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 colla...

<ol>
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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. 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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. 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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 border="0" cellpadding="0" cellspacing="0" width="770">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Collagen type I</td>
			<td style="width:156px;"></td>
			<td style="width:116px;"></td>
			<td style="width:217px;">Collagen extracted from calf hide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrocloric Acid (HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffer Solution (PBS Tablets)</td>
			<td>MP Biomedical</td>
			<td>U5378</td>
			<td>1 tablet per 100 mL makes 1XPBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumium from bovine serum (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glutaraldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>G5882</td>
			<td>Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lard</td>
			<td>Fields</td>
			<td>3090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Molds</td>
			<td></td>
			<td></td>
			<td>Milled using Microlution Machine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Air Brush Kit</td>
			<td>Central Pneumatic</td>
			<td>47791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Small O ring for double syringe</td>
			<td>Medmix</td>
			<td>PPB-X05-04-02SM</td>
			<td>Piston B, 5mL, 4:1, PE natural</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe cap&#160;</td>
			<td>Medmix</td>
			<td>VLX002-SM</td>
			<td>Cap, 4:1/10:1, PE brown, med</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Double Syringe&#160;</td>
			<td>Medmix</td>
			<td>SDL X05-04-50M</td>
			<td>Double syringe, 5 mL, 4:1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe Dispenser</td>
			<td>Medmix</td>
			<td>DL05-0400M</td>
			<td>Dispenser, 5 mL, 4:1, med , plain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminim</td>
			<td></td>
			<td></td>
			<td>3.6 mg/mL- extracted USC lab</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20 mL Syringe Luer Lock Tip</td>
			<td>BD</td>
			<td>302830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer Lock Caps</td>
			<td>Fisher</td>
			<td>JGTCBLLX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma -Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gibco Minimum Essential Media 10X (MEM)</td>
			<td>&#160;Life Technologies</td>
			<td>1143-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Life Technologies</td>
			<td>27250-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV Crosslinker&#160;</td>
			<td>Spectroline UV</td>
			<td>XLE1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Cloride (NaCl)</td>
			<td>Fisher</td>
			<td>S271-10</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride (KCl)</td>
			<td>Sigma -Aldrich</td>
			<td>P5405-250</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher</td>
			<td>BP328-500</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma -Aldrich</td>
			<td>G-8270</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Phosphate didasic (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S-7907</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 ...

