This work was supported by NIH-NIDCR IRO1DE019355 (MJ Yost, PI), and NSF-EPSCoR (EPS-0903795).

1. Determine the Percentage of Solids in the Collagen Batch
<ol>
	<li>Extract the collagen following a previously published procedure13. Thaw a minimum of 20 ml of collagen. Determine the initial percentage of collagen solids in the batch in order to manipulate the collagen concentration in the formed hydrogels.
	<ol>
		<li>Cut three pieces of aluminum foil (about 6 x 6 cm) and shape each one as a pan by using the bottom of a 25 ml beaker. Record the weight of each pan.</li>
		<li>Add a small amount of collagen to each pan and record the total weight of the aluminum pan and collagen. Add 0.5 - 0.8 g of collagen on each aluminum pan. 
		Note: After this step, there are three aluminum foil pans and each one should have a small amount of collagen. Make sure to record the weight of each (total 3) empty aluminum pan and the weight of the pan after the addition of collagen.&#160;</li>
		<li>Calculate the weight of the collagen in each pan using the following formula: 
		Collagen weight = Pan and collagen weight - Pan weight</li>
		<li>Place the three aluminum pans that hold the collagen in the oven at 100 &#176;C for 24 hr.</li>
		<li>After 24 hr, record the weight of each aluminum pan and the dehydrated collagen.</li>
		<li>Calculate the weight of the dehydrated collagen using the following equation: 
		Dehydrated collagen weight = Pan and dehydrated collagen weight - Pan weight</li>
		<li>Calculate the percentage of solid for the three samples to determine the collagen solid concentration using the formula below: 
		<img alt="Equation 2" src="/files/ftp_upload/53578/53578eq2.jpg" /></li>
		<li>Calculate the average collagen solid content of the batch using the percentage of collagen solids for each of the three samples. 
		Note: The collagen that will be used is what is left of the hydrated collagen. None of the dehydrated collagen will be used.</li>
		<li>After determining the percentage of collagen solid (initial collagen concentration) of the batch, continue using the remaining hydrated collagen. Use a calibrated pH meter to adjust the pH of the collagen batch to 3.
		<ol>
			<li>Add small amounts (2-5 &#181;l at a time) of 12 N Hydrochloric Acid (HCl). Keep on ice at all times. Do not add the hydrochloric acid directly to the collagen&#160;&#8212; add the acid to the side of the tube. After adding the acid, use the spatula to push the acid to the collagen and quickly stir the mixture.</li>
		</ol>
		</li>
		<li>Once it reaches a pH of 3, let the collagen sit O/N at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of the BSA Rubber
<ol>
	<li>Prepare the Bovine Serum Albumin (BSA) solution following the procedure listed below.
	<ol>
		<li>Prepare 2x Phosphate Buffered Saline solution (PBS). Add two PBS tablets to 100 ml of water to make 0.02 M PBS solution.</li>
		<li>Combine BSA with 2x PBS to create a 30% BSA solution using the procedure listed below.
		<ol>
			<li>For example, to make a 30% BSA with 30 ml of 2x PBS, use 12.9 g of BSA. Add 1/3 of the 2x PBS (e.g., 10 ml) to a flask with a stir bar. Add 1/2 of the BSA (e.g., 6.45 g BSA) to the flask and using a spatula, wet the dry solute.</li>
			<li>Repeat this process of adding PBS and then BSA until all the solute and solvent is in the flask. Use the spatula to wet all the solute. It will look like a mixture of some liquid and clumps. Let it sit for approximately 30 min.</li>
			<li>Then, turn the stirrer on a low cycle and make sure that there are no clumps around the stir bar. It should take 90 min to get everything in solution or it can be left stirring O/N at 4 &#176;C. After the solute is all dissolved, place the solution in a 20 ml syringe and capped with a 0.20 &#181;m syringe filter. Press the plunger to expel liquid through filter and collect the sterilized solution in a new tube. Store at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Prepare 3% glutaraldehyde solution by diluting 25% glutaraldehyde solution with sterile filtered 2x PBS. For example, for 10 ml of 3% Glutaraldehyde solution, use 2 ml of 25% glutaraldehyde and 8 ml of 2x PBS. Store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Molds Treatment
