Name: 3d printed porous cellulose nanocomposite hydrogel scaffolds
Uploaded: 2019-04-24T13:00:00
Description: Watch this Scientific Journal Video about 3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds at JoVE.com

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 ...

<ol>
	<li>Moon, R. J., Martini, A., Nairn, J., Simonsen, J., Youngblood, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose+nanomaterials+review:+structure,+properties+and+nanocomposites.">Cellulose nanomaterials review: structure, properties and nanocomposites.</a> Chemical Society Reviews. 40 (7), 3941-3994 (2011).</li><li>Dufresne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanocellulose:+a+new+ageless+bionanomaterial.">Nanocellulose: a new ageless bionanomaterial.</a> Materials Today. 16 (6), 220-227 (2013).</li><li>Chinga-Carrasco, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+and+limitations+of+nanocelluloses+as+components+in+biocomposite+inks+for+three-dimensional+bioprinting+and+for+biomedical+devices.">Potential and limitations of nanocelluloses as components in biocomposite inks for three-dimensional bioprinting and for biomedical devices.</a> Biomacromolecules. 19 (3), 701-711 (2018).</li><li>Naseri, N., Poirier, J., Girandon, L., Fr&#246;hlich, M., Oksman, K., Mathew, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Dimensional+porous+nanocomposite+scaffolds+based+on+cellulose+nanofibers+for+cartilage+tissue+engineering:+tailoring+of+porosity+and+mechanical+performance.">3-Dimensional porous nanocomposite scaffolds based on cellulose nanofibers for cartilage tissue engineering: tailoring of porosity and mechanical performance.</a> Royal Society of Chemistry Advances. 6 (8), 5999-6007 (2016).</li><li>Naseri, N., Deepa, B., Mathew, A. P., Oksman, K., Girandon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanocellulose-Based+Interpenetrating+Polymer+Network+(IPN)+Hydrogels+for+Cartilage+Applications.">Nanocellulose-Based Interpenetrating Polymer Network (IPN) Hydrogels for Cartilage Applications.</a> Biomacromolecules. 17 (11), 3714-3723 (2016).</li><li>Naseri, N., Mathew, A. P., Girandon, L., Fr&#246;hlich, M., Oksman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+electrospun+nanocomposite+mats+based+on+chitosan-cellulose+nanocrystals+for+wound+dressing:+effect+of+surface+characteristics+of+nanocrystals.">Porous electrospun nanocomposite mats based on chitosan-cellulose nanocrystals for wound dressing: effect of surface characteristics of nanocrystals.</a> Cellulose. 22 (1), 521-534 (2015).</li><li>Xing, Q., Zhao, F., Chen, S., McNamara, J., DeCoster, M. A., Lvov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+biocompatible+three-dimensional+scaffolds+of+cellulose+microfiber/gelatin+composites+for+cell+culture.">Porous biocompatible three-dimensional scaffolds of cellulose microfiber/gelatin composites for cell culture.</a> Acta Biomaterialia. 6 (6), 2132-2139 (2010).</li><li>Nandgaonkar, A., Krause, W., Lucia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cellulosic+composite+scaffolds+for+cartilage+tissue+engineering.">Fabrication of cellulosic composite scaffolds for cartilage tissue engineering.</a> Nanocomposites for musculoskeletal tissue regeneration. , 187-212 (2016).</li><li>Gross, B. C., Erkal, J. L., Lockwood, S. Y., Chen, C., Spence, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+3D+printing+and+its+potential+impact+on+biotechnology+and+the+chemical+sciences.">Evaluation of 3D printing and its potential impact on biotechnology and the chemical sciences.</a> Analytical Chemistry. 86 (7), 3240-3253 (2014).</li><li>Markstedt, K., Mantas, A., Tournier, I., Marti&#769;nez A&#769;vila, H., Ha&#776;gg, D., Gatenholm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+human+chondrocytes+with+nanocellulose-alginate+bioink+for+cartilage+tissue+engineering+applications.">3D bioprinting human chondrocytes with nanocellulose-alginate bioink for cartilage tissue engineering applications.</a> Biomacromolecules. 16 (5), 1489-1496 (2015).</li><li>Sultan, S., Mathew, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printed+scaffolds+with+gradient+porosity+based+on+a+cellulose+nanocrystal+hydrogel.">3D printed scaffolds with gradient porosity based on a cellulose nanocrystal hydrogel.</a> Nanoscale. 10, 4421-4431 (2018).</li><li>Sultan, S., Siqueira, G., Zimmermann, T., Mathew, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printing+of+nano-cellulosic+biomaterials+for+medical+applications.">3D printing of nano-cellulosic biomaterials for medical applications.