Name: ceramic omnidirectional bioprinting in cell-laden suspensions for the generation of bone analogs
Uploaded: 2022-08-08T13:00:00
Description: Watch this Scientific Journal Video about Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs at JoVE.com

The authors would like to acknowledge the National Health and Medical Research Council (Grant no. GNT1111694 and GNT1141602) and the Australian Research Council (Grant no. FT180100417, FL150100060, and CE14100036). The authors would like to acknowledge the Biomedical Imaging Facility at the University of New South Wales. Figures were created with Biorender.com, Adobe Photoshop, and Adobe Illustrator and have been exported under a paid subscription.

1. Bone-ink fabrication
<ol>
	<li>Synthesis of &#945;-tricalcium phosphate
	<ol>
		<li>Weigh out calcium hydrogen phosphate (CaHPO4) and calcium carbonate (CaCO3) powders in a 3:2 Ca:P molar ratio. Using a spatula, thoroughly homogenize the two powders.</li>
		<li>Add the calcium hydrogen phosphate-calcium carbonate powder mixture to a zirconia crucible such that it is no more than 75% full. 
		NOTE: To avoid contamination, use a new crucible or a crucible previously used to ...

<ol>
	<li>Bates, P., Ramachandran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+injury,+healing+and+grafting.">Bone injury, healing and grafting.</a> Basic Orthopaedic Sciences. The Stanmore Guide. , 123-134 (2007).</li><li>Lin, X., 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=The+bone+extracellular+matrix+in+bone+formation+and+regeneration.">The bone extracellular matrix in bone formation and regeneration.</a> Frontiers in Pharmacology. 11, 757 (2020).</li><li>Reznikov, N., 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=A+materials+science+vision+of+extracellular+matrix+mineralization.">A materials science vision of extracellular matrix mineralization.</a> Nature Reviews Materials. 1, 16041 (2016).</li><li>Kang, H. W., 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=A+3D+bioprinting+system+to+produce+human-scale+tissue+constructs+with+structural+integrity.">A 3D bioprinting system to produce human-scale tissue constructs with structural integrity.</a> Nature Biotechnology. 34 (3), 312-319 (2016).</li><li>Lin, 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=3D+printing+of+bioceramic+scaffolds-Barriers+to+the+clinical+translation:+From+promise+to+reality,+and+future+perspectives.">3D printing of bioceramic scaffolds-Barriers to the clinical translation: From promise to reality, and future perspectives.</a> Materials. 12 (17), 2660 (2019).</li><li>Qu, M., 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=Multi-dimensional+printing+for+bone+tissue+engineering.">Multi-dimensional printing for bone tissue engineering.</a> Advanced Healthcare Materials. 10 (11), 2001986 (2021).</li><li>Lode, A., 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=Fabrication+of+porous+scaffolds+by+three-dimensional+plotting+of+a+pasty+calcium+phosphate+bone+cement+under+mild+conditions.">Fabrication of porous scaffolds by three-dimensional plotting of a pasty calcium phosphate bone cement under mild conditions.</a> Journal of Tissue Engineering and Regenerative Medicine. 8 (9), 682-693 (2014).</li><li>Bernal, P. N., 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=Volumetric+bioprinting+of+complex+living-tissue+constructs+within+seconds.">Volumetric bioprinting of complex living-tissue constructs within seconds.</a> Advanced Materials. 31 (42), 1904209 (2019).</li><li>Diloksumpan, P., 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=Combining+multi-scale+3D+printing+technologies+to+engineer+reinforced+hydrogel-ceramic+interfaces.">Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces.</a> Biofabrication. 12 (2), 025014 (2020).</li><li>Thrivikraman, 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=Rapid+fabrication+of+vascularized+and+innervated+cell-laden+bone+models+with+biomimetic+intrafibrillar+collagen+mineralization.">Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization.</a> Nature Communications. 10 (1), 3520 (2019).</li><li>Romanazzo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+bone-like+structures+through+omnidirectional+ceramic+bioprinting+in+cell+suspensions.">Synthetic bone-like structures through omnidirectional ceramic bioprinting in cell suspensions.</a> Advanced Functional Materials. 31 (13), 2008216 (2021).</li><li>Hinton, T. 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=Three-dimensional+printing+of+complex+biological+structures+by+freeform+reversible+embedding+of+suspended+hydrogels.">Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.</a> Science Advances. 1 (9), 1500758 (2015).</li><li>Phromsopha, T., Baimark, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+starch/gelatin+blend+microparticles+by+a+water-in-oil+emulsion+method+for+controlled+release+drug+delivery.">Preparation of starch/gelatin blend microparticles by a water-in-oil emulsion method for controlled release drug delivery.</a> International Journal of Biomaterials. 2014, 829490 (2014).</li><li>Moreno, D., 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=Solid-state+synthesis+of+alpha+tricalcium+phosphate+for+cements+used+in+biomedical+applications.">Solid-state synthesis of alpha tricalcium phosphate for cements used in biomedical applications.</a> Bolet&#237;n de la Sociedad Espa&#241;ola de Cer&#225;mica y Vidrio. 59 (5), 193-200 (2020).</li></ol>

