2017-04254).

1. Preparation of precursors
<ol>
	<li>Preparation of cellulose nanocrystals suspension 
	NOTE: Isolation of cellulose nanocrystals is done according to the procedure reported by Mathew, et al30.
	<ol>
		<li>Dilute 17 wt% suspension of cellulose nanocrystals to 2 wt% by adding distilled water to make a total volume of 2 L. Mix thoroughly using ultra sonication and use smaller batches (250-300 mL) for efficient mixing.</li>
		<li>Pass the sonified suspension through the homogenizer 10 times at a pressure of 500-600 bar. At this point, a thick transparent gel of 2 wt% cellulose nanocrystals is obtained.</li>
		<li>Concentrate 2 wt% cellulose nanocrystals gel to 11 wt% through centrifugations at 24,500 x g for 1.5 h. Decant water out in between every 30 min. 
		&#8203;NOTE: Experiment can be paused here.</li>
	</ol>
	</li>
	<li>Preparation of matrix phases
	<ol>
		<li>Prepare homogeneous solution of 6 wt% sodium alginate (SA) in distilled water at 60 &#176;C under continuous stirring.</li>
		<li>Prepare homogeneous solution of 12 wt% gelatin (Gel) in distilled water at 60 &#176;C under continuous stirring. 
		NOTE: Prepare a volume of 20 mL for matrix solutions and store in refrigerator.</li>
	</ol>
	</li>
	<li>Preparation of crosslinkers
	<ol>
		<li>Prepare the solution of 3 wt% calcium chloride in distilled water at room temperature under continuous stirring.</li>
		<li>Prepare the solution of 3 wt% glutaraldehyde in distilled water at room temperature under continuous stirring. 
		NOTE: Prepare a volume of 50 mL for crosslinking solutions and store in room temperature. Refer to the Table of Materials for vendor information. Experiment can be paused here.</li>
	</ol>
	</li>
</ol>
2. Preparation of hydrogel ink
<ol>
	<li>Prepare 40 mL of hydrogel ink in a polystyrene container by mixing 11 wt% CNC, 6 wt% SA and 12 wt% Gel to obtain a wet (wt%) composition of CNC/SA/Gel/Water: 6.87/1.50/1.50/90.12.</li>
	<li>Heat the mixture to 40 &#176;C and mix with a spatula until a smooth paste is obtained.</li>
	<li>Transfer the mixture into a 60 mL syringe. Pass the mixture through a series of nozzles with different diameters into another 60 mL syringe, with the help of mechanical clamp. Repeat the process until smoothly extruded filaments of hydrogel ink are obtained. Start with nozzle with biggest diameter of 800 &#181;m, followed by 600 &#181;m and 400 &#181;m.</li>
	<li>Gently centrifuge (4,000 x g) the syringe filled with hydrogel ink to remove trapped air. 
	NOTE: Experiment can be paused here.</li>
</ol>
3. Measurement of rheological properties of hydrogel
NTE: Perform the rheological properties by using a smooth cone-on-plate geometry, CP25-2-SN7617, diameter 25 mm, 2&#176; nominal angle and gap height 0.05 mm at 25 &#176;C.
<ol>
	<li>Turn on the rheometer, air compressor and temperature control box. Initialize the software.</li>
	<li>Mount the measuring tool in the rheometer and set zero-gap.</li>
	<li>Extrude approximately 1 mL of the hydrogel ink onto the rheometer platform.</li>
	<li>Measure the viscosity as a function of shear rate. Select the shear rate range from 0.001 to 1,000.</li>
	<li>After the measurement is done, clean the rheometer platform and measuring tool. Extrude 1 mL of fresh hydrogel ink again on the rheometer platform.</li>
	<li>Measure storage moduli (G&#8242;) and loss moduli (G&#8243;) as a function of shear stress at a frequency of 1 Hz. Select the shear stress range from 103 to 107.</li>
	<li>Once the tests are completed, copy the data into text file and plot rheological curves in logarithmic scale.</li>
</ol>
4. File preparation for 3D Printing
NOTE: Cura 2.4.0 software is used for designing 3D scaffolds (20 mm3) having three types of pores. 1- Uniform pores of 0.6 mm, 2- uniform pores of 1.0 mm and 3- gradient pores of range 0.5-1 mm.
<ol>
	<li>Download stereolithography (stl) file of a solid cube from thingsinverse.com and open the file in Cura.</li>
	<li>Click the loaded model and move it to X/Y/Z : 0/0/0 mm. Click Scale, uncheck box for Uniform scaling and set the dimensions to&#160; X/Y/Z : 20/20/20 mm. Click Rotate and rotate the cube by 45&#176; in XY plane.</li>
	<li>In the side panel, in Nozzle &#38; Material, select 0.4 mm and paste the profile. Select Discov3ry complete as the printer. &#160;</li>
