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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+tissue+engineering+in+articular+cartilage+repair+and+regeneration.">The role of tissue engineering in articular cartilage repair and regeneration.</a> Critical Reviews&#8482; in Biomedical Engineering. 37 (1-2), (2009).</li><li>Athanasiou, K., Rosenwasser, M., Buckwalter, J., Malinin, T., Mow, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+comparisons+of+in+situ+intrinsic+mechanical+properties+of+distal+femoral+cartilage.">Interspecies comparisons of in situ intrinsic mechanical properties of distal femoral cartilage.</a> Journal of Orthopaedic Research. 9 (3), 330-340 (1991).</li><li>Schinagl, R. M., Gurskis, D., Chen, A. C., Sah, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth-dependent+confined+compression+modulus+of+full-thickness+bovine+articular+cartilage.">Depth-dependent confined compression modulus of full-thickness bovine articular cartilage.</a> Journal of Orthopaedic Research. 15 (4), 499-506 (1997).</li><li>Athanasiou, K., Niederauer, G., Schenck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+topography+of+human+ankle+cartilage.">Biomechanical topography of human ankle cartilage.</a> Annals Biomedical Engineering. 23 (5), 697-704 (1995).</li><li>Athanasiou, K. A., Liu, G. T., Lavery, L. A., Lanctot, D. R., Schenck, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+topography+of+human+articular+cartilage+in+the+first+metatarsophalangeal+joint.">Biomechanical topography of human articular cartilage in the first metatarsophalangeal joint.</a> Clinical Orthopaedics and Related Research. 348, 269-281 (1998).</li><li>Guilak, F., Jones, W. R., Ting-Beall, H. P., Lee, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deformation+behavior+and+mechanical+properties+of+chondrocytes+in+articular+cartilage.">The deformation behavior and mechanical properties of chondrocytes in articular cartilage.</a> Osteoarthritis and Cartilage. 7 (1), 59-70 (1999).</li><li>Mathew, A. P., Oksman, K., Karim, Z., Liu, P., Khan, S. A., Naseri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+scale+up+and+characterization+of+wood+cellulose+nanocrystals+hydrolysed+using+bioethanol+pilot+plant.">Process scale up and characterization of wood cellulose nanocrystals hydrolysed using bioethanol pilot plant.</a> Industrial Crops and Products. 58, 212-219 (2014).</li><li>Compton, B. G., Lewis, J. 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-printing+of+lightweight+cellular+composites.">3D-printing of lightweight cellular composites.</a> Advanced Materials. 26 (34), 5930-5935 (2014).</li><li>Sarem, M., Moztarzadeh, F., Mozafari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+genipin+assist+gelatin/carbohydrate+chitosan+scaffolds+to+act+as+replacements+of+load-bearing+soft+tissues.">How can genipin assist gelatin/carbohydrate chitosan scaffolds to act as replacements of load-bearing soft tissues.</a> Carbohydrate Polymers. 93 (2), 635-643 (2013).</li><li>Chia, H. N., Hull, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compressive+moduli+of+the+human+medial+meniscus+in+the+axial+and+radial+directions+at+equilibrium+and+at+a+physiological+strain+rate.">Compressive moduli of the human medial meniscus in the axial and radial directions at equilibrium and at a physiological strain rate.</a> Journal of orthopaedic research. 26 (7), 951-956 (2008).</li><li>Zhang, K., Fan, Y., Dunne, N., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+microporosity+on+scaffolds+for+bone+tissue+engineering.">Effect of microporosity on scaffolds for bone tissue engineering.</a> Regenerative biomaterials. 5 (2), 115-124 (2018).</li><li>Lin, N., 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+in+biomedicine:+Current+status+and+future+prospect.">Nanocellulose in biomedicine: Current status and future prospect.</a> European Polymer Journal. 59, 302-325 (2014).</li><li>Domingues, R. M., Gomes, M. E., Reis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+cellulose+nanocrystals+in+tissue+engineering+strategies.">The potential of cellulose nanocrystals in tissue engineering strategies.</a> Biomacromolecules. 15 (7), 2327-2346 (2014).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:1217px;" width="1215">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<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>
		</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 ...

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.

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 ...

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 ...

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 ...

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 ...

3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, ...

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 ...

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 ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

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

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