Note: The prototype described in this paper uses a custom made stainless steel Y mold piece. The mold contains an inflow and two outflow channels of 4 and 3 mm, respectively. First, clean molds, spray them with unsaturated lard, and sterilize them. Prepare the molds following the procedure described below.
<ol>
	<li>Clean stainless steel molds using sonicator at a frequency of 35 kHz. Place the molds in the sonicator and submerge them with water and ice. Keep the molds cold at all times while the sonicator is running. Run the sonicator for 2 periods of 90 min.
	<ol>
		<li>After each period, use a needle to make sure that there is no material in the Luer lock stainless steel or brass connector. Use soap and water to clean the entire surface of the two sides of the molds. Verify that there is no obstruction in the channels.</li>
	</ol>
	</li>
	<li>Place the molds, screws, and Luer lock connector in an autoclave bag and autoclave it.</li>
	<li>Fill the bottle that attaches to an air sprayer half way with commercially available lard (mixed fatty acid release agent). Replace the cap with a regular cap bottle. Place it in an autoclave bag and autoclave it. 
	Note: Lard is used to facilitate the release of the material that will be reaction injected later on (BSA rubber). Do not place the sprayer bottle lid in the autoclave- it can damage the internal seal.</li>
	<li>Warm up the lard for 45 sec or until its clear and liquid in a microwave. Screw the air sprayer lid to the lard bottle. Connect the lid with the sprayer. Attach the sprayer to the air source at the lab bench. Open the air valve, and open the nozzle of the sprayer until it starts wetting the surface of a paper towel.</li>
	<li>Spray lard perpendicular to the mold surface until the surface is fully covered. After each piece has been sprayed, place them in a Petri dish and seal it. Place the molds at 4 &#176;C for 2 hr.</li>
	<li>Proceed to sterilize the molds by exposing the surface to UV light for 30 min. Place them back at 4 &#176;C until they are ready to be reaction injected.</li>
</ol>
4. Reaction Injection of the BSA Rubber
Note: All the materials and solution should be keep cold until ready to use to prevent premature setting of the BSA rubber in the next steps.
<ol>
	<li>Prepare the dispenser for delivering the BSA rubber to the molds following these steps.
	<ol>
		<li>Sterilize all of the mixing components (two O rings, syringe cap, double syringe, mix tip, and 4:1 dispenser) by exposing them to UV light for 30 min in the polymerase chain reaction (PCR) hood. 
		Note: A PCR hood was used because this procedure involves fixatives. These chemicals cannot be used in the cell/tissue culture hood due to the risk of exposure and toxicity to the cells. Any other hood that contains a UV light will be suitable.&#160;</li>
		<li>Place the tip cap on the solution holder.</li>
		<li>Perform the mixing and injection at a 4:1 ratio of BSA:Glutaraldehyde. Add the 30% BSA to the double syringe chamber that will deliver the highest amount of solution (It will take approximately 4 ml to fill). Make sure to leave enough space to place the O ring in order to prevent overflowing and contamination of the adjacent chamber.</li>
		<li>Add 3% glutaraldehyde solution to the other chamber (it will take about 1 ml to fill). Make sure to leave enough space to place the O ring to prevent overflowing and contamination of the adjacent chamber.</li>
		<li>Place the double syringe on the dispenser. Tilt the assembly vertically so that the syringe cap is on top. Replace the cap with the mixing tip.</li>
