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.

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			<td height="21" style="height:21px;width:283px;">60 mL syringe</td>
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			<td height="21" style="height:21px;width:283px;">Clamps, three pronged, Talon</td>
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			<td style="width:551px;">102 mm, Dual adjustment clamp, large, clamp extension 127 mm</td>
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			<td height="21" style="height:21px;width:283px;">Cura 2.4.0</td>
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			<td height="21" style="height:21px;width:283px;">Discov3ry Complete</td>
			<td style="width:209px;">Structur3D Printing</td>
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			<td style="width:551px;">Ultimaker 2+ 3D printer integrated with Discov3ry paste extruder</td>
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			<td height="21" style="height:21px;width:283px;">Gelatin from bovine skin</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">9000-70-8</td>
			<td style="width:551px;"></td>
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			<td height="21" style="height:21px;width:283px;">Glutaraldehyde solution 50 wt. % in H2O</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">111-30-8</td>
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			<td height="21" style="height:21px;width:283px;">homogenizer</td>
			<td style="width:209px;">SPX</td>
			<td style="width:175px;">APV-2000</td>
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			<td height="21" style="height:21px;width:283px;">Instron 5960</td>
			<td style="width:209px;">Instron</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">Instron 5960, Biopuls Bath, 100 N load cell, 37 &#176;C,</td>
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			<td height="21" style="height:21px;width:283px;">Physica MCR 301 rheometer</td>
			<td style="width:209px;">Anton Paar</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">CP25-2-SN7617, gap height 0.05 mm, 25 &#176;C</td>
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			<td height="21" style="height:21px;width:283px;">Sorvall Lynx 6000 centrifuge</td>
			<td style="width:209px;">AB Ninolab</td>
			<td style="width:175px;">s/n 41881692</td>
			<td style="width:551px;">F12-rotor (6x500 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:283px;">stainless steel nozzle</td>
			<td style="width:209px;">Structur3D Printing</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">800, 600 and 400 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">thingsinverse</td>
			<td style="width:209px;">MakerBot&#39;s</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">&#160;sharing and downloading 3D printable things in form of stl files</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">ultra sonication</td>
			<td style="width:209px;">Qsonica, LLC</td>
			<td style="width:175px;">Q500</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Unbarked wood chips</td>
			<td style="width:209px;">Norway spruce(Picea abies)</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">dry matter content of 50&#8211;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

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Bioengineering

9.4K 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

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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 ...

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 ...

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict regional aerosol delivery. Leveraging 3D printing to generate patient-specific lung models, we outline the design of a high-throughput, in vitro experimental setup for quantifying lobular pulmonary deposition. This system is made with a combination of commercially available and 3D printed components and allows the flow rate through each lobe of the lung to be independently controlled. Delivery of fluorescent aerosols to each lobe is measured using fluorescence microscopy. This protocol has the potential to promote the growth of personalized medicine for respiratory diseases through its ability to model a wide range of patient demographics and disease states. Both the geometry of the 3D printed lung model and the air flow profile setting can be easily modulated to reflect clinical data for patients with varying age, race, and gender. Clinically relevant drug delivery devices, such as the endotracheal tube shown here, can be incorporated into the testing setup to more accurately predict a device&#8217;s capacity to target therapeutic delivery to a diseased region of the lung. The versatility of this experimental setup allows it to be customized to reflect a multitude of inhalation conditions, enhancing the rigor of preclinical therapeutic testing.

We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles.

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict ...

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 ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

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 ...

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 ...

Bioprinting Bacteria in Hydrogel Matrices for the Study of Motility and Growth

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. However, the majority of previous work has focused on studies of cells in bulk liquids or at flat surfaces, which do not fully recapitulate the complexity of many natural bacterial habitats. Here, this gap in knowledge is addressed by describing the development of a method to 3D-print dense colonies of bacteria into jammed granular hydrogel matrices. These matrices have tunable pore sizes and mechanical properties; they physically confine the cells, thus supporting them in 3D. They are optically transparent, allowing for direct visualization of bacterial spreading through their surroundings using imaging. As a proof of this principle, here, the capability of this protocol is demonstrated by 3D printing and imaging non-motile and motile Vibro cholerae, as well as non-motile Escherichia coli, in jammed granular hydrogel matrices with varying interstitial pore sizes.

Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes.

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. ...

Analyzing Cell-Seeded Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as&#160;its ability to effectively support osteogenic differentiation. The present study further&#160;examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then&#160;assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and&#160;energy-dispersive spectroscopy (EDS)&#160;confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young&#39;s modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived&#160;scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Author Spotlight: Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering 

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.

Methods Article

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds