The authors are grateful for support from NIH Director's New Innovator Awards (1DP2 OD006499-01 to A.A. and 1DP2 OD006460-01 to A.J.E.), and King Abdulaziz City for Science and Technology (UC San Diego Center of Excellence in Nanomedicine). We would like to thank Ms. Jessica Moore for her critical comments on the manuscript.

1. Synthesis of Fluorescently Labeled Acryloyl-PEG-RGDS
<ol>
 <li>React the RGDS peptide with acryloyl-PEG- succinimidyl carboxymethyl ester (aPEG-SCM, PEG MW: 3400 g/mol) and N-N diisopropylethylamine (DIPEA) at 1.2:1:2 molar ratios in dimethyl sulfoxide (DMSO) under argon at room temperature overnight. </li>
 <li>Confirm conjugation by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Add 1 &mu;l of aPEG-RGDS reaction solution to a sample spot on the MALDI target and dry. Prepare a saturated solution of Universal MALDI Matrix in tetrahydrofuran (THF) and vortex for 1 min. Add ~1 &mu;l of this solution to the same sample spot. Repeat the procedure for aPEG-SCM for comparison. Load and analyze. The molecular weight of aPEG-RGDS should be greater than aPEG-SCM (Figure 2).</li>
 <li>To conjugate the fluorophore, add an equimolar amount of Alexa Fluor 350 carboxylic acid (succinimydyl ester), dissolved in a minimal volume of DMSO, to the aPEG-RGDS reaction solution from 1.1 and react under argon at room temperature overnight. </li>
 <li>Purify aPEG-RGDS-350 by dialyzing (MW 3500 Da) against DI-H2O at 4 &deg;C for 48 hr at 1,000:1 volumetric ratio, exchanging dialysate at least twice per day. </li>
 <li>Freeze dry the purified aPEG-RGDS-350 in a Labconco Freezone Plus or equivalent freeze dry system and store at -20 &deg;C. </li>
</ol>
2. Preparation of a 2D Mold and Fabrication of a 2D PEG Gel with Alternating RGDS-350 Layers
<ol>
 <li>Prepare hydrophobic glass slides. Place clean glass slides into a glass dish in a vacuum oven. Heat to 80 &deg;C for 30 min to completely dry the surfaces. Place dish with slides in a fume hood and add 250 &mu;l Sigmacote to each slide, gently rocking for 30 sec to coat entire surface. Thoroughly rinse the coated slides with 100% methanol, followed by washing in distilled water, soaking twice for 5 min in at least 10 ml. </li>
 <li>Cut silicone spacers (0.8 mm thick) with 10 mm biopsy punches. </li>
 <li>Autoclave the silicone spacers and Sigmacote-treated glass slides. </li>
 <li>Formulate solutions for each respective layer in individual microfuge tubes by mixing PEG diacrylate (PEGda) precursor (final concentration 15% w/v) with different amounts of iodixanol (60% stock solution in water) to yield varying final concentrations (e.g. 40%, 30%, 20% and 10%), supplementing the remaining volume with phosphate buffered saline (PBS) to obtain solutions of graded densities. In a similar manner, for alternating layers, mix aPEG-RGDS-350 (final concentration 8 mM) with iodixanol and PBS to yield various concentrations (e.g. 35%, 25%, and 15%). </li>
 <li>Add photoinitiator (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 333mg/ml stock in N-vinyl pyrrolidone) to each solution for various layers (10 &mu;l of stock solution per ml of each layer solution). Photoinitiator is added last to prevent polymerization before layering of the gels in the mold, as it is sensitive to light. </li>
 <li>Subsequent steps of this protocol will be performed in a biosafety cabinet to ensure sterility.</li>
 <li>Filter-sterilize each solution using a sterile 1 ml syringe and 0.2 &mu;m filter. Assemble the mold setup by sandwiching the spacer between two Sigmacore-treated glass slides and secure with clamps placing as depicted in Figure 1. </li>
 <li>Cast the layered gels by adding the most dense solution (e.g. PEGda with 40% iodixanol) first, followed by a less dense solution (e.g. aPEG-RGDS-350 with 35% iodixanol). Repeat the alternate layering to achieve several layers of desired composition and densities as shown in Figure 1. </li>
 <li>Irradiate the mold with 365 nm light for 3 min using a portable UVR-9000 lamp. Allow the polymerized gels to cure for 5 min. Remove the clamps, then gently lift the top glass slide and the mold; the stratified DGMP gels will remain on the slides. Using a sterile spatula, carefully place the gels in a 50 ml tube containing sterile PBS or culture medium for washing.</li>
 <li>Wash the polymerized gels in PBS at 1,000:1 volumetric ratio, exchanging buffer at least twice per day to remove the density modifier, photoinitiator, and unreacted polymer. Alternatively, PBS may be exchanged with cell growth medium. Store the DGMP gels in PBS or growth medium for the cell culture experiment outlined in Step 3.</li>
 <li>To visualize alternating layers, arrange DGMP gel (these gels will not be usable for cell culture) along a ruler on the sample tray of a VersaDoc gel documentation unit. Expose the gels in 350 nm; the exposure time will vary depending on the concentration of the fluorophore. Alternating dark and blue bands in the DGMP hydrogel demonstrate formation of discrete layers of distinct chemical composition (Figure 3). </li>
</ol>
3. 2D Cell Culture on DGMP Gels
<ol>
 <li>For gels incorporating RGDS peptide, use adhesion dependent cells, such as C2C12 myoblasts.
 </li>
 <li>Gently insert DGMP gels (stored in PBS) into the wells of 48-well cell culture plates using a sterile cell scraper in a biosafety cabinet. 
 </li>
 <li>Pre-warm growth medium (Dulbecco's modified Eagle's medium or DMEM supplemented with 10% v/v fetal bovine serum and 1% v/v 100x penicillin-streptomycin solution) and PBS in a water bath set at 37 &deg;C. </li>
 <li>Wash a 60% confluent plate (10 mm) of C2C12 cells three times with PBS. Aspirate off PBS and harvest cells by adding 1 ml of 0.25% Trypsin-EDTA and incubating at 37 &deg;C for 2 min. Resuspend the cells in growth medium and count cells. Seed the DGMP gel-containing cell culture well with C2C12 myoblasts (20,000 cells/ cm2). Incubate the cells at 37 &deg;C in 5%CO2/95% relative humidity. Gently exchange medium after 4 hr, careful not to remove lightly adhered cells.</li>
 <li>After 24 hr, the attachment of C2C12 myoblasts on the RGDS-containing layers of DGMP gels may be confirmed by epifluorescence and phase contrast microscopy (Zeiss Axiovert 200). </li>
</ol>

