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>
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Y., Whitlock, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspheres+containing+decellularized+cartilage+induce+chondrogenesis+and+remain+functional+after+incorporation+within+a+poly(caprolactone)+filament+useful+for+fabricating+a+3D+scaffold.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+changes+caused+by+decellularization+may+compromise+mechanical+integrity+of+tracheal+scaffolds.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+in+tissue+engineering+bone+and+cartilage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+biodegradable+lumbar+interbody+fusion+cage+design+and+fabrication+using+integrated+global-local+topology+optimization+with+laser+sintering.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+optimization+of+three+dimensional+tissue+engineering+scaffold+architectures+for+prescribed+bulk+modulus+and+diffusivity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+bone+engineering+using+ex+vivo.+gene+therapy+and+topology-optimized,+biodegradable+polymer+composite+scaffolds.">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. -. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ornamenting+3D+printed+scaffolds+with+cell-laid+extracellular+matrix+for+bone+tissue+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+interface+microstructure+on+interfacial+shear+strength+for+osteochondral+scaffolds+based+on+biomimetic+design+and+3D+printing.">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. 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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 past2,3,14,15. We have previously noted the increased migration, adhesion, proliferation, and overall maintenance of resulting tissues when compared to traditional techniques2,15,16,17,18. Many have attributed these desirable results to the process of dynamic reciprocity through which the host cells receive cues from the decellularized matrix, dynamically respond, and replicate the cues for new cells by laying more extracellular matrix that typically resembles what is already present19,20,21,22. While this has been studied for many applications, many of the processes are not easy to replicate and cannot be adapted for different uses, unable to successfully create highly patient-specific constructs, unable to create complex morphologies, and unable to withstand in vivo forces2,3,4,13,14,15,16.
The innovative approach proposed herein avoids both transient and prolonged exposure to high temperatures that are typically required by 3D printing when using traditional mechanical extrusion-based FDM printers with a new carrier vehicle. Moreover, the carrier vehicle (PLA microspheres) helps protect the encapsulated biologic for the relatively short period of time it is exposed to heat and provides an all-in-one treatment option for fast turnover in the clinic2. The methods proposed herein demonstrate how to create biologically active filaments for 3D printing and scaffolds via 3D printing where a critical step is the extrusion of the filament and the printing of those filaments at low temperatures (65 &#176;C). The ability of the encapsulated proteins to remain functional was demonstrated by using ALP as a surrogate for DM throughout the process. ALP was used as the enzyme must be in a very specific functional conformation in order to catalyze the colorimetric reaction assessed in this protocol23. If the filament is not extruded with careful attention to diameter, temperature, and speed, the biologic activity and utility for 3D printing would be sacrificed.
In this protocol, microspheres containing decelluarized matrices (PLA-DM) were co-extruded with PCL to make 3D printable filaments and 3D printed scaffolds for osteochondral repair (PLA-DM/PCL). As mentioned in the protocol steps, continuous monitoring of the filament production process is essential for high quality of the filament. Adjustments must be made to extrusion speed, spooler speed, and extrusion temperature in order to maintain the desired filament diameter (typically 1.75 mm). The presence of the microspheres in the scaffolds is confirmed by SEM imaging and the maintenance of enzyme functionality is demonstrated by an alkaline phosphatase assay. Note that this protocol is limited by the large amount of microspheres required for production and the relatively lower resolution of fused deposition modeling to other 3D printing modalities. Nevertheless, the increased biologic activity is a major advancement. Although not the focus of this protocol, subsequent studies will concentrate on the impact of the microspheres on mechanical strength, cell migration and differentiation, and further characterizations of the scaffolds. Overall, the technique described herein allows decellularized matrix and other proteins to be printed at lower temperatures than previously allowed and in thermally protective barriers in order to maintain function and mechanical strength2,3.