</a> Current Opinion in Biomedical Engineering. 2, 29-34 (2017).</li><li>Sultan, S., Abdelhamid, H. N., Zou, X., Mathew, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CelloMOF:+Nanocellulose+Enabled+3D+Printing+of+Metal-Organic+Frameworks.">CelloMOF: Nanocellulose Enabled 3D Printing of Metal-Organic Frameworks.</a> Advanced Functional Materials. , 1805372-1805384 (2018).</li><li>Siqueira, G., 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=Cellulose+Nanocrystal+Inks+for+3D+Printing+of+Textured+Cellular+Architectures.">Cellulose Nanocrystal Inks for 3D Printing of Textured Cellular Architectures.</a> Advanced Functional Materials. 27 (12), 1604619-1604629 (2017).</li><li>Wang, 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=All-in-One+Cellulose+Nanocrystals+for+3D+Printing+of+Nanocomposite+Hydrogels.">All-in-One Cellulose Nanocrystals for 3D Printing of Nanocomposite Hydrogels.</a> Angewandte Chemie International Edition. 57 (9), 2353-2356 (2018).</li><li>Hausmann, M. K., 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=Dynamics+of+Cellulose+Nanocrystal+Alignment+during+3D+Printing.">Dynamics of Cellulose Nanocrystal Alignment during 3D Printing.</a> ACS Nano. 12 (7), 6926-6937 (2018).</li><li>Liu, Y., 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=Nanoscale+assembly+of+cellulose+nanocrystals+during+drying+and+redispersion.">Nanoscale assembly of cellulose nanocrystals during drying and redispersion.</a> ACS Macro Letters. 7 (2), 172-177 (2018).</li><li>Murphy, S. V., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+tissues+and+organs.">3D bioprinting of tissues and organs.</a> Nature Biotechnology. 32 (8), 773-785 (2014).</li><li>Pati, F., 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=Printing+three-dimensional+tissue+analogues+with+decellularized+extracellular+matrix+bioink.">Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink.</a> Nature communications. 5, 3935 (2014).</li><li>Xia, Z., Jin, S., Ye, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+and+organ+3D+bioprinting.">Tissue and organ 3D bioprinting.</a> SLAS TECHNOLOGY: Translating Life Sciences Innovation. 23 (4), 301-314 (2018).</li><li>Zhang, Q., Lu, H., Kawazoe, N., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore+size+effect+of+collagen+scaffolds+on+cartilage+regeneration.">Pore size effect of collagen scaffolds on cartilage regeneration.</a> Acta Biomaterialia. 10 (5), 2005-2013 (2014).</li><li>Loh, Q. L., Choong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+scaffolds+for+tissue+engineering+applications:+role+of+porosity+and+pore+size.">Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size.</a> Tissue Engineering, Part B: Reviews. 19 (6), 485-502 (2013).</li><li>Bru&#382;auskait&#279;, I., Bironait&#279;, D., Bagdonas, E., Bernotien&#279;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+and+cells+for+tissue+regeneration:+different+scaffold+pore+sizes-different+cell+effects.">Scaffolds and cells for tissue regeneration: different scaffold pore sizes-different cell effects.</a> Cytotechnology. 68 (3), 355-369 (2016).</li><li>Zhang, L., Hu, J., Athanasiou, K. 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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>
			<td style="width:209px;">Structur3D Printing</td>
			<td style="width:175px;"></td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Alginic acid sodium salt</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">9005-38-3</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Anhydrous calcium chloride</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">10043-52-4</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Clamps, three pronged, Talon</td>
			<td style="width:209px;">VWR</td>
			<td style="width:175px;">241-0404</td>
			<td style="width:551px;">102 mm, Dual adjustment clamp, large, clamp extension 127 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cura 2.4.0</td>
			<td style="width:209px;">Ultimaker</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">Free slicing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Discov3ry Complete</td>
			<td style="width:209px;">Structur3D Printing</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">Ultimaker 2+ 3D printer integrated with Discov3ry paste extruder</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">homogenizer</td>
			<td style="width:209px;">SPX</td>
			<td style="width:175px;">APV-2000</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
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			<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 ...