The 3D printing technique COBICS was developed to enable the fabrication of mineralized bone-like structures via extrusion into a crosslinkable microgel suspension containing live cells. The technique has been applied to a degradable microgel suspension, and cells show good viability, spreading, and osteogenic differentiation ability within the system11. A key determinant of the success of constructs created using this technique is the proper synthesis of the &#945;-TCP-based bone-ink. First, the ...

Bone has remarkable regeneration abilities as one of the few structures in the body that can heal by recreating its normal cellular composition, orientation, and mechanical strength up until a critical defect size, when endogenous healing capacity is compromised1. Bone, together with cartilage and ligament, supports and facilitates body movement, while also storing minerals and fats and producing blood cells. As a hard, dense connective tissue, bone is mainly composed of an inorganic phase, water, and organic material composed primarily of collagen fibers2. Cells are embedded within this highly mineralized matrix of collagen I fibers and hydroxyapatite (HA) crystals, forming a hierarchical structure3.
The complex organization of this tissue makes the fabrication of synthetic alternatives to replicate the heterogeneous bone micro- and nano-environments exceptionally challenging3. For this purpose, a variety of materials, including bioceramics, cell-laden hydrogels, and synthetic materials have been proposed as solutions to create bone matrices. Among the scaffold fabrication techniques, 3D printing-based techniques have recently emerged and received much attention from the tissue engineering community owing to their remarkable ability to allow the fabrication of highly sophisticated and precise structures with great promise of patient-specific treatment4,5,6. Hydrogels have been the most popular choice of matrix mimics and bio-inks since they can be printed together with cells and bioactive molecules, generating functional constructs6. However, hydrogels lack the functional properties of bone, such as mechanical strength and a highly calcified, inorganic phase containing metabolically active cells.
3D printed ceramic scaffolds typically require postprocessing steps, including sintering, high-temperature treatments, or using harsh chemicals that must be thoroughly washed before in vitro or in vivo applications5. To address these limitations, Lode et al.7 recently developed an &#945;-tricalcium phosphate-based paste formed by hydroxyapatite, which can be printed and set under physiological conditions. However, this material still cannot be printed together with live cells as it requires post-treatment in a humid environment and subsequent aqueous solution immersion for a long period.
Alternatively, cell-laden hydrogels with inorganic particles incorporated have been proposed as a replacement for 3D bone matrix8,9. Despite their great ability to support cell viability, they are not able to recapitulate the densely mineralized bone tissue environment. Thrivikarman et al.10 adopted a biomimetic approach in which a supersaturated calcium and phosphate medium was used with a non-collagenous protein analog to better mimic the nanoscale apatite deposition. However, their constructs still cannot generate rigid 3D constructs with micro- and macro-scale architecture resembling bone.
The present study addresses these shortcomings through the development of a printing strategy to fabricate bone-mimicking constructs, in inorganic and organic phases, that are able to integrate both cells and growth factors11. COBICS uniquely recapitulates the mineral and cellular structure of bone using a microgel-based bioprinting technique. The protocol herein describes the process of synthesizing the ceramic bone-ink and gelatin-based microgels and then combining cells that enable COBICS. The process begins with the synthesis of the main precursor material of the bone-ink. The cross-linkable hydrogel is then synthesized and formed into microgels. Lastly, the bone-ink is deposited omnidirectionally in a support bath of the microgels laden with cells (Figure 1).
The bone-ink may be printed into any suspension of microgels that have the appropriate yield-stress characteristics, that is, the ability to fluidize at a specific shear rate and subsequently support the deposited structure. Two flexible approaches have been demonstrated: a suspension consisting of gelatin microgels and a suspension consisting of gelatin methacrylate (GelMA) microgels. The former suspension dissolves when the temperature is raised to 37 &#176;C, the freeform reversible embedding of suspended hydrogels (FRESH) technique12, while the latter can be photocrosslinked after printing, effectively &#34;stitching&#34; the microgels together and locking the printed bone-ink in place. The present study focuses on using GelMA as the matrix as it provides the unique advantage of being able to support cell growth with in situ printing of complex bone mimetic structures. Ultimately, this approach enables the generation of complex tissue models with high levels of biomimicry and broad implications for disease modeling, drug discovery, and regenerative engineering.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63943/63943fig01.jpg" /> 
