Name: Novel Process for 3D Printing Decellularized Matrices (Video) | JoVE Journal
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Learn a novel 3D printing method for tissue-engineered scaffolds using polycaprolactone and decellularized porcine cartilage matrices. This co-printing technique creates mechanically sound structures that replicate native tissue environments for improved cell culture.

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5 minutes

This project was partially funded by a grant from the Pediatric Orthopaedic Society of North America (POSNA) and the National Institutes of Health grant NIBIB R21EB025378-01 (Exploratory Bioengineering Research Grant).

1. Obtaining and Preprocessing Microspheres
<ol>
	<li>Produce microspheres with the desired matrix encapsulated (PLA-DM)2. 
	NOTE: It is imperative that the microspheres are of uniform size. For this reason, sieving the microspheres prior to use is essential. Although matrix decellularization and encapsulation have been detailed in previous publications2, a brief summary of the process follows.
	<ol>
		<li>First, harvest cartilage plugs from porcine hind limbs. Decellularize the cartilage in a series of washes with 0.05% trypsin/0.5 mm tetrasodium ethylenediaminetetraacetic acid (EDTA), Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), and 1.5% peracetic acid and 2.0% Triton X-100 for 4 h each with distilled water washes before and after each step2.</li>
		<li>Drain the decellularized matrix, freeze it, lyophilize, grind, and dissolve into pepsin solution. Following dissolution, mix the pepsin solution with PLA which has been dissolved in dichloromethane.</li>
		<li>Add the mixture dropwise into a 3% polyvinyl alcohol in water solution. Centrifuge the resulting microspheres, rinse, drain, and lyophilize again. 
		NOTE: For full details on the process see the previously published protocol2.</li>
	</ol>
	</li>
	<li>Sieve the microspheres.
	<ol>
		<li>Ensure that all sieve plates have been thoroughly cleaned and are dry prior to use. If necessary, clean sieves using ultrasonic cleaner to ensure that all spheres are removed from the sieve.</li>
		<li>Assemble the sieve shaker with the 106 &#181;m sieve tray at the top, the 53 &#181;m tray after that, and the sieve pan at the bottom.</li>
		<li>Place dry microspheres in the topmost sieve tray and place the lid on the top tray. Turn on coarse sieving for 8 to 10 min. Repeat on fine for 8 to 10 min. 
		NOTE: The sieve times may need to be increased or decreased depending on the batch.</li>
		<li>Carefully remove the sieve plates one by one and place them upside down on a large weigh paper. Tap the sides gently to ensure that most of the spheres have fallen out of the sieve and onto the paper.</li>
		<li>Discard the oversized spheres (&#62;106 &#181;m) and undersized spheres (&#60;53 &#181;m). Add spheres that are in the 53 to 106 &#181;m size range to a labeled centrifuge tube with the type and batch number then place in a -20 &#176;C freezer until further use.</li>
	</ol>
	</li>
</ol>
2. Microsphere Quality Control Assessments
Note: See Figure 1.
<ol>
	<li>Perform macroscopic/visual assessment to check that the microspheres are uniform and spherical, with no aggregates present.</li>
	<li>Assess the microspheres using a scanning electron micrograph (SEM).
	<ol>
		<li>For this, place microspheres onto an SEM chuck and sputter coat with gold-palladium in argon atmosphere using a sputter coater to a thickness of 4 nm.</li>
		<li>Observe surface features, morphology, and diameters of the microspheres using a 10 kV accelerating voltage and a 10 mm working distance to ensure that production and sieving of the microspheres was successful.</li>
	</ol>
	</li>
</ol>
3. Filament Creation for 3D Printing
<ol>
	<li>Measure and record the mass of the microspheres obtained from steps 2 and 3; at least 25 g is needed.</li>
	<li>Add polycaprolactone (PCL) powder to the microspheres for a 1:4 weight ratio of microspheres to PCL.</li>
	<li>Mix the powder mixture on a miniature rolling mixer at 20 rpm for 5 min then flip the container and mix at 20 rpm for an additional 5 min (see Figure 2).</li>
	<li>Many commercially available extruders (see the Table of Materials) have insulating jackets because their intended working temperatures are for traditional fused deposition modeling (FDM) filaments. Modify the extruder (if necessary) by removing the insulating material and use it in combination with desktop fans (which blow ambient air onto the extruder and extruded filament) to use of the extruder at lower temperatures. 
	NOTE: Desktop fans which blow ambient air to cool the extruder and filament are useful for this procedure.</li>
	<li>Setup the equipment setup for extrusion. See Figure 3.
	<ol>
		<li>Setup the extruder so that its outlet is ~60 cm from the inlet to the spooler, with a direct path from the extrusion outlet to the spooler inlet. 