This is the first report on 3D printed nanocellulose hydrogel scaffolds where we have a gradient pore structure and mechanical properties to mimic natural structures as cartilage. The two main advantages of using 3D printing techniques are customization and design freedom. That opens up an unlimited opportunity of fabricating new and unexplored geometric designs.This protocol is user-friendly, and newcomers can easily reproduce the results. The choice of the slicing software and the nozzle movement have significant impact on the final product. To begin, prepare 40 milliliters of hydrogel ink by mixing 11%by weight CNC, 6%by weight sodium alginate, and 12%by weight gelatin in a container.Heat the mixture to 40 degrees Celsius, and mix with a spatula until a smooth paste is obtained. Transfer the mixture into a 60-milliliter syringe. Next, with the help of a mechanical clamp, pass the mixture through a series of nozzles with different diameters into another 60-milliliter syringe.Repeat the process until smoothly extruded filaments of hydrogel ink are obtained. Gently centrifuge the syringe filled with the hydrogel ink at 4, 000 times g to remove trapped air. From the SD card, select the saved files for uniform and gradient porosity scaffolds, and start printing.If needed, adjust the speed and flow rate accordingly. To cross-link the scaffold after the 3D printing is complete, gently add drops of 3%by weight calcium chloride solution to the scaffold until it becomes completely wet. Wait for five minutes.Very carefully transfer the scaffold from the printer bed to a 50-milliliter container filled with 3%by weight calcium chloride solution. Leave it overnight. Wash thoroughly with distilled water, and transfer the scaffold to a 50-milliliter container filled with 3%by weight glutaraldehyde solution.Leave it overnight. Wash thoroughly, and store the 3D printed scaffold in distilled water. For compression testing, fill the container equipped with a submersible compression base plate with two liters of water, and start the heating system to reach 37 degrees Celsius.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. Set the strain rate to two millimeters per minute and end of result as 80%compressive strain together with 90-newton force.In the Measurement section, select Force, Displacement, Compressive Stress, and Compressive Strain. Choose the option to export data as text files for future plotting. Set the zero extension point by using the jog controls to lower the crosshead plate as close as possible to the base plate.Measure and record the dimensions of the samples to be tested. When the water temperature reaches 37 degrees Celsius, place the sample on the base plate. Secure the sample by moving the crosshead plate so that it starts to touch the sample.Move the water bath up so that the plates with the sample in between them are immersed in water. Enter the sample name and dimensions, and start the test. After the test is complete, first move the water bath down, and then raise the crosshead plate.Remove the sample and its pieces, if any, clean both the plates, and load a new sample. After all the samples are tested, export the raw data. Plot compressive stress versus compressive strain curves, and determine the compressive tangent modulus at strain values of one to 5%and 25 to 30%CNCs-based nanocomposite hydrogel ink shows a strong non-Newtonian shear thinning behavior with a five order of magnitude drop of the apparent viscosity.The hydrogel ink exhibits a viscoelastic solid behavior, as the storage modulus is an order of magnitude greater than the loss modulus at low shear stress. At low strain rates of one to 5%the compressive modulus is similar for all types of porous scaffolds in comparison to the reference scaffold with no porosity, showing that the elastic nature of the hydrogel ink is preserved even in the presence of the macropores. However, at high strain rates of 25 to 30%the highest modulus is obtained for the reference scaffold with no porosity.As soon as the pore size increases, the modulus decreases due to the decrease in density, indicating the expected relationship between porosity of the scaffolds and the corresponding mechanical properties. Furthermore, the compressive modulus of the 3D hydrogel scaffolds increases as the compression rate increases, exhibiting and mimicking the viscoelasticity of natural cartilage tissues. The homogeneous and the continuous flow of the ink during 3D printing are the most important things.This method will be used by researchers to expand to other application areas by using nanocellulose hydrogel as a 3D printable platform. For example, we have already developed nanocellulose-based hybrids for controlled drug release following the same procedure.

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

Research

JoVE Journal

Bioengineering

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 ...

Assembly and Priming of the 3D-Printed Bioreactor for Perfusion Culture

Begin assembling the bioreactor under laminar flow by first assembling the two 3D printed easy flow inserts. Then using one insert as the reservoir, and the other as the reaction chamber, proceed to assemble the perfusion system. Attach two connected three-way valves to the reservoir R1 port using a system tube equipped with a one-way valve.Connect the resulting outlet with the pump head. Then connect the pump head to the reaction chamber's C4 port using similar tubing with a one-way valve. Next, connect the reaction chamber port C1 via soft wall tubing to the resistance channel with a smaller inner diameter.Create a perfusion return channel by extending the resistance channel with the system tubing, and closing the luminal circulation loop by connecting to the reservoir at R5.Create an overflow channel by connecting the reaction chamber port C3 to the reservoir port R3 using system tubing, Attach a ventilator filter through R2.Construct a pressure dampener by connecting a syringe filled with air and media to the reaction chamber C6.Using a perfusion media, prime the system through the media exchange ports and the reservoir.

Perfusion Culture of Blood Vessels in the 3D-Printed Bioreactor

To prepare the sample for perfusion culture using the 3D-printed bioreactor. In a laminar flow cabinet, place the isolated porcine carotid artery samples in cold transport media. Using a scalpel blade, remove excess connective tissue and trim the ends of the tissue.Then wash the tissue twice in cold transport media. Next, place the tissue in transport media on an orbital shaker for a minimum of 30 minutes for a thorough final washing. After that, connect a non-branching segment of the washed artery to the fabricated bioreactor system using two barbed lure connections.And secure it using a vessel bond made of vascular silicone ties. Using a small syringe attached to the first lure connection, gently flow media through the artery to ensure its patency. After securing the artery to the fabricated insert, fill the reaction space with perfusion media.Next, carefully fill the luminal circulation loop with media, removing any remaining air from the system. Lastly, connect the reaction space to the assembled perfusion system, completing the circulation. To perform the perfusion culture, place the perfusion system in an incubator.After connecting it to a peristaltic pump, connect any additional acquisition systems, such as pressure sensors. Allow the system to equilibrate overnight with a low media flow rate of approximately 10 to 15 milliliters per minute. The following day, incrementally increase the flow until a final flow rate of 35 milliliters per minute is achieved.Every three days, replace 50%of the media by connecting one syringe with fresh media to the media exchange port closer to the pump and an empty syringe to the port closer to the reservoir to collect the spent medium. After the experiment, using sterile surgical scissors, harvest the tissue from the reaction chamber by trimming the tissue ends connected to the bioreactor. Confocal imaging using endothelial and smooth muscle-specific markers revealed that the normal structure of an artery was well preserved and maintained throughout, starting from the time of harvest, to cleaning and processing, and even after perfusion culture.However, incorrect handling during processing resulted in the loss of the endothelium ahead of the culture, and the application of non-physiological culture conditions like abrupt initiation of high flow showed luminal damage. Histological evaluation of the tissue using hematoxylin and eosin staining and immunofluorescence staining at the time of harvest and after seven days of perfusion culture revealed that the morphology and overall distribution of the cells in the vessel wall were maintained throughout.