	<li>In the side panel, select Custom for Print Setup. Under Quality section, enter 0.2 mm for all sub sections. Under Shell section, enter 0 mm for all sub sections. Under Material section, enter 26 &#176;C for temperature, 1 mm Diameter and 100 % Flow. Under Speed section, enter 30 mm/s as Print Speed and 120 mm/s as Travel Speed. Under Support section, uncheck the box for Enable Support. Under Build Plate Adhesion section, select Skirt, enter 3 mm as Skirt Distance and 150 mm as Skirt/Brim Minimum length.</li>
	<li>For scaffolds with uniform pore size, enter 0.6 or 1 mm Infill Line Distance and select Grid Infill Pattern.</li>
	<li>For gradient porosity scaffolds, Merging and Grouping tool is used. Right click the loaded model, select Multiple Models, enter 2 and press OK. Scale each model as X/Y/Z : 20/20/7 mm. Place the models on top of each other. Enter Infill Line Distance as 0.3, 0.5 and 0.7 mm for bottom, middle and top model, respectively. Select all three models (Ctrl + A), right click and click Group Models.</li>
	<li>Save the models on the Sure Digital (SD) card. Cura automatically save the file as gcode that is read by the printer.</li>
</ol>
5. 3D printing porous scaffolds
<ol>
	<li>Insert the transfer tube into the nozzle holder and connect 400 &#181;m nozzle to it. Level the build plate to get the correct distance between the build plate and nozzle.</li>
	<li>Load the centrifuged syringe into the cartridge and connect it to the other side of the transfer tube.</li>
	<li>Insert the SD card into the printer, select Purge fast and start purging the hydrogel ink until it starts to extrude from the nozzle. Continue purging for 2-3 min to obtain a homogeneous flow.</li>
	<li>From the SD card, select the saved files for uniform and gradient porosity scaffolds and start printing. Keep an eye on the extrusion rate. If needed, adjust the speed and flow rate accordingly. For smaller pore size, use faster speed combined with low flow rate (50 mm/s and 70%). 
	NOTE: Do not touch the 3D printed scaffolds.</li>
</ol>
&#160;6. Crosslinking of 3D printed scaffolds
<ol>
	<li>After the 3D printing is complete, gently add drops of 3 wt% CaCl2 to the scaffold until it becomes completely wet. Wait for 5 min.</li>
	<li>Very carefully transfer the scaffold from the printer to a 50 mL container filled with 3 wt% CaCl2. Leave it overnight.</li>
	<li>Wash thoroughly with distilled water and transfer the scaffold to a 50 mL container filled with 3 wt% glutaraldehyde. Leave it overnight.</li>
	<li>Wash thoroughly and store the 3D printed scaffold in distilled water.</li>
</ol>
7. Compression testing
NOTE: Perform compression tests with 100 N load cell in water at 37 &#176;C.
<ol>
	<li>Fill the container equipped with submersible compression base plate with 2 L of water and start the heating system to reach 37 &#176;C.</li>
	<li>Initialize Bluehill Universal software and set up the testing method. Select rectangular specimen geometry and choose the option to enter dimensions before testing each sample. &#160;Set the strain rate to 2 mm/min and end of result as 80% compressive strain together with 90 N force.</li>
	<li>In the Measurement section, select force, displacement, compressive stress and compressive strain. Choose the option to export data as text files for future plotting.</li>
	<li>Set the zero extension point by using the jog controls to lower crosshead plate as close as possible to base plate.</li>
	<li>Measure and note the dimensions of the samples to be tested.</li>
	<li>When the water temperature reaches to 37 &#176;C, place the sample on the base plate.&#160; Secure the sample by moving the crosshead plate so that it starts to touch the sample.</li>
	<li>Move the water bath up, so that the plates with the sample in-between them are immersed in water.</li>
	<li>Enter sample name and dimensions. Start the test.</li>
	<li>After the test is complete, first move the water bath down and then raise the crosshead plate.</li>
	<li>Remove the sample and its pieces, if any, clean both the plates and load a new sample.</li>
	<li>After all the samples are tested, export the raw data. Plot compressive stress vs. compressive strain curves&#160;and determine the compressive tangent modulus at strain values of 1-5 % and 25-30 %. 
	NOTE: Place the gradient cube in such a way that the larger holes face the stationery base plate. 
	First secure the scaffold in between the grips and then start/stop the measurement.</li>
</ol>

