Name: three-dimensional biomimetic technology: novel biorubber creates defined micro- and macro-scale architectures in collagen hydrogels
Uploaded: 2016-02-12T14:00:00
Description: 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

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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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 (Video) | JoVE

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.

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 ...

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