The overall goal of this protocol is to engineer three dimensional collagen scaffolds that can resemble the architectural features, composition, and mechanical properties of in-vivo tissue. This method can help answer key questions in the tissue engineering field, such as ECM cell interactions, the influence of architectural features in the creation of engineered tissues, and the development of tissue replacements, among others. The main advantage of this technique is that it provides a viable procedure to recapitulate the microgeometry of interior flow channels and paths with high fidelity in a biodegradable material.Demonstrating the procedure will be Veronica Rodriguez-Rivera, a post-doctoral associate in my laboratory. The materials used to prepare the BSA rubber must be stored and kept cold until they are ready to use so that the BSA rubber does not prematurely set. Sterility is also very important to this procedure.First, sterilize the dispenser. In a non-cell culture hood, lay out the O rings, the syringe, its cap, the mix tip, and the four-to-one dispenser. UV sterilize these materials for 30 minutes.First, screw the two stainless steel mold pieces together and attach the Luer lock. Before assembling the dispenser, spray pipettes and reagents prior to introducing them to the hood with 70%ethanol. First attach the tip cap to the solution holder.Next, load the 30%BSA solution into the larger chamber without contaminating the other chamber. Leave enough room to attach the O ring. Then, load the smaller dispenser chamber with three percent glutaraldehyde and attach another O ring.Now, attach the syringe to the dispenser. Then tilt the assembly so the cap is on top, and replace the cap with a mixing tip. Then gently tap the sides to allow the air bubbles to travel upwards.Next, place the dispenser inside of an autoclaved bag to contain the exhaust solution. In the bag, with the dispenser upright, dispense a small amount of solution to clear all the air from the syringe. Removing the air is very important.Then, quickly, attach the stainless steel Y mold's Luer lock to the syringe tip. Now, with the mold in your non-dominant hand, alternate ejecting solution into the left and right exhausts of the outflow channels. Press the exhausts, thus filling the internal voids with solution.Then, position the mold horizontally and inject it with more solution until half the dispenser volume has been used. The mold should now be placed into a dish, wrapped in parafilm, and stored at four degrees Celsius overnight. This procedure is scaled to four grams of acidified collagen.Keep it chilled until it is used. After the collagen has been weighted, proceed to sterilize the solution by gamma radiation. In preparation, make fresh 0.2 normal HEPES solution in water.Then transfer the contents to a 50 milliliter conical tube. Adjust the pH to nine using concentrated sodium hydroxide. Be sure to UV-sterilize the hood and autoclave the forceps, spatula, and scalpel.In the hood, mix 1.5 milliliters of the HEPES solution with 1.5 milliliters of 10x MEM. Store this mixture on ice. Also, chill a 12 well plate and a 20 milliliter syringe in the freezer for 10 minutes.For added sterility, spray the tubes and plates involved with 70%ethanol prior to placing them in the hood. Spray the mold plate, and place it in the hood. Then, remove the BSA rubber molds with forceps.Then, cut the exhaust channels off the mold using a scalpel. Next, briefly vortex the HEPES-MEM mixture and add one milliliter to the collagen. Then, thoroughly mix the solution with a sterile spatula.Follow with a brief vortex, and quickly transfer the solution to the cold, 20 milliliter syringe. Now, while securing the BSA rubber inside of the well, dispense half the collagen hydrogel solution into the well bottom. Next, using tweezers, position the mold so that the rubber inflow and outflow ends are touching the walls of the well.Then, cover the mold with collagen hydrogel. Now, seal the well with parafilm, and load the plate into a 37 degree Celsius incubator. Allow one hour for the collagen to polymerize.During this time, keep the UV light in the hood on. Also, set the UV crosslinking apparatus to deliver 630, 000 microjoules per square centimeter. Now, spray down your gloved hands with 70%ethanol, and load the plate with the collagen covered molds into the crosslinker, and start the irradiation process.After the irradiation, spray your gloves again, and unload the plate from the crosslinker. Then, using a sterile spatula, remove the gel from the well flip it over, and crosslink the bottom of the hydrogel. After the final crosslinking cycle, proceed with the enzyme digestion.To remove the BSA rubber without disturbing the collagen scaffold, use sterile 0.25%trypsin solution at pH 7.8 and press it through a 0.20 micron filter. Transfer enough filtered trypsin solution to a new 50 milliliter conical tube. Then, transfer the collagen hydrogel to the conical tube that contains the enzyme solution.Make sure that the hydrogel is completely covered with the solution. Seal the tube with parafilm, and vortex it gently for about a minute. Then, incubate the tube at 30 degrees Celsius and let the digestion go for 15 to 24 hours.For the first four hours, remove the tube every 15 to 30 minutes and gently vortex it to help complete the BSA rubber digestion. The next day, the digestion is considered complete when no rubber floats in the solution or there are no broken down pieces of the rubber in the hydrogel. Now, replace the trypsin solution with sterile Mosconas solution, and let the tube shake at four degrees Celsius for 30 minutes.Later, aspirate the Mosconas solution and repeat this step two more times to fully clean the collagen scaffold. An extensive number of tests were used to optimize the use of the biorubber and described in detail in the text protocol. Using the optimized protocol and three solid mold pieces, collagen hydrogels were fabricated and examined in detail.The Y mold needed was crafted on a Microlution machine. The Y mold was injected with 30%BSA and three percent glutaraldehyde, which reacted overnight at four degrees Celsius. Next, the BSA rubber was casted with the collagen and heated to 37 degrees Celsius for an hour.The rubber was enzyme digested for 15 hours, which weakened it to the point of falling off without affecting the geometry of the collagen hydrogel. The four millimeter inflow channel cast by the mold was measured with calipers to be precisely four millimeters in the collagen hydrogel. Testing the stability of the mold, it was found that dimensions as small as 300 microns could be reliably engineered.In addition, the scaffolds were tested for residual glutaraldehyde after the Mosconas washes, and no residue was found. After watching this video, you should have a good understanding of how to fabricate 3D collagen scaffolds with the potential to mimic in-vivo geometries. Following this procedure, other methods that complement this technique, such as creating 3D models of in-vivo tissues and fabricating molds with specific architectural features can be performed to answer additional questions.After its development, this technique paved the way for researchers in the field of tissue engineering to explore the interaction between internal architectural features and matrix components with cells in in-vitro models.

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Research

JoVE Journal

Bioengineering

9.1K vistas.  Medical University of South Carolina. The overall goal of this protocol is to engineer three dimensional collagen scaffolds that can resemble the architectural features, composition, and mechanical properties of in-vivo tissue. This method can help answer key questions in the tissue engineering field, such as ECM cell interactions, the influence of architectural features in the creation of engineered tissues, and the development of tissue replacements, among others. The main advantage of this technique is that it provides a viabl...