<ol>
	<li>Place, E. S., Evans, N. D., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+in+biomaterials+for+tissue+engineering.">Complexity in biomaterials for tissue engineering.</a> Nat. Mater. 8, 457-470 (2009).</li><li>Liu, V. A., Jastromb, W. E., Bhatia, 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=Engineering+protein+and+cell+adhesivity+using+PEO-terminated+triblock+polymers.">Engineering protein and cell adhesivity using PEO-terminated triblock polymers.</a> J. Biomed. Mater. Res. 60, 126-134 (2002).</li><li>Sharma, B., 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=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Eng. 13, 405-414 (2007).</li><li>Hahn, M. 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=Photolithographic+patterning+of+polyethylene+glycol+hydrogels.">Photolithographic patterning of polyethylene glycol hydrogels.</a> Biomaterials. 27, 2519-2524 (2006).</li><li>Kizilel, S., Sawardecker, E., Teymour, F., Perez-Luna, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+formation+of+covalently+bonded+hydrogel+multilayers+through+surface+initiated+photopolymerization.">Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization.</a> Biomaterials. 27, 1209-1215 (2006).</li><li>Harley, B. 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=Design+of+a+multiphase+osteochondral+scaffold.+II.+Fabrication+of+a+mineralized+collagen-glycosaminoglycan+scaffold.">Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen-glycosaminoglycan scaffold.</a> J. Biomed. Mater. Res. A. 92, 1066-1077 (2010).</li><li>Cuchiara, M. P., Allen, A. C., Chen, T. M., Miller, J. S., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+microfluidic+PEGDA+hydrogels.">Multilayer microfluidic PEGDA hydrogels.</a> Biomaterials. 31, 5491-5497 (2010).</li><li>Roam, J. L., Xu, H., Nguyen, P. K., Elbert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+protein+concentration+gradients+mediated+by+density+differences+of+poly(ethylene+glycol)+microspheres.">The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres.</a> Biomaterials. 31, 8642-8650 (2010).</li><li>Gleghorn, J. P., Lee, C. S., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+laminated+alginate+gels+for+tissue+engineering+of+layered+structures.">Adhesive properties of laminated alginate gels for tissue engineering of layered structures.</a> J. Biomed. Mater. Res. A. 85, 611-618 (2008).</li><li>Karpiak, J. V., Ner, Y., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+gradient+multilayer+polymerization+for+creating+complex+tissue.">Density gradient multilayer polymerization for creating complex tissue.</a> Adv. Mater. 24, 1466-1470 (2012).</li><li>Pierschbacher, M. D., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+attachment+activity+of+fibronectin+can+be+duplicated+by+small+synthetic+fragments+of+the+molecule.">Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule.</a> Nature. 309, 30-33 (1984).</li><li>Kaushik, G., Fuhrmann, A., Cammarato, A., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Mechanical+Analysis+of+Myofibrillar+Perturbation+and+Aging+on+Soft,+Bilayered+Drosophila+Myocardium."> In Situ Mechanical Analysis of Myofibrillar Perturbation and Aging on Soft, Bilayered Drosophila Myocardium.</a> Biophysical Journal. 101, 2629-2637 (2011).</li></ol>