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The authors have nothing to disclose.

3D printing requires suitable rheological properties of the hydrogel ink. The high viscosity ink will require extreme pressures for its extrusion while low viscosity ink will not maintain its shape after extrusion. The viscosity of the hydrogel ink can be controlled through the concentration of the ingredients. As compared to our previous work11, the solid content of the hydrogel ink is increased from 5.4 to 9.9 wt% resulting in concentrated hydrogel ink which helps to improve the resolution of th...

Cellulose is a polysaccharide consisting of linear chains of &#946; (1-4) linked D-glucose units. It is the most abundant natural polymer on Earth and is extracted from a variety of sources, including marine animals (e.g., tunicates), plants (e.g., wood, cotton, wheat straw), and bacterial sources, such as algae (e.g., Valonia), fungi, and even amoeba (protozoa)1,2. Cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) with at least one dimension on nanoscale are obtained through mechanical treatments and acid hydrolysis from cellulose. They not only possess the properties of cellulose, such as potential for chemical modification, low toxicity, biocompatibility, biodegradable and renewable, but it also has nanoscale characteristics like high specific surface area, high mechanical properties, rheological and optical properties. These attractive properties have made CNFs and CNCs suitable for biomedical applications, mainly in the form of 3-dimensional (3D) hydrogel scaffolds3. These scaffolds require customized dimensions with controlled pore structure and interconnected porosity. Our group and others have reported 3D porous cellulose nanocomposites prepared through casting, electrospinning and freeze-drying4,5,6,7,8. However, control on the pore structure and fabrication of complex geometry is not achieved through these traditional techniques.
3D printing is an additive manufacturing technique, in which 3D objects are created layer by layer through the computer-controlled deposition of the ink9. The advantages of 3D printing over traditional techniques includes design freedom, controlled macro and micro dimensions, fabrication of complex architectures, customization and reproducibility.&#160; In addition, 3D printing of CNFs and CNCs also offers shear-induced alignments of nanoparticles, preferred directionality, gradient porosity and can easily be extended to 3D bioprinting10,11,12,13,14,15. Recently, the dynamics of CNCs alignment during 3D printing has been reported16,17. Advances in the field of bioprinting have enable 3D printed tissues and organs despite the involved challenge such as choice and concentration of living cells and growth factors, composition of the carrier ink, printing pressures and nozzle diameters18,19,20.
The porosity and compressive strength of cartilage regenerative scaffolds are important properties that dictates its efficiency and performance. Pore size plays an important role for the adhesion, differentiation, and proliferation of cells as well as for the exchange of nutrients and metabolic waste21. However, there is no definite pore size that can be considered as an ideal value, some studies showed higher bioactivity with smaller pores while others showed better cartilage regeneration with larger pores. Macropores (&#60;500 &#181;m) facilitate tissue mineralization, nutrient supply and waste removal while micropores (150-250 &#181;m) facilitate cell attachment and better mechanical properties22,23. The implanted scaffold must have sufficient mechanical integrity from the time of handling, implantation and until the completion of its desired purpose. The aggregate compressive modulus for natural articular cartilage is reported to be in the range of 0.1-2 MPa depending on age, sex and tested location4,24,25,26,27,28,29. &#160;
In our previous work11, 3D printing was used to fabricate porous bioscaffolds of a double crosslinked interpenetrating polymer network (IPN) from a hydrogel ink containing reinforced CNCs in a matrix of sodium alginate and gelatin. The 3D printing pathway was optimized to achieve 3D scaffolds with uniform and gradient pore structures (80-2,125 &#181;m) where nanocrystals orient preferably in the printing direction (degree of orientation between 61-76%). Here, we present the continuation of this work and demonstrates the effect of porosity on the mechanical properties of 3D printed hydrogel scaffolds in simulated body conditions. CNCs used here, were earlier reported by us to be cytocompatible and non-toxic (i.e., cell growth after 15 days of incubation was confirmed30). Moreover, scaffolds prepared via freeze-drying using the same CNCs, sodium alginate and gelatin showed high porosity, high uptake of phosphate buffer saline and cytocompatibility toward mesenchymal stem cells5. The goal of this work is to demonstrate the hydrogel ink processing, 3D printing of porous scaffolds and the compression testing. Schematics of the processing route is shown in Figure 1.