		NOTE: The spooler can optionally be raised 3-4 inches from the bench if it is found that the filament is drooping to the point of touching the benchtop.</li>
		<li>Place a desktop fan ~15 cm from the heating jacket and direct it towards the heating jacket to offer cooling with ambient air throughout filament production. Place a second cooling fan approximately halfway between the extruder and spooler and direct it towards the extrudate to assist in cooling the filament with ambient air.</li>
		<li>Adjust the positioning as needed throughout the process.</li>
	</ol>
	</li>
	<li>Set the modified extruder heating element to 52 &#176;C, turn on the desktop cooling fans, and allow the instrument to come to equilibrium for 20 to 30 min. Ensure that the proper nozzle is attached to the extruder.</li>
	<li>Just before beginning, fill the extruder hopper with the microsphere/PCL mixture from step 3.3. Turn on the spooler and the extruder auger to initiate extrusion of filament.</li>
	<li>When the initial filament is extruded, manually pull the extrudate from the extrusion outlet with forceps and feed it to the filament spooler.</li>
	<li>The desired filament will take some time to come out of the spooler. Using separate spools or tape, clearly mark when the filament composition visually appears uniform.</li>
	<li>Monitor the process closely and modify parameters as necessary. Adjust the extruder temperature, extrusion auger speed, and spooler speed to obtain a 1.75 mm diameter filament as measured by calipers. Adjust the fans as needed to cool the filament properly to avoid non-circular filament cross-sections. Mix and refill the hopper as necessary. 
	NOTE: Close attention is required during this process to obtain adequate filament for subsequent 3D printing. The above parameters will change depending on the ambient conditions, the fill level and uniformity of the mixture in the hopper, and the thermodynamics and flow dynamics of the specific batches of PCL and microspheres.</li>
	<li>Continue extruding until all of the powder has been used and the hopper is almost empty. Add PCL powder (without microspheres) to the hopper to flush out the microsphere mixture that is currently in the extruder. Continue adding PCL powder to the hopper until no more microspheres are visible in the extrudate.</li>
	<li>Be sure to label and separate the filament which contains the microspheres in the desired concentration, as after the filament is cooled it is harder to distinguish the uniform filament from non-uniform filament.</li>
	<li>Continue extruding until there is minimal powder left in the hopper, then turn off the spooler, extruder auger, extruder heating element, and fans.</li>
</ol>
4. Printing with the Filament
<ol>
	<li>Design a geometry of the desired shape and form using a computer aided design software. Then slice the model and dictate the toolpath using slicing software that is compatible with the 3D printing machine being used.</li>
	<li>Load the filament from step 3 onto any standard FDM printer, fitted with standard nozzles of the desired diameter (typically 0.4 mm). Begin the print (typically at 65-70 &#176;C and 300 mm/min linear speed) as the custom filament is deposited layer-by-layer by the machine.</li>
	<li>Make sure to pay special attention to the first layer and adjust settings as needed to get a good quality print. 
	NOTE: Adjustments may be made to the print speed, print temperature, platform temperature, extrusion multiplier, and other parameters. Refer to the printer and slicing manufacturer&#39;s troubleshooting guide for further assistance.</li>
</ol>
5. Quality Control Assessment
<ol>
	<li>Place the printed constructs on SEM chucks and sputter coat with gold-palladium in argon atmosphere using a sputter coater to a thickness of 4 nm.</li>
	<li>Observe under the microscope using a 10 kV accelerating voltage and a 10 mm working distance to check surface features and for the presence or absence of microspheres if applicable.</li>
</ol>
6. Functional Testing of the Printed Constructs
NOTE: Alkaline phosphatase (ALP) can be used as a surrogate for decellularized matrix to determine if encapsulated proteins are biologically active after the filament production process. ALP is used because it catalyzes a reaction from a substrate, p-nitrophenyl phosphate, to change from colorless to yellow byproducts, p-nitrophenol and inorganic phosphate, but only if ALP is in the functional conformation.
<ol>
	<li>Print a geometry (n = 3) that has an end mass of at least 400 mg with the ALP microsphere filament (PLA-ALP/PCL) using identical print parameters as the PLA-DM/PCL scaffolds. Also print PCL-only (PCL(-)) scaffolds of the same geometry as the PLA-ALP/PCL scaffolds. Submerge them in 1 mL Tris-HCl buffer and incubate for 24 h at 37 &#176;C and 110 rpm rotation to allow enzyme diffusion.</li>
	<li>Add 1 mL of 1 mg/mL p-nitrophenyl phosphate, disodium hexahydrate in Tris-HCl. Incubate at 37 &#176;C, 110 rpm for an additional 10 h. Read the supernatant absorbance at 415 nm.</li>
</ol>