Figure 1: Schematic of the workflow. (A) The bone-ink is synthesized starting from &#945;-tricalcium phosphate synthesis and its subsequent combination with glycerol, polysorbate 80, and ammonium phosphate dibasic. (B) GelMA microgels are fabricated by the water-in-oil emulsion method. The obtained microgels are then (C) hydrated and (D) combined with cells. Cell-microgel composites are then used as a granular bath in which the bone-ink is deposited. (E) The whole construct is then UV-crosslinked and transferred to the incubator for culture. Abbreviations:&#160;&#945;-TCP =&#160;&#945;-tricalcium phosphate; GelMA =gelatin methacrylate.&#160;<a href="https://www.jove.com/files/ftp_upload/63943/63943fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Printer Extruder</td>
			<td>Hyrel3D</td>
			<td>EMO-25</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Falcon</td>
			<td>BDAA352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol 100% Denatured</td>
			<td>Chem-Supply</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Chem-Supply</td>
			<td>154871</td>
			<td></td>
		</tr>
		<tr>
			<td>Alumina crucible</td>
			<td>Coors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium phosphate dibasic (NaHPO4)</td>
			<td>Sigma</td>
			<td>A5764</td>
			<td></td>
		</tr>
		<tr>
			<td>Autodesk Fusion 360</td>
			<td>Autodesk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet level 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium carbonate</td>
			<td>Sigma</td>
			<td>239216</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hydrogen phosphate (CaHPO4)</td>
			<td>Sigma</td>
			<td>C7263</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>various volumes used</td>
		</tr>
		<tr>
			<td>Cellulose Dialysis Tubes, 14 kDa cut-off</td>
			<td>Sigma</td>
			<td>D9777</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td>3-16KL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensing Tip, 23 G</td>
			<td>Nordson</td>
			<td>7018302</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate</td>
			<td>Thermo FIsher</td>
			<td>11885084</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo FIsher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Elevator furnace</td>
			<td>Labec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Engine HR Multihead Printer</td>
			<td>Hyrel3D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Bovogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin type A, from porcine skin</td>
			<td>Sigma</td>
			<td>G2500</td>
			<td></td>
		</tr>
		<tr>
			<td>General Purpose Stainless Steel Tips</td>
			<td>Nordson EF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Human adipose derived stem cells</td>
			<td>ATCC</td>
			<td>PCS-500-011</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 800 Confocal Microscope</td>
			<td>ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer (Alpha 1-4 LDplus)</td>
			<td>Christ</td>
			<td>101541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic hot plate and stirrer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrylic anhydride</td>
			<td>Sigma</td>
			<td>276685</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini 2 Desktop 3D Printer</td>
			<td>LulzBot</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm sealing film</td>
			<td>Parafilm</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo FIsher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary ball mill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary ball mill jar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylenesorbitan monooleate Tween-80</td>
			<td>Sigma</td>
			<td>P6224</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>FEI Nova NanoSEM 450 FE-SEM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Science Kimwipes Delicate Task Wipers</td>
			<td>Kimtech</td>
			<td>18813156</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel standard test sieve</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sunflower Oil</td>
			<td>Community Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25% phenol red</td>
			<td>Thermo FIsher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Microscope Software</td>
			<td>ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead viability/ cytotoxicity kit for mammalian cells </td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, low glucose, no phenol red </td>
			
			<td>Thermo Fisher</td>
			<td>11054020</td>
<td></td>
		</tr>
	</tbody>
</table>

ceramic omnidirectional bioprinting in cell-laden suspensions for the generation of bone analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering) which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. To demonstrate COBICS formation of complex bone structures and the co-printing of cells in microgel suspensions, representative images of bone-like composites made of the bone-ink were taken, and a semi-quantitative analysis of ADSC viability...

Watch this Scientific Journal Video about Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs at JoVE.com

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

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