		<li>Screw the two stainless steel mold pieces together.</li>
		<li>Place the assembly inside of an autoclave bag.</li>
		<li>To remove any air in the dispenser, hold it in the upright position and quickly squeeze the handle one time to release a small amount of the BSA mixed with the glutaraldehyde. Then quickly attach the stainless steel Y mold&#39;s Luer lock connector to the syringe tip.</li>
		<li>Hold the stainless steel Y mold with the left hand and the BSA-Glutaraldehyde mixture dispenser on the right. Alternate covering each of left and right exhaust of the outflow channels by pressing the exhaust to the sides of the autoclave bag to make sure the inside voids are filled with solution. Then, place the molds horizontally and inject again.</li>
		<li>Detach the molds from dispenser and place in a 25 mm Petri dish.</li>
		<li>Place Parafilm around the Petri dish to prevent dehydration of the rubber.</li>
		<li>Place the mold in the 4 &#176;C fridge O/N.</li>
	</ol>
	</li>
</ol>
5. Adjusting the Collagen Concentration
Note: The collagen should be kept on ice at all times during the process.
<ol>
	<li>Modify the collagen concentration using the percentage of collagen solids.
	<ol>
		<li>Make 10 ml of 1.75% collagen by adjusting the initial collagen concentration with cold water.</li>
		<li>Using a calibrated pH meter, adjust the pH to 3 using 12 N Hydrochloric Acid. Do not add the hydrochloric acid directly to the collagen- add the acid to the side of the tube. After adding the acid, use the spatula to push the acid into the collagen and quickly stir the mixture.</li>
		<li>Weigh 4 g of collagen in a separate conical tube.</li>
		<li>Centrifuge the collagen to remove air at 4 &#176;C and 9,343 x g for 20-30 min.</li>
		<li>UV sterilize the cell culture hood for 30 min, and add 14 &#181;l of laminin to the 4 ml collagen. This will result in a final laminin concentration of 10 &#181;g/&#181;l. Note: Laminin provides structural integrity, adhesion, and promotes various cellular responses.</li>
		<li>Turn the PCR UV light on for 20-30 min prior to using the hood for sterilization.</li>
		<li>Place the Luer lock cap, to attach to a 20 ml syringe, in ethanol for a 2 hr. Then, allow it to dry and place it in UV light.</li>
		<li>Gamma irradiate the collagen for 8.6 min to reach 1,200 cGy. 
		Note: The time will depend on the decay of the Cesium source. Adjust the time to reach the same dosage.</li>
	</ol>
	</li>
</ol>
6.Casting Collagen on BSA Rubber
<ol>
	<li>To polymerize the collagen, use an 8:1:1 ratio (collagen:HEPES:MEM). The following procedure is based on an initial 4 g of acidified collagen (from Step 5.1.8).
	<ol>
		<li>Make a 0.2 N HEPES solution in water, and adjust the pH to 9 by adding small amounts (1-5 &#181;l) of 1-5 M sodium hydroxide (NaOH) solution. Using a calibrated pH meter, monitor the pH of the solution after each addition. Store at 4 &#176;C or keep on ice.</li>
		<li>Turn on the UV light of the tissue culture hood for 20-30 min to sterilize the hood.</li>
		<li>Autoclave forceps, spatula, and scalpel for sterilization.</li>
		<li>Mix 1.5 ml of 0.2 N HEPES (pH 9) and 1.5 ml of 10x MEM using the tissue culture hood. Make sure to keep it on ice and vortex it for 5 sec prior to use. Store at 4 &#176;C or keep on ice.</li>
		<li>To sterilize the PCR hood, turn on the UV light for 30 min.</li>
		<li>Place a 12 well plate and a 20 ml syringe in -20 &#176;C for 10 min, or until ready to continue. Note: Keep all materials cold until ready to use. The increase in temperature induces premature collagen fibrillogenesis.</li>
		<li>Spray all tubes and well plates that are going to be in the hood with 70% ethanol and let them dry for sterilization. Place a 20 ml syringe on ice to cool for later use.</li>
		<li>In the PCR hood, open the stainless steel molds to release the BSA rubber and using a scalpel, cut the exhaust channels of the BSA rubber mold.</li>
		<li>Under the PCR hood, open the sterile collagen tubes and add 1 ml of the HEPES-MEM solution (make sure that before extracting the HEPES-MEM solution, that it is well mixed and there are no solid deposits).</li>
		<li>Using the sterile spatula, thoroughly mix the collagen and buffer solution.</li>
		<li>Close and vortex it quickly to ensure a well-mixed hydrogel.</li>
		<li>Transfer to a cold 20 ml syringe.</li>
		<li>With one hand, hold the BSA Rubber inside of the well and, using the other, dispense half of the collagen hydrogel solution onto the bottom of the well.</li>
		<li>Using the sterile tweezers, ensure that the rubber inflow and outflow ends are touching the sides of the well.</li>
		<li>Pour collagen solution on top of the rubber until is completely covered.</li>
		<li>Ensure that the BSA rubber is suspended within the collagen and that there are no bubbles, especially near the ends of the rubber.</li>
		<li>Place the cover and wrap Parafilm around the circumference of the well.</li>
		<li>Put in the incubator for 1 hr at 37 &#176;C. Keep the PCR UV light on.</li>
	</ol>
	</li>
	<li>After the polymerization of the collagen, UV crosslink the hydrogel via the following procedure. 
	Note: The crosslinking of the collagen will be done using a UV crosslinker apparatus in which the amount of energy can be controlled.
	<ol>
		<li>Turn on the UV crosslinker and use the energy setting to irradiate the empty chamber with 630,000 &#181;J/cm2.</li>
		<li>Remove the gels from the incubator.</li>
		<li>Spray hands with ethanol, and, inside the chamber, remove the lid as quickly as possible.</li>
		<li>Close the chamber and UV crosslink the hydrogels by selecting the energy setting and irradiating 630,000 &#181;J/cm2.</li>
		<li>After the crosslinking cycle, turn off the UV light on the PCR hood</li>
		<li>Spray hands with ethanol and open the chamber, quickly placing the lid back onto the well plate. Move the well plate to the PCR hood.</li>
		<li>Using the sterile spatula, gently loosen and remove the gel from the well. Flip the gels under the hood to crosslink the bottom of the hydrogel. Repeat step 6.2.3 and 6.2.4.</li>
	</ol>
	</li>
</ol>
7. Enzyme Digestion of the BSA Rubber
<ol>
	<li>In order to have a hollow collagen scaffold, remove BSA Rubber in a way that does not affect the dimensions embedded in the hydrogel. The procedure is described below.
	<ol>
		<li>Turn on the UV light for 20-30 min to sterilize the tissue culture hood.</li>
		<li>Make 0.25% trypsin solution pH 7.8. For example, for 15 ml of water, add 0.0376 g of trypsin in a 50 ml conical tube. Adjust the pH to 7.8 by adding small amounts (2-5 &#181;l) of 1 M NaCl. Place the solution in a 20 ml syringe and capped with a 0.20 &#181;m syringe filter. Press the plunger to expel liquid through filter and collect the sterile solution in a new tube.</li>
		<li>Turn on the water bath and set the temperature to 30 &#176;C.</li>
		<li>After 30 min, turn off the UV light. Spray ethanol on all the tubes and materials that will be used in the hood.</li>
		<li>Transfer the collagen hydrogel under the hood and place in separate conical tube.</li>
		<li>Add around 3-5 ml (just enough to cover the gels) of 0.25% trypsin solution with a pH of 7.8 to each tube.</li>
		<li>Seal the tubes with Parafilm and vortex lightly for approximately 1 min.</li>
		<li>Place in the 30 &#176;C water bath for 15-24 hr. While in the water bath, lightly vortex the gels frequently until the BSA rubber has been digested or removed from the hydrogel. 
		Note: In order to determine if the BSA rubber has been removed, either the rubber is floating in the trypsin solution or there are broken-down pieces. There must be no visual dark areas within the hydrogel.</li>
	</ol>
	</li>
	<li>To ensure that all the BSA rubber and trypsin has been removed from the hydrogels, rinse it as described below.
	<ol>
		<li>Turn on the PCR hood UV light for 20-30 min.</li>
		<li>Prepare Mosconas solution. Combine potassium chloride (KCl, 28.6 mM), (NaHCO3, 11.9 mM), glucose (9.4 mM) and (NaH2PO4, 0.08 mM) in water. Adjust pH to 7.4 with 1 M NaOH or 12 M HCl solution. Place the solution in a 20 ml syringe and use a syringe filter of 0.20 &#181;m to sterilize the solution.</li>
		<li>Spray everything with ethanol prior placing them on the hood and allow the ethanol to dry.</li>
		<li>Open the tube and transfer 5-10 ml of sterile Mosconas solution to a new 50 ml conical tube.</li>
		<li>Transfer the collagen hydrogel to the conical tube that contains the enzyme solution. Make sure that the hydrogel is completely covered with the solution. Leave the tube in the shaker in the fridge at 4 &#176;C for 30 min.</li>
		<li>Aspirate the Mosconas solution and repeat step 7.2.5 twice.</li>
		<li>Store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kundu, J., Pati, F., Hun Jeong, Y., Cho, D. W., Cho, D. W., Forgacs, G. a. b. o. r., Sun, W. e. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biofabrication. , 23-46 (2013).</li><li>Vats, A., Tolley, N. S., Polak, J. M., Gough, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+and+biomaterials+for+tissue+engineering:+a+review+of+clinical+applications.">Scaffolds and biomaterials for tissue engineering: a review of clinical applications.</a> Clin. Otolaryngol. 28, 165-172 (2003).</li><li>Chan, B. P., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+in+tissue+engineering:+general+approaches+and+tissue-specific+considerations.">Scaffolding in tissue engineering: general approaches and tissue-specific considerations.</a> Eur Spine J. 17, s467-s479 (2008).</li><li>Salerno, A., Di Maio, E., Iannace, S., Netti, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+the+pore+structure+of+PCL+scaffolds+for+tissue+engineering+prepared+via+gas+foaming+of+multi-phase+blends.">Tailoring the pore structure of PCL scaffolds for tissue engineering prepared via gas foaming of multi-phase blends.</a> J. Porous Mater. 19, 181-188 (2011).</li><li>Hasan, 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=Electrospun+scaffolds+for+tissue+engineering+of+vascular+grafts.">Electrospun scaffolds for tissue engineering of vascular grafts.</a> Acta Biomater. 10, 11-25 (2014).</li><li>Huang, Z. -. M., Zhang, Y. Z., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+polymer+nanofibers+by+electrospinning+and+their+applications+in+nanocomposites.">A review on polymer nanofibers by electrospinning and their applications in nanocomposites.</a> Compos. Sci. Technol. 63, 2223-2253 (2003).</li><li>Sell, S. A., McClure, M. J., Garg, K., Wolfe, P. S., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+collagen/biopolymers+for+regenerative+medicine+and+cardiovascular+tissue+engineering.">Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering.</a> Adv. Drug. Deliv. Rev. 61, 1007-1019 (2009).</li><li>Zheng, W., Zhang, W., Jiang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+Collagen+Nanofibrous+Materials+for+Bone+Tissue+Engineering.">Biomimetic Collagen Nanofibrous Materials for Bone Tissue Engineering.</a> Adv. Eng. Mater. 12, B451-B466 (2010).</li><li>Cao, H., Kuboyama, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biodegradable+porous+composite+scaffold+of+PGA/beta-TCP+for+bone+tissue+engineering.">A biodegradable porous composite scaffold of PGA/beta-TCP for bone tissue engineering.</a> Bone. 46, 386-395 (2010).</li><li>Ankam, 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=Substrate+topography+and+size+determine+the+fate+of+human+embryonic+stem+cells+to+neuronal+or+glial+lineage.">Substrate topography and size determine the fate of human embryonic stem cells to neuronal or glial lineage.</a> Acta Biomater. 9, 4535-4545 (2013).</li><li>Bose, S., Vahabzadeh, S., Bandyopadhyay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+tissue+engineering+using+3D+printing.">Bone tissue engineering using 3D printing.</a> Mater. Today. 16, 496-504 (2013).</li><li>Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S., Dubruel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+trends+and+limitations+in+hydrogel-rapid+prototyping+for+tissue+engineering.">A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering.</a> Biomaterials. 33, 6020-6041 (2012).</li><li>Yost, M. 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=A+novel+tubular+scaffold+for+cardiovascular+tissue+engineering.">A novel tubular scaffold for cardiovascular tissue engineering.</a> Tissue Eng. 10, 273-284 (2004).</li><li>Habeeb, A. J., Hiramoto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+of+proteins+with+glutaraldehyde.">Reaction of proteins with glutaraldehyde.</a> Arch Biochem Biophys. 126, 16-26 (1968).</li><li>Tanford, C., Buzzell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Viscosity+of+Aqueous+Solutions+of+Bovine+Serum+Albumin+between+pH+4.3+and+10.5.">The Viscosity of Aqueous Solutions of Bovine Serum Albumin between pH 4.3 and 10.5.</a> J. Phys. Chem. 60, 225-231 (1956).</li><li>Yadav, S., Shire, S. J., Kalonia, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscosity+analysis+of+high+concentration+bovine+serum+albumin+aqueous+solutions.">Viscosity analysis of high concentration bovine serum albumin aqueous solutions.</a> Pharm Res. 28, 1973-1983 (2011).</li><li>Tobitani, A., Ross-Murphy, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intrinsic+viscosity+of+polyelectrolytes+revisited.">The intrinsic viscosity of polyelectrolytes revisited.</a> Polym. Int. 44, 338-347 (1997).</li><li>Arakawa, T., Timasheff, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+protein+solubility.">Theory of protein solubility.</a> Methods Enzymol. 114, 49-77 (1985).</li><li>Migneault, I., Dartiguenave, C., Bertrand, M. J., Waldron, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutaraldehyde:+behavior+in+aqueous+solution,+reaction+with+proteins,+and+application+to+enzyme+crosslinking.">Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.</a> Biotechniques. 37, 790-796 (2004).</li><li>Burmeister, J. 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=Glutaraldehyde+cross-linked+glutamate+oxidase+coated+microelectrode+arrays:+selectivity+and+resting+levels+of+glutamate+in+the+CNS.">Glutaraldehyde cross-linked glutamate oxidase coated microelectrode arrays: selectivity and resting levels of glutamate in the CNS.</a> ACS Chem Neurosci. 4, 721-728 (2013).</li><li>Chatterji, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin+with+hydrophilic/hydrophobic+grafts+and+glutaraldehyde+crosslinks.">Gelatin with hydrophilic/hydrophobic grafts and glutaraldehyde crosslinks.</a> J. Appl. Polym. Sci. 37, 2203-2212 (1989).</li><li>Freije, J. R., 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=Chemically+modified,+immobilized+trypsin+reactor+with+improved+digestion+efficiency.">Chemically modified, immobilized trypsin reactor with improved digestion efficiency.</a> J Proteome Res. 4, 1805-1813 (2005).</li><li>Cunha-Filho, M. S., Alvarez-Lorenzo, C., Martinez-Pacheco, R., Landin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+gels+for+intratumoral+delivery+of+beta-lapachone:+effect+of+cyclodextrins+and+ethanol.">Temperature-sensitive gels for intratumoral delivery of beta-lapachone: effect of cyclodextrins and ethanol.</a> ScientificWorldJournal. 2012, 126723 (2012).</li><li>Basak, R., Bandyopadhyay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+hydrophobic+drugs+in+Pluronic+F127+micelles:+effects+of+drug+hydrophobicity,+solution+temperature,+and+pH.">Encapsulation of hydrophobic drugs in Pluronic F127 micelles: effects of drug hydrophobicity, solution temperature, and pH.</a> Langmuir. 29, 4350-4356 (2013).</li></ol>

The authors have nothing to disclose.

Biofabrication is a highly multidisciplinary field in which biology and engineering principles merge to generate complex materials that mimic native tissue. In order to achieve this, there is a need to develop techniques that use the information gathered from in vivo tissue and translate it into an in vitro scaffold. In this way, a platform can be engineered that closely resembles the architectural, functional, and mechanical properties of the in vivo tissue. The optimal scaffolding material sh...

In the tissue engineering field the ability to fabricate tissue scaffolds is vital. A suitable tissue scaffold has a 3D structure, is composed of biocompatible materials, and mimics in vivo tissue architecture to facilitate cell and tissue growth and remodeling. This scaffold must allow the transport of nutrients and the removal of wastes1-4. One of the main obstacles in the production of these scaffolds is the ability to recapitulate specific geometrical features into a biocompatible material. Several biofabrication techniques have been reported to control the geometrical features of these scaffolds, examples are electrospinning5-8, solvent-casting9, stereolithography10, and 3D-printing11, among others. These techniques fall short in providing a relatively easy transfer of controllable internal and external architectural features, are expensive, are limited by their resolution and printability (e.g., nozzle gauge, material restriction), or require post-fabrication techniques which demands a long period of time to produce viable scaffolds12.
In many commercial fabrication systems, the creation of internal voids, channels, and features is achieved using sand or other suitable removable or sacrificial materials. The metal or plastic part is formed around the sand mold, and once it is solidified, the sand is removed. In much the same manner, the next generation of biomaterials needs the biosand equivalent. Therefore, the BSA rubber was developed as a substitute for biosand. The BSA rubber is a newly formulated material that consists of bovine serum albumin crosslinked with glutaraldehyde. The ultimate goal is to recreate specific architectural features into a biodegradable collagen scaffold. The characteristics of the sacrificial biorubber that maintains dimensional fidelity with the mold of the original tissue are described.
Several combinations of BSA and glutaraldehyde concentrations were tested using a variety of solvents. This material was created by the reaction between BSA and glutaraldehyde. BSA rubber can be reaction injected into the intricate geometries of the tissue molds. Crosslinked BSA is trypsin labile and readily digested by the enzyme at mild pH and temperature conditions. Conversely, intact type I collagen is very resistant to trypsin digestion. These features were capitalized to selectively remove the BSA rubber leaving the collagen behind. The present work consisted of determining the ideal parameters needed to obtain a labile mold that can deliver specific architectural features to a biocompatible scaffold. The specific features that were evaluated included mixability, enzyme digestion, load bearing, and ability to be reaction injected into a negative mold. The combination of 30% BSA and 3% glutaraldehyde fulfills these requirements. This protocol provides the necessary guidelines to create these three-dimensional scaffolds. The prototype consists of a collagen scaffold that represents a branched architecture with one inflow and two outflow channel with diameters of 4- and 3-mm, respectively. This technique has the potential to mimic macro- and micro-environments of the tissue of interest. This technology provides a viable technique to deliver a specific geometrical instructive to a biodegradable material in a relatively easy and timely matter with high fidelity, which can be tuned to mimic the in vivo tissue elasticity and other characteristics of the tissue of interest.

<table><tbody><tr><td>Collagen type I</td><td></td><td></td><td>Collagen extracted from calf hide</td></tr><tr><td>Hydrocloric Acid (HCl)</td><td>Sigma-Aldrich</td><td>7647-01-0</td><td></td></tr><tr><td>Phosphate Buffer Solution (PBS Tablets)</td><td>MP Biomedical</td><td>U5378</td><td>1 tablet per 100 ml makes 1x PBS</td></tr><tr><td>Albumium from bovine serum (BSA)</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma -Aldrich</td><td>G5882</td><td>Toxic</td></tr><tr><td>Lard</td><td>Fields</td><td>3090</td><td></td></tr><tr><td>Stainless Steel Molds</td><td></td><td></td><td>Milled using Microlution Machine</td></tr><tr><td>Air Brush Kit</td><td>Central Pneumatic</td><td>47791</td><td></td></tr><tr><td>Mixing Tip for double syringe</td><td>Medmix</td><td>ML2.5-16-LLM</td><td>Mixer, DN2,5X16, 4:1 brown, med</td></tr><tr><td>Small O ring for double syringe</td><td>Medmix</td><td>PPB-X05-04-02SM</td><td>Piston B, 5 ml, 4:1, PE natural</td></tr><tr><td>Double Syringe cap&amp;nbsp;</td><td>Medmix</td><td>VLX002-SM</td><td>Cap, 4:1/10:1, PE brown, med</td></tr><tr><td>Big O ring for double syringe</td><td>Medmix</td><td>PPA-X05-04-02SM</td><td>Piston A, 5 ml, 4:1</td></tr><tr><td>Double Syringe</td><td>Medmix</td><td>SDL X05-04-50M</td><td>Double syringe, 5 ml, 4:1</td></tr><tr><td>Double Syringe Dispenser</td><td>Medmix</td><td>DL05-0400M</td><td>Dispenser, 5 ml, 4:1, med , plain</td></tr><tr><td>Laminim</td><td></td><td></td><td>3.6 mg/ml - extracted USC lab</td></tr><tr><td>20 ml Syringe Luer Lock Tip</td><td>BD</td><td>302830</td><td></td></tr><tr><td>Luer Lock Caps</td><td>Fisher</td><td>JGTCBLLX</td><td></td></tr><tr><td>HEPES</td><td>Sigma -Aldrich</td><td>H4034</td><td></td></tr><tr><td>Gibco Minimum Essential Media 10x (MEM)</td><td>Life Technologies</td><td>1143-030</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>27250-018</td><td></td></tr><tr><td>UV Crosslinker&amp;nbsp;</td><td>Spectroline UV</td><td>XLE1000</td><td></td></tr><tr><td>Sodium Cloride (NaCl)</td><td>Fisher</td><td>S271-10</td><td>To prepare Mosconas</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma -Aldrich</td><td>P5405-250</td><td>To prepare Mosconas</td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher</td><td>BP328-500</td><td>To prepare Mosconas</td></tr><tr><td>Glucose</td><td>Sigma -Aldrich</td><td>G-8270</td><td>To prepare Mosconas</td></tr><tr><td>Sodium Phosphate didasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S-7907</td><td>To prepare Mosconas</td></tr><tr><td>Sterile Filter for syringes</td><td>Corning</td><td>431224</td><td></td></tr></tbody></table>

three-dimensional biomimetic technology: novel biorubber creates defined micro- and macro-scale architectures in collagen hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

The results demonstrate that this biofabrication technique is efficient in generating 3D scaffolds that can mimic the spatial arrangement seen in in vivo tissue. The architectural features are vital parameters for tissue engineering application, playing a crucial role in the in vivo cell interaction and functionality of the tissue.
The consistency and mixability of the BSA rubber was an important parameter in p...

Watch this Scientific Journal Video about Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels at JoVE.com

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

three-dimensional-biomimetic-technology-novel-biorubber-creates

Research

JoVE Journal

Bioengineering

9.2K Views.  Medical University of South Carolina. An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Video: Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Cancer Research

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

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 ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

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

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

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

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

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