<ol><li>Hutchmaker, D., Teoh, S., Zein, I., Ng, K. W., Schantz, J. -T., Leahy, J. C. <a target="_blank" href="https://www.astm.org/DIGITAL_LIBRARY/STP/PAGES/STP15307S.htm">Design and Fabrication of a 3D Scaffold for Tissue Engineering Bone.</a> Synthetic Bioabsorbable Polymers and Implants. 15 (2), 845-847 (1988).</li>
<li>Ghosh, P., Gruber, S. M. S., Lin, C. -Y., Whitlock, P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/29394158">Microspheres containing decellularized cartilage induce chondrogenesis and remain functional after incorporation within a poly(caprolactone) filament useful for fabricating a 3D scaffold.</a> Biofabrication. , (2018).</li>
<li>Partington, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+changes+caused+by+decellularization+may+compromise+mechanical+integrity+of+tracheal+scaffolds%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds.</a> Acta Biomaterialia. 9 (2), 5251-5261 (2013).</li>
<li>Hutmacher, D. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/11071603">Scaffolds in tissue engineering bone and cartilage.</a> Biomaterials. 21 (24), 2529-2543 (2000).</li>
<li>Kang, H., Hollister, S. J., La Marca, F., Park, P., Lin, C. -Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porous+biodegradable+lumbar+interbody+fusion+cage+design+and+fabrication+using+integrated+global-local+topology+optimization+with+laser+sintering%5BTitle%5D)+AND+%22Journal+of+biomechanical+engineering%22%5BJournal%5D)">Porous biodegradable lumbar interbody fusion cage design and fabrication using integrated global-local topology optimization with laser sintering.</a> Journal of biomechanical engineering. 135 (10), 101013-101018 (2013).</li>
<li>Kang, H., Lin, C. Y., Hollister, S. J. <a target="_blank" href="https://link.springer.com/article/10.1007/s00158-010-0508-8">Topology optimization of three dimensional tissue engineering scaffold architectures for prescribed bulk modulus and diffusivity.</a> Structural and Multidisciplinary Optimization. 42 (4), 633-644 (2010).</li>
<li>Lin, C. -Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+bone+engineering+using+ex+vivo.+gene+therapy+and+topology-optimized%2C+biodegradable+polymer+composite+scaffolds%5BTitle%5D)+AND+%22Tissue+Engineering%22%5BJournal%5D)">Functional bone engineering using ex vivo. gene therapy and topology-optimized, biodegradable polymer composite scaffolds.</a> Tissue Engineering. 11 (9-10), 1589-1598 (2005).</li>
<li>Lin, C. -Y., Hsiao, C. -C., Chen, P. -Q., Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interbody+Fusion+Cage+Design+Using+Integrated+Global+Layout+and+Local+Microstructure+Topology+Optimization%5BTitle%5D)+AND+%22Spine%22%5BJournal%5D)">Interbody Fusion Cage Design Using Integrated Global Layout and Local Microstructure Topology Optimization.</a> Spine. 29 (16), 1747-1754 (2004).</li>
<li>Zopf, D., Hollister, S., Nelson, M., Ohye, R., Green, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioresorbable+Airway+Splint+Created+with+a+Three-Dimensional+Printer%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Bioresorbable Airway Splint Created with a Three-Dimensional Printer.</a> New England Journal of Medicine. 368 (21), 2043-2045 (2013).</li>
<li>Pati, F., Song, T. H., Rijal, G., Jang, J., Kim, S. W., Cho, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ornamenting+3D+printed+scaffolds+with+cell-laid+extracellular+matrix+for+bone+tissue+regeneration%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.</a> Biomaterials. 37, 230-241 (2015).</li>
<li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+interface+microstructure+on+interfacial+shear+strength+for+osteochondral+scaffolds+based+on+biomimetic+design+and+3D+printing%5BTitle%5D)+AND+%22Materials+Science+and+Engineering+C%22%5BJournal%5D)">The effect of interface microstructure on interfacial shear strength for osteochondral scaffolds based on biomimetic design and 3D printing.</a> Materials Science and Engineering C. 46, 10-15 (2015).</li>
<li>Williams, J. M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15763261">tissue engineering using polycaprolactone scaffolds fabricated via. selective laser sintering.</a> Biomaterials. 26 (23), 4817-4827 (2005).</li>