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 border="0" cellpadding="0" cellspacing="0" style="width:877px;" width="877">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Sieve machine</td>
			<td style="width:396px;">Haver &#38; Boecker Tyler</td>
			<td style="width:132px;">Ro-Tap RX 29-E Pure</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sieve 90 um</td>
			<td>Fisherbrand</td>
			<td>170328156</td>
			<td>No. 170</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sieve 53 um</td>
			<td>Fisherbrand</td>
			<td>162513588</td>
			<td>No. 270</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sieve 106 um</td>
			<td>Fisherbrand</td>
			<td>162018121</td>
			<td>No. 140</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sputter coater</td>
			<td>Leica</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanning Electron Microscope</td>
			<td>Hitachi, USA</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filabot EX2</td>
			<td>Filabot.com</td>
			<td>FB00061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filabot Spooler</td>
			<td>Filabot.com</td>
			<td>FB00073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CAPA 6506</td>
			<td>Perstorp</td>
			<td>24980-41-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline, PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6&#34; Fan</td>
			<td>Comfort Zone, Amazon</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic Water Bath</td>
			<td>Cole Parmer</td>
			<td>SK-08895-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dreamer</td>
			<td>FlashForge</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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&#38;persist=true&#38;ip= 
			no&#38;gclid=CjwKCAjw- 
			dXaBRAEEiwAbwCi5khGDMz0 
			dTjsraEsBGfhMEH7ytx 
			LQWGUPNgUJYQ1p3vj_yxkYoI_ 
			ixoC9GwQAvD_BwE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro Balance</td>
			<td>Mettler Toledo, Fisher Scientific</td>
			<td>01-913-851</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Simplify3D</td>
			<td>Simplify3D</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SolidWorks</td>
			<td>SolidWorks</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microspheres</td>
			<td>Produced in-house, see concurrently submitted JoVE submission</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">p-nitrophenyl phosphate, disodium salt, hexahydrate</td>
			<td>Millipore</td>
			<td>4876-5GM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphatase, alkaline</td>
			<td>Roche Diagnostics GmbH</td>
			<td>10 713 023 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absorbance Reader</td>
			<td>Tecan</td>
			<td>Sunrise</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T6455-100ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Excella E24</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 filament would also feel coarser to the touch than the PCL(-) filament. Scaffolds should be printed in the desired geometry which was dictated by the software in step 4.1. The scaffold quality and shape should be repeatable and uniform from one print to another. After printing, scaffolds with and without microspheres will be difficult to distinguish macroscopically, but under SEM, microspheres should be visible on the surface and throughout the constructs. Under SEM, PCL(-) filament will appear smooth, with some striations as an artifact of the extrusion process (Figure 4B). Microspheres should be visible both protruding through and under the surface of the PLA-DM/PCL scaffolds (see Figure 4C). When using ALP as a surrogate for DM, the functionality of the enzyme within the scaffold should be maintained with significantly higher absorbance (t-test, p &#60; 0.05) at 415 nm than those of blank PCL(-) scaffolds, 0.297 &#177; 0.023 and 0.166 &#177; 0.012, respectively, Figure 5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58720/58720fig1.jpg" /> 
Figure 1.&#160;Representative macroscopic (left) and SEM (right) images of microspheres after preparation and sieving2. Note that the microspheres are spherical and in the appropriate size range (53-106 &#181;m diameter). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58720/58720fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58720/58720fig2.jpg" /> 
Figure 2. Custom made rolling mixer. The custom-made rolling mixer is used for combining the microspheres with PCL powder. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58720/58720fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58720/58720fig3.jpg" /> 
Figure 3. Filament production setup. The outlet of the extruder is set approximately 60 cm from the inlet of the spooler. Desktop fans are located near the heating element and approximately half way between the extruder and spooler. The spooler can optionally be elevated 3-4 inches above the benchtop. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58720/58720fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58720/58720fig4.jpg" /> 
Figure 4. Quality Assessments. (A) PCL(-) (left) and PLA-DM/PCL (right) scaffolds are difficult to distinguish macroscopically. (B) Under SEM, the PCL(-) scaffold appears mostly smooth, with a few striations as artifacts of the printing process. (C) Under SEM, microspheres are visible in the PLA-DM/PCL samples. Some of the microspheres are indicated using arrows. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58720/58720fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58720/58720fig5.jpg" /> 
Figure 5.&#160;Representative results of an ALP colorimetric assay. The absorbance of ALP-containing scaffolds (PLA-ALP/PCL) is significantly higher than that of the PCL-only (PCL(-)) scaffolds, indicating that the ALP enzyme catalyzed the reaction from colorless p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate. This demonstrates that the ability to print functional proteins with the process described in this manuscript. * significantly different (p &#60; 0.05) from all other groups. Error bars indicate standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58720/58720fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Novel Process for 3D Printing Decellularized Matrices at JoVE.com

Novel Process for 3D Printing Decellularized Matrices

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 ...

novel-process-for-3d-printing-decellularized-matrices

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.0K Views.  University of Cincinnati. 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.

Video: Novel Process for 3D Printing Decellularized Matrices

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

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 cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

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

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 complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

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.

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

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...