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 ...

This protocol allows fabrication of bone analogs without requiring heating or toxic chemicals. In this way, bone structures are formed with the potential for repairing a bone defect in clinical settings using a patient's own cells. The main advantage of the technique is that the bone ink can be printed with living cells to form any complex bone-like structure and to direct differentiation toward bone forming lineage.Further, drugs can be loaded into the bone-ink to simultaneously enhance wound healing, bone regeneration, and to treat pathologies like cancer. Demonstrating the procedure will be Gagan Jalandhra, a PhD candidate in my lab, and Dr.Sara Romanazzo, who has worked with me and Dr.Jalandhra on various projects, including development of this technology. Begin by adding the calcium hydrogen phosphate and calcium carbonate powder mixture to a zirconia crucible such that it is no more than 75%full.Transfer the crucible to a furnace, heat it to 1, 400 degrees Celsius at a rate of five degrees Celsius per minute and hold for three hours. Quench the reaction by removing the crucible from the furnace and leaving at a top of refractory block. Allow it to cool completely before handling.Use a mortar and pestle to break and grind the alpha-tricalcium phosphate cake such that the resulting granules have a maximum size of 200 micrometers. Add three millimeter yttria-stabilized zirconia balls to the grinded mixture, followed by 100%ethanol. Secure the lid and grind for two hours at 180 rotations per minute.Collect the suspension and separate the balls, using 100%ethanol for washing. Dry the suspension in an oven at 120 degrees Celsius for 24 hours. Add the dried powder to the milling jars with one millimeters zirconia balls and 100%ethanol in the same weight ratios as in the first stage.Grind for two hours at 180 rotations per minute. Then separate and dry in the oven. Remove the sample from the oven.To make the bone-ink, add 630 microliters of glycerol and 130 microliters of polysorbate 80 to a ball mil jar. Then add 100 milligrams of ammonium phosphate dibasic and two grams of alpha-tricalcium phosphate powder to the solution and stir continuously with a spatula to combine. Add a 25 millimeters zirconia ball, secure the lid and place it inside a planetary mill for 60 minutes at 180 rotations per minute, pausing halfway to scrape down the sides of the jar with a spatula using a spatula.Load the ink into a one milliliter syringe. Make a 10%solution of gelatin type A in 1X PBS as described in the text manuscript. Place the conical flask on the stirrer.Then add 5.796 milliliters of methacrylic anhydride and continue stirring in the dark at 50 degrees Celsius for 90 minutes. Quench the reaction by diluting the conical flasks contents two fold with PBS. Decant into 50 milliliter tubes and remove the unreacted methacrylic anhydride by centrifugation at 3000 RCF at room temperature for three minutes and collecting the supernatant.Dialyze the supernatant inside 14 kilodalton cutoff cellulose dialysis tubes against deionized water at 40 degrees Celsius for five days while gently stirring. Replace the deionized water every day. Prepare for storage by decanting into 50 milliliter tubes, securing the cap and placing it in the refrigerator for 12 hours.Then freeze the samples using liquid nitrogen and immediately lyophilize for five days at minus 54 degrees Celsius and 0.4 millibars. Store the resulting foam in a freezer at minus 20 degrees Celsius. To synthesize GelMA microgels, make a 10%weight by weight GelMA solution in PBS by weighing the lyophilized GelMA, adding it to a tube with PBS and heating in a water bath at 50 degrees Celsius until fully hydrated.Add 37 milliliters of oil per one milliliter of GelMA solution to a beaker, ensuring it is no more than 65%full. Set up a double beaker system on a hot plate with magnetic stirring by placing the beaker containing oil inside a larger beaker. Heat to 40 degrees Celsius while stirring.Load the GelMA solution into a syringe and add it dropwise into the stirring oil through a 0.45 micrometer filter. Allow the emulsion to equilibrate for 10 minutes. Reduce the temperature of the emulsion to 15 degrees Celsius to thermally.Stabilize the spheres by adding crushed ice into the space between the two beakers. Add acetone to the spinning GelMA emulsion. Decant the contents of the beaker into 50 milliliter tubes, making sure to wash the walls of the beaker with acetone.Let it rest for 20 minutes to allow the dehydrated micro gels to settle to the bottom. Discard the supernatant and wash the microgels twice with acetone. Consolidate into one tube, top up with acetone and sonicate for 10 seconds.Again, wash it with acetone twice. Store in acetone at room temperature until required for printing. To prepare the GelMA microgel suspension for printing, formulate a 1%weight by weight solution of GelMA in DMEM as described in the text manuscript.Evaporate the acetone from the dehydrated microgels and weigh the resulting powder into a tube. Add acetone and transfer to a sterile environment. To form the microgel suspension, evaporate the acetone from the powder inside the biosafety cabinet.After evaporation, add DMEM, 1%GelMA solution in DMEM and 2.5%LAP initiator solution to achieve a final packing fraction of 30%Allow to fully hydrate for at least 12 hours at room temperature. Culture the adipose-derived mesenchymal stem cells in low glucose DMEM supplemented with 10%FBS and 1%penicillin-streptomycin at 37 degrees Celsius and 5%carbon dioxide until confluent. Remove the adipose-derived mesenchymal stem cells from the tissue culture flask by removing the medium and washing with sterile PBS.Pellet the cells by centrifuging at 150 RCF at room temperature for five minutes. Count the cells and calculate at least 5 x 10 to the five cells for each milliliter of GelMA microgels. Allocate the required volume of the cell suspension to a separate tube and pellet as demonstrated earlier.Carefully remove as much supernatant as possible using a pipette, leaving only the cell pellet. Add the required volume of microgel suspension to the pellet and gently pipette up and down to ensure even distribution. Load the cell-laden in microgel suspension into a reactor using a pipette.Using a one milliliter syringe fitted with a 23 gauge needle, deposit the bone-ink, crosslink the cell and bone-ink laden GelMA microgel constructs with a UV crosslinker lamp at 405 nanometers for 90 seconds. Immediately transfer the microgel suspension to an appropriately sized well plate containing complete DMEM. Incubate at 37 degrees Celsius and 5%carbon dioxide.Replace the culture medium after 24 hours, then every 48 to 72 hours as required. The bone-ink was analyzed by scanning electron microscopy to determine its micro structure, which showed the formation of hydroxyapatite crystal after ink setting, particularly visible in dry conditions. In this study, cells were mixed with GelMA microgels and kept in culture for up to five days, either in micro gel suspension only or in the COBICS system.The cells showed good viability in the presence of the bone-ink, both on day one and day five, confirming that the leaching products of the ink were not detrimental to the cells. It is crucial to control the stirrig speed while synthesizing microgel as a slight changes stirring can cause size differences. It is also important to ensure homogenous distribution of the cells within microgel suspension.The same protocol can be used to print inside gelatin microgel suspensions, which act as sacrificial supports, leaving behind standalone bone constructs. But using GelMA opens up the avenue for one pot multiphasic constructs, such as osteochondral or bone tendon interfaces.

Research

JoVE Journal

Bioengineering

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...