<li>Monibi, F. A., Cook, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-Derived+Extracellular+Matrix+Bioscaffolds%3A+Emerging+Applications+in+Cartilage+and+Meniscus+Repair%5BTitle%5D)+AND+%22Tissue+Engineering+Part+B%3A+Reviews%22%5BJournal%5D)">Tissue-Derived Extracellular Matrix Bioscaffolds: Emerging Applications in Cartilage and Meniscus Repair.</a> Tissue Engineering Part B: Reviews. , (2017).</li>
<li>Wiles, K., Fishman, J., Coppi, P., Birchall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Host+Immune+Response+to+Tissue-Engineered+Organs%3A+Current+Problems+and+Future+Directions%5BTitle%5D)+AND+%22Tissue+Engineering+Part+B%3A+Reviews%22%5BJournal%5D)">The Host Immune Response to Tissue-Engineered Organs: Current Problems and Future Directions.</a> Tissue Engineering Part B: Reviews. 22 (3), (2016).</li>
<li>Sutherland, A. J., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioactive+Microsphere-Based+Scaffolds+Containing+Decellularized+Cartilage%5BTitle%5D)+AND+%22Macromolecular+Bioscience%22%5BJournal%5D)">Bioactive Microsphere-Based Scaffolds Containing Decellularized Cartilage.</a> Macromolecular Bioscience. , (2015).</li>
<li>Whitlock, P. W., Smith, T. L., Poehling, G. G., Shilt, J. S., Van Dyke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+naturally+derived%2C+cytocompatible%2C+and+architecturally+optimized+scaffold+for+tendon+and+ligament+regeneration%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration.</a> Biomaterials. , (2007).</li>
<li>Whitlock, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+cyclic+strain+on+tensile+properties+of+a+naturally+derived%2C+decellularized+tendon+scaffold+seeded+with+allogeneic+tenocytes+and+associated+messenger+RNA+expression%5BTitle%5D)+AND+%22Journal+of+surgical+orthopaedic+advances%22%5BJournal%5D)">Effect of cyclic strain on tensile properties of a naturally derived, decellularized tendon scaffold seeded with allogeneic tenocytes and associated messenger RNA expression.</a> Journal of surgical orthopaedic advances. 22 (3), 224-232 (2013).</li>
<li>Whitlock, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+process+for+optimizing+musculoskeletal+allograft+tissue+to+improve+safety%2C+ultrastructural+properties%2C+and+cell+infiltration%5BTitle%5D)+AND+%22Journal+of+Bone+and+Joint+Surgery+-+Series+A%22%5BJournal%5D)">A novel process for optimizing musculoskeletal allograft tissue to improve safety, ultrastructural properties, and cell infiltration.</a> Journal of Bone and Joint Surgery - Series A. 94 (16), 1458-1467 (2012).</li>
<li>Schultz, G. S., Davidson, J. M., Kirsner, R. S., Herman, I. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21362080">Dynamic Reciprocity in the Wound Microenvironment.</a> Wound Repair Regeneration. 19 (2), 134-148 (2012).</li>
<li>Benders, K. E. M., van Weeren, P. R., Badylak, S. F., Saris, D. B. F., Dhert, W. J. A., Malda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix+scaffolds+for+cartilage+and+bone+regeneration%5BTitle%5D)+AND+%22Trends+in+Biotechnology%22%5BJournal%5D)">Extracellular matrix scaffolds for cartilage and bone regeneration.</a> Trends in Biotechnology. 31 (3), 169-176 (2013).</li>
<li>Crapo, P., Gilbert, T., Badylak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+tissue+and+whole+organ+decellularization+processes%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li>
<li>Guan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porcine+kidneys+as+a+source+of+ECM+scaffold+for+kidney+regeneration%5BTitle%5D)+AND+%22Materials+Science+and+Engineering+C%22%5BJournal%5D)">Porcine kidneys as a source of ECM scaffold for kidney regeneration.</a> Materials Science and Engineering C. 56, 451-456 (2015).</li>
<li>Dean, R. L. <a target="_blank" href="https://iubmb.onlinelibrary.wiley.com/doi/abs/10.1002/bmb.2002.494030060138">Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and divalent cations.</a> Biochemistry and Molecular Biology Education. 30 (6), 401-407 (2002).</li>
</ol>