<table><tbody><tr><td>60 mL syringe</td><td>Structur3D Printing</td><td></td><td></td></tr><tr><td>Alginic acid sodium salt</td><td>Sigma-Aldrich</td><td>9005-38-3</td><td></td></tr><tr><td>Anhydrous calcium chloride</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td></td></tr><tr><td>Clamps, three pronged, Talon</td><td>VWR</td><td>241-0404</td><td>102 mm, Dual adjustment clamp, large, clamp extension 127 mm</td></tr><tr><td>Cura 2.4.0</td><td>Ultimaker</td><td></td><td>Free slicing software</td></tr><tr><td>Discov3ry Complete</td><td>Structur3D Printing</td><td></td><td>Ultimaker 2+ 3D printer integrated with Discov3ry paste extruder</td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma-Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>Glutaraldehyde solution 50 wt. % in H2O</td><td>Sigma-Aldrich</td><td>111-30-8</td><td></td></tr><tr><td>homogenizer</td><td>SPX</td><td>APV-2000</td><td></td></tr><tr><td>Instron 5960</td><td>Instron</td><td></td><td>Instron 5960, Biopuls Bath, 100 N load cell, 37 &amp;deg;C,</td></tr><tr><td>Physica MCR 301 rheometer</td><td>Anton Paar</td><td></td><td>CP25-2-SN7617, gap height 0.05 mm, 25 &amp;deg;C</td></tr><tr><td>Sorvall Lynx 6000 centrifuge</td><td>AB Ninolab</td><td>s/n 41881692</td><td>F12-rotor (6x500 ml)</td></tr><tr><td>stainless steel nozzle</td><td>Structur3D Printing</td><td></td><td>800, 600 and 400 &amp;micro;m</td></tr><tr><td>thingsinverse</td><td>MakerBot&#039;s</td><td></td><td>&amp;nbsp;sharing and downloading 3D printable things in form of stl files</td></tr><tr><td>ultra sonication</td><td>Qsonica, LLC</td><td>Q500</td><td></td></tr><tr><td>Unbarked wood chips</td><td>Norway spruce(Picea abies)</td><td></td><td>dry matter content of 50&amp;ndash;55%</td></tr></tbody></table>

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;

CNCs based nanocomposite hydrogel ink shows a strong non-Newtonian shear thinning behavior (Figure 2a). The apparent viscosity of 1.55 &#215; 105 Pa.s at a low shear rate (0.001 s-1) drops by five orders of magnitude to a value of 22.60 Pa.s at a shear rate of 50 s-1 (&#8776;50 s-1 being a typical shear rate experienced during 3D printing)31. The hydrogel ink exhibits a viscoelastic solid behavior, as the st...

Watch this Scientific Journal Video about 3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds at JoVE.com

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

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 ...

3d-printed-porous-cellulose-nanocomposite-hydrogel-scaffolds

Research

JoVE Journal

Bioengineering

9.5K Views.  Stockholm University. 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.

Video: 3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

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 ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

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 ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

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 ...