The authors have no conflicting interests to disclose.

DGMP is a simple strategy for preparing multilayered gels that does not rely on expensive instrumentation. This protocol can be adapted for creating scaffolds using other biocompatible materials, such as collagen and hyaluronic acid. Bioactive small molecules, for example cell adhesion-promoting RGDS peptide, can be tethered to the polymer matrix to prevent mixing of cues between layers. Proteins can be encapsulated in distinct layers without the need for chemical conjugation as they, depending on the matrix mesh size, are less prone to diffuse through hydrogels10. Here we used iodixanol (Nycoprep), an inert density modifier, which has previously been used for viable cell applications. Other density modifiers such as sucrose and dextrose can also be used. By varying the settling time (ts), one can fine-tune the interfaces between two layers to produce smooth or sharp transitions as needed (longer settling time gives smoother transitions)10. For example, smoother transitions between DGMP gel layers could be used to generate a continuous gradient of a biological cue to study cell processes such as chemotaxis. 
The effect of density modifier on gel stiffness is shown in Figure 5 for a 15% aPEGda gel; a more complete characterization of stiffness and porosity as a function of PEGda and iodixanol concentrations is currently being evaluated. While the PEGda concentration in this example is relatively high, we observed a 60% greater elastic modulus in gels with 30% iodixanol compared to gels without. The change in gel stiffness can be adjusted for by modulating the macromer concentration or crosslinking density.
We have also applied the DGMP technique to create 3D multilayered gels using polyacrylamide and PEG precursors10. Varying the concentration or the degree of crosslinking of the prepolymer allows structural variation in the scaffolds, which can be used to explore cell behavior such as polarized growth and migration in 3D. 
In summary, DGMP is an adaptable technique that can be applied to fabricate 2D and 3D scaffolds from a variety of biocompatible materials for a broad range of biomedical and basic research applications.

<table border="1">
 <tbody>
 <tr>
 <td>Reagent or Instrument</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Polyethylene glycol succinimydyl carboxymethyl (a-PEG-SCM)</td>
 <td>Laysan</td>
 <td>120-64</td>
 </tr>
 <tr>
 <td>Polyethelyene glycol diacrylate (PEGda)</td>
 <td>Dajac Labs</td>
 <td>9359</td>
 </tr>
 <tr>
 <td>Arginine-Glycine-Aspartic acid-Serine (RGDS)</td>
 <td>American Peptide</td>
 <td>49-01-4</td>
 </tr>
 <tr>
 <td>N,N- Diisopropylethylamine (DIPEA)</td>
 <td>Sigma</td>
 <td>D125806</td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D2438</td>
 </tr>
 <tr>
 <td>N,N- dimethylformamide (DMF)</td>
 <td>Fisher</td>
 <td>D119-4</td>
 </tr>
 <tr>
 <td>Tetrahydrofuran (THF)</td>
 <td>Fisher</td>
 <td>T397</td>
 </tr>
 <tr>
 <td>Dialysis cassette (3500 Da)</td>
 <td>Thermo Scientific</td>
 <td>66330</td>
 </tr>
 <tr>
 <td>Alexa Fluor 350 carboxylic acid succinimydyl ester</td>
 <td>Life Technologies</td>
 <td>A-10168</td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>Sigma</td>
 <td>SL2</td>
 </tr>
 <tr>
 <td>Silicone spacers</td>
 <td>Grainger</td>
 <td>1MWA4</td>
 </tr>
 <tr>
 <td>Biopsy punches</td>
 <td>Acuderm</td>
 <td>P1025 (10 mm) 
 P850 (8 mm)</td>
 </tr>
 <tr>
 <td>Dulbecco's phosphate buffered saline (DPBS)</td>
 <td>Hyclone</td>
 <td>SH30028</td>
 </tr>
 <tr>
 <td>Iodixanol (NycoPrep)</td>
 <td>Fisher</td>
 <td>NC9388846</td>
 </tr>
 <tr>
 <td>2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone </td>
 <td>Sigma </td>
 <td>410896</td>
 </tr>
 <tr>
 <td>Dulbecco's modified Eagle's medium (DMEM)</td>
 <td>Life Technologies</td>
 <td>11054</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Life Technologies</td>
 <td>10082</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Life Technologies</td>
 <td>15140</td>
 </tr>
 <tr>
 <td>C2C12 myoblasts</td>
 <td>ATCC</td>
 <td>CRL-1772</td>
 </tr>
 <tr>
 <td>MALDI</td>
 <td>Bruker</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>UVR-9000</td>
 <td>Bayco</td>
 <td>UVR-9000</td>
 </tr>
 <tr>
 <td>VersaDoc</td>
 <td>Bio-Rad</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

density gradient multilayered polymerization (dgmp): a novel technique for creating multi-compartment, customizable scaffolds for tissue engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

MALDI-TOF analysis confirms the conjugation of RGDS peptide to acryloyl-PEG (Figure 2). Gel imaging reveals alternating RGDS- 350 (blue) layers after photopolymerization (Figure 3A). As shown in Figure 3A, 2D DGMP gel size can be varied based on the diameter of the silicone molds (10 mm, left ; 8 mm, right), and therefore are easily customizable for use in multiple assays &#x2013; in this case to fit a 48 well cell culture plate (Figure 3B). Epifluorescence and phase contrast microscopy of C2C12 myoblasts cultured on a DGMP gel shows selective attachment on RGDS-350-containing PEG layers (Figure 4), demonstrating compartmentalization of the cell adhesion peptide (RGDS).
<img src="/files/ftp_upload/50018/50018fig1.jpg" alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> 
Figure 1. Molecular weight analysis by MALDI-TOF comparing aPEG-SCM to aPEG-RGDS obtained after conjugation of RGDS peptide. 
<img src="/files/ftp_upload/50018/50018fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> 
Figure 2. Schematic representation of DGMP gel fabrication. After the gradients are layered, they can be allowed to settle for varying periods of time (ts) to create graduated interfaces, followed by photopolymerization. Stratified DGMP gels can be easily extracted from the mold for further use. <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/50018/50018fig3.jpg" alt="Figure 3" /> 
Figure 3. A) 2D multilayered gels obtained after photopolymerization imaged using 350 nm and white light channels of VersaDoc gel documentation unit. The grayscale image reveals alternating layers containing RGDS in white. B) Insertion of DGMP gel into 48-well cell culture dishes.
<img src="/files/ftp_upload/50018/50018fig4.jpg" alt="Figure 4" /> 
Figure 4. Merged phase contrast and epifluorescence image of C2C12 myoblasts grown on DGMP gels (scale bar 50 &mu;m). 
<img src="/files/ftp_upload/50018/50018fig5.jpg" alt="Figure 5" /> 
Figure 5. Effect of iodixanol on gel surface elasticity. Atomic force microscopy measurements of static samples of crosslinked PEG substrates using previously established methods with a 2 nN force trigger 12. *p&lt; 0.05 and **p&lt;0.01.

Watch this Scientific Journal Video about Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering at JoVE.com

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

density-gradient-multilayered-polymerization-dgmp-novel-technique-for

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Bioengineering

12.4K Views.  University of California, San Diego. Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Video: Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

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

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

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

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

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

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

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

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...