The authors have nothing to disclose.

Both decellularized matrices and 3D printed PCL scaffolds have independently been shown to allow adhesion and proliferation of cells, validating their use for osteochondral repair10,11,12. The use of decellularized matrix in engineering approaches to tissue repair has been a subject of much interest and success in the recent past2,3,14<...

Current tissue engineering techniques for clinical applications such as bone, cartilage, tendon, and ligament reconstruction use auto- and allografts to repair damaged tissue. Each of these techniques is performed routinely as a &#34;gold standard&#34; in clinical practice by first harvesting the donor tissue either from the patient or a cadaveric match, and then placing the donor tissue into the defect site2. However, these strategies are limited by donor site morbidity, donor site scarcity for large defects, risk of infection, and difficulty finding grafts that match the desired geometry. In addition, studies have shown that allografts used for reconstruction have reduced mechanical and biologic properties when compared with native tissue3. With these considerations in mind, tissue engineers have recently turned to three dimensional (3D) bioprinting to produce custom, complex geometries that are biologically active and designed to accommodate defect size and shape while providing sufficient mechanical properties until biologic remodeling is complete.
Ideally, a 3D-printed scaffold would consist of a polymeric backbone that can retain the required mechanical stability of native tissue while the incorporated biologics offer biochemical cues to surrounding cells, leading to their migration, proliferation, differentiation, and tissue production2,5. Unfortunately, most constructs that contain biologic components are made with gels or polymers that are too weak to withstand in vivo forces experienced by the targeted tissues for auto/allograft reconstruction. Other polymers such as polylactic acid (PLA) are bioresorbable, 3D printable and structurally sound, but are printed at temperatures at or above 210 &#176;C - making it impossible for biologics to be co-printed during fabrication. Polycaprolactone (PCL) is another FDA-cleared, bioresorbable polymer that can be 3D printed at a lower temperature (65 &#176;C), which has become increasingly popular in fabricating patient-specific implants with complex morphologies5,6,7,8,9. However, most bioprinters using pneumatic technology make it impossible to print PCL at lower temperatures where biological activities can remain unharmed. To date, the integration of these polymers with auto/allografts into a novel printable biomaterial has yet to be accomplished. In the absence of such a material, a true tissue engineered approach to tissue reconstruction is unlikely. Therefore, we have sought to combine PLA, PCL, and decellularized allograft matrices (DM) to utilize the advantages of each material in order to manufacture a viable construct capable of reconstructing complex tissues. This process would provide the initial mechanical strength necessary to resist in vivo forces and the thermal stability to accommodate additive manufacturing in a construct that induces formation of the desired tissue.
In a recent attempt to address the aforementioned hurdles, we showed that it is feasible to encapsulate decellularized cartilage extracellular matrix within a thermally protective PLA barrier that can be extruded within PCL filaments, maintaining the ability of DM to influence surrounding host cells2. This has inspired us to seek clinically effective approaches for tissue reconstruction. In the current study, we utilize the platform technology to build all-in-one scaffolds that include PLA, DM, and PCL (PLA-DM/PCL).
Our goal is to improve the efficacy and utility of allografts using the proposed novel biofabrication technique to more accurately recapitulate native tissue, to ultimately use them in various applications.

<table><tbody><tr><td>Sieve machine</td><td>Haver &amp;amp; Boecker Tyler</td><td>Ro-Tap RX 29-E Pure</td><td></td></tr><tr><td>Sieve 90 um</td><td>Fisherbrand</td><td>170328156</td><td>No. 170</td></tr><tr><td>Sieve 53 um</td><td>Fisherbrand</td><td>162513588</td><td>No. 270</td></tr><tr><td>Sieve 106 um</td><td>Fisherbrand</td><td>162018121</td><td>No. 140</td></tr><tr><td>Sputter coater</td><td>Leica</td><td>n/a</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>Hitachi, USA</td><td>n/a</td><td></td></tr><tr><td>Filabot EX2</td><td>Filabot.com</td><td>FB00061</td><td></td></tr><tr><td>Filabot Spooler</td><td>Filabot.com</td><td>FB00073</td><td></td></tr><tr><td>CAPA 6506</td><td>Perstorp</td><td>24980-41-4</td><td></td></tr><tr><td>Phosphate buffered saline, PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>6&quot; Fan</td><td>Comfort Zone, Amazon</td><td>n/a</td><td></td></tr><tr><td>Ultrasonic Water Bath</td><td>Cole Parmer</td><td>SK-08895-13</td><td></td></tr><tr><td>Dreamer</td><td>FlashForge</td><td>n/a</td><td></td></tr><tr><td>Drum Mixer</td><td>Custom made</td><td>n/a</td><td>Similar piece of equipment: https://www.coleparmer.com/i/argos-technologies-flexiroll-digital-tube-roller-shaker-120-vac/0439744?PubID=UX&amp;amp;persist=true&amp;amp;ip=&lt;br/&gt; no&amp;amp;gclid=CjwKCAjw-&lt;br/&gt; dXaBRAEEiwAbwCi5khGDMz0&lt;br/&gt; dTjsraEsBGfhMEH7ytx&lt;br/&gt; LQWGUPNgUJYQ1p3vj_yxkYoI_&lt;br/&gt; ixoC9GwQAvD_BwE</td></tr><tr><td>Micro Balance</td><td>Mettler Toledo, Fisher Scientific</td><td>01-913-851</td><td></td></tr><tr><td>Simplify3D</td><td>Simplify3D</td><td>n/a</td><td></td></tr><tr><td>SolidWorks</td><td>SolidWorks</td><td>n/a</td><td></td></tr><tr><td>Microspheres</td><td>Produced in-house, see concurrently submitted JoVE submission</td><td></td><td></td></tr><tr><td>p-nitrophenyl phosphate, disodium salt, hexahydrate</td><td>Millipore</td><td>4876-5GM</td><td></td></tr><tr><td>Phosphatase, alkaline</td><td>Roche Diagnostics GmbH</td><td>10 713 023 001</td><td></td></tr><tr><td>Absorbance Reader</td><td>Tecan</td><td>Sunrise</td><td></td></tr><tr><td>Tris-HCl Buffer</td><td>Sigma-Aldrich</td><td>T6455-100ML</td><td></td></tr><tr><td>Heated shaker</td><td>New Brunswick Scientific</td><td>Excella E24</td><td></td></tr></tbody></table>

novel process for 3d printing decellularized matrices

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

After sieving, microspheres should appear uniform and be free from aggregates. Under SEM, the sieved microspheres may have small pores on their surface, but will otherwise be spherical and smooth, as shown in Figure 1. All extruded filaments should be of uniform diameter and circular cross-section. A filament that contains microspheres (PLA-DM/PCL) will have a slightly more matte finish while a PCL-only (PCL(-)) filament would look more glossy. The PLA-DM/PCL...

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

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Research

JoVE Journal

Bioengineering

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

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

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (FcεRI).

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

Fabrication of Decellularized Spleen Matrix Derived from Rats

This protocol describes the fabrication of 3D bone constructs using human osteocytes embedded in methacrylated gelatin (GelMA) hydrogels supplemented with decellularized extracellular matrix. The method is low-cost, scalable, and compatible with basic laboratory equipment, providing a translational platform for studying musculoskeletal disease processes.

A 3D Bone Culture Platform Using Human Osteocytes and Decellularized Extracellular Matrix for Modeling Musculoskeletal Diseases

Learn a novel 3D printing method for tissue-engineered scaffolds using polycaprolactone and decellularized porcine cartilage matrices. This co-printing technique creates mechanically sound structures that replicate native tissue environments for improved cell culture.

Novel Process for 3D Printing Decellularized Matrices

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