This work was supported by the Singapore National Research Foundation administered by one of its Research Centers of Excellence, the Mechanobiology Institute, Singapore. MFAC is supported by the Agency for Science, Technology and Research (Singapore) and National Agency for Research (France) joint program under project number 1122703037. BKKT is supported by the Mechanobiology Institute. We thank Mr. Daniel HC Wong for proof-reading the manuscript and Ms. Dawn JH Neo for assisting in the video production.

1. Preparation of Polyelectrolyte Solutions
<ol>
	<li>Purify chitosan, as detailed in Liao et al. Briefly, create a 1% (w/v) solution of chitosan in 2% (v/v) acetic acid and vacuum filter using grade 93 filter paper. Neutralize the filtrate using 5M NaOH until the pH stabilized to 7. Centrifuge the precipitated chitosan at 1,200 x g for 10 min. Decant the supernatant and add deionized water to wash the chitosan. Repeat the centrifugation and washing step two more times. Freeze the precipitated chitosan at -80 &#176;C and lyophilize O/N to obtain the purified form. Store purified chitosan in a dehumidified cabinet.</li>
	<li>Weigh out 1 g of purified chitosan into a sterile tissue culture dish. Place the chitosan in the tissue culture dish as close as possible to the UV lamp in the biological safety cabinet and expose to UV light for 15 min. Using sterile forceps, place the sterilized chitosan into a glass container. Dissolve chitosan using filtered 0.15M acetic acid to a final concentration between 0.5% and 1% (w/v).</li>
	<li>Weigh out 0.1 g of alginic acid sodium salt and dissolve in 10 ml distilled deionized (DDI) water to obtain a 1% (w/v) solution. Mix the alginic acid sodium salt for at least 2 hr on the vortex mixer to ensure complete dissolution. Filter the alginate solution through 0.2 &#181;m syringe filter. Store the alginate solution at 4 &#176;C.</li>
	<li>Reconstitute human recombinant growth factors such as vascular endothelial growth factor (VEGF) or beta - nerve growth factor (NGF), as recommended by manufacturer.</li>
</ol>
2. Drawing of IPC Fibers
<ol>
	<li>Mix proteins, growth factors or other biomolecules into 10-20 &#181;l aliquot of the polyelectrolyte solution that has a similar net charge. Biological molecules with net negative charge (eg bovine serum albumin [BSA]) should be mixed with alginate solution. Biological molecules with net positive charge (eg VEGF) should be mixed with chitosan solution.</li>
	<li>Place small aliquots (10-20 &#181;l) of chitosan and alginate on a stable flat surface that is covered with parafilm. The droplets of chitosan and alginate should be placed in close proximity but not in contact with each other.</li>
	<li>Lightly dip each tip on a pair of forceps into the chitosan and alginate droplets. Bring the droplets of polyelectrolytes together by pinching the forceps. When the droplets come into contact with each other, slowly pull the forceps vertically upward to draw the IPC fiber from the interface of the two droplets (Figure 1A).</li>
	<li>Carefully place the end of the drawn IPC fiber on the forceps on a collector, such as a flat polymeric scaffold affixed on a rotating mandrel (see section 3 and 4). Rotate the mandrel at a fixed speed of 10 mm/sec to allow formation of uniform and beadless IPC fibers. Increasing the speed of drawing the IPC fibers will form beads, which will cause a burst release of incorporated biochemical and premature fiber termination.10</li>
	<li>To determine incorporation efficiency, collect all the remaining liquids left on the parafilm by diluting with 500 &#181;l of 1X phosphate buffered saline (PBS). Measure the protein or growth factor content in the residue through BCA assay (for BSA), ELISA (for VEGF and NGF) or an appropriate assay to detect incorporated biomolecule.</li>
</ol>
3. Fabrication of&#160;Composite&#160;Hydrogel Scaffold of Pullulan-Dextran (PD) Polysaccharide and IPC Fibers
<ol>
	<li>Fabricating sacrificial pullulan frame for IPC fiber collection
	<ol>
		<li>Weigh out pullulan polysaccharide and mix with distilled deionized (DDI) water to create a 20% (w/v) aqueous solution. Mix the pullulan solution O/N to ensure homogeneity.</li>
		<li>Cast 15 g of pullulan solution into a 10&#160;cm diameter tissue culture polystyrene (TCPS) dish. Dry the pullulan solution O/N at 37&#160;&#176;C. Cut the pullulan films into 7 mm x 7 mm square frames.</li>
	</ol>
	</li>
	<li>Preparing pullulan-dextran polysaccharide solution
	<ol>
		<li>Create a 30% (w/v) solution of the polysaccharides pullulan and dextran (3:1 ratio) in DDI water. Mix O/N to ensure homogeneity of the polysaccharide solution. Slowly add in sodium bicarbonate to the polysaccharide solution to achieve a final concentration of 20% (w/v). Mix O/N to ensure homogeneity of the solution. Store the polysaccharide solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Collecting IPC fibers on pullulan frame
	<ol>
		<li>Affix the sacrificial pullulan frame (section 3.1) using an alligator clip. Stick the alligator clip and pullulan frame on the end of the rotating mandrel using plastic-coated adhesive tape. Rotate the mandrel with the affixed frame at a constant speed of 10 mm/sec.&#160;The pullulan frame can be affixed onto the rotating mandrel in desired orientations.</li>
	</ol>
	</li>
	<li>Draw the IPC fibers using a pair of forceps (section 1) and attach the drawn end of the IPC fibers onto the rotating pullulan frame. Draw the IPC fibers at a constant speed. Upon reaching the terminal end of the IPC fiber, dry the fibers-on-frame construct O/N at RT.</li>
	<li>Embedding IPC fibers into PD hydrogel scaffold</li>
	<li>To crosslink every gram of pullulan-dextran solution, add 100 &#181;l of 11% (w/v) sodium trimetaphosphate aqueous solution and 100 &#181;l 10M sodium hydroxide.7 Mix the solution at 60 rpm using a stirplate for 1 to 2 min. After the addition of sodium trimetaphosphate and sodium hydroxide, the polysaccharide solution will crosslink almost immediately. Pour the viscous polysaccharide solution onto the fibers-on-frame construct to fully embed the IPC fibers. Incubate the combined pullulan-dextran-IPC fibers (PD-IPC) at 60 &#176;C for 30 min&#160;to form a chemically crosslinked composite scaffolds (Figure 1B).</li>
	<li>CAUTION: Perform step 3.3.2 in the fume hood and use proper protective equipment as acetic acid is a corrosive and flammable.</li>
	<li>To induce pore formation in the PD-IPC scaffold, submerge the whole scaffold in 20% (w/v) acetic acid for 20 min.</li>
	<li>Remove unreacted reagents by washing PD-IPC scaffolds in 1X PBS for 5 min&#160;while shaking at 100 rpm. Repeat this step 2 times.</li>
	<li>Remove the excess PBS and immediately freeze the PD-IPC scaffolds at -80 &#176;C O/N. Lyophilize the scaffolds at least 24 hr before use in any controlled release or bioactivity assays.</li>
</ol>
4. Fabrication of Composite Scaffold of PCL and IPC Fibers
CAUTION: Dichloromethane is a hazardous material. Use the fume hood and personal protective equipment when handling dichloromethane.&#160;
<ol>
	<li>Creating pristine and patterned PDMS substrates
	<ol>
		<li>Create a pristine polydimethylsiloxane (PDMS) elastomeric substrate using a piece of TCPS of desired dimension using soft lithography process. Create patterned PDMS substrates by using standard soft lithographic methods on poly(methyl methacrylate) templates with the desired topography.12</li>
	</ol>
	</li>
	<li>Fabricating sacrificial PCL frame for IPC fiber collection
	<ol>
		<li>Weigh out PCL and dissolve in dichloromethane to create a 0.9% (w/v) solution. For every 1 cm2 area of the PDMS substrate, drop 500 &#181;l of 0.9% PCL solution. Allow all of the dichloromethane solvent to fully evaporate in the fume hood. Repeat the process of casting 0.9% PCL to thicken the film to the desired thickness. Remove the dried PCL film from the PDMS substrate. Create a hole in the PCL frame using a suitably-sized puncher.2</li>
	</ol>
	</li>
	<li>Collecting IPC fibers on the PCL frame
	<ol>
		<li>Affix the sacrificial PCL frame with hole (from 4.2.1) on an alligator clip. Stick the alligator clip onto the rotating mandrel by using plastic-coated adhesive tape. Attach the drawn end of the IPC fiber onto the PCL frame before starting the rotation at a constant speed of 10mm/sec (section 2). After the end of IPC fiber drawing, dry the fiber-on-frame construct O/N at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Embedding fiber-on-frame construct into patterned PCL substrate
	<ol>
		<li>Drop 500 &#181;l of 0.9% PCL solution onto the PDMS substrate to create a pristine or patterned PCL base, as required. Cast multiple layers of 0.9% PCL solution to obtain a scaffold with the desired thickness. Allow all of the dichloromethane solvent to fully evaporate in the fume hood.</li>
		<li>Place the fiber-on-frame construct (section 4.3.1) on top of the PCL base. Add 0.9% PCL solution on the fiber-on-frame construct multiple times to get the desired thickness and fully embed the IPC fibers, fabricating a PCL-IPC composite scaffold (Figure 1C).</li>
	</ol>
	</li>
</ol>
5. Measurement of Release of Biological Agents from Composite IPC Scaffolds
<ol>
	<li>Place composite PD-IPC or PCL-IPC scaffolds and stand-alone IPC fibers separately in a 24-well plate.</li>
	<li>Immerse the scaffold and stand-alone IPC fibers with 500 &#181;l of 1X PBS. Incubate the samples at 37 &#176;C. Collect PBS at various time points (release media) and replace with 500 &#181;l of 1X PBS.</li>
	<li>Measure the amount of protein or growth factor in the release media using a BCA assay (BSA), ELISA (VEGF and NGF) or other appropriate assay to calculate the cumulative release profile for the incorporated biomolecule.</li>
</ol>
6. Seeding of Cells on Composite IPC Scaffolds to Test Bioactivity of Released Biological Agents
<ol>
	<li>Sterilize the lyophilized PD-IPC or PCL-IPC composite scaffolds using UV light in the biological safety cabinet for at least 20 min.</li>
	<li>Use standard cell culture techniques to obtain a cell suspension of 2 x 105 cells in 200 &#181;l growth media. Seed the concentrated cell suspension onto the composite scaffolds. After 20 min, top-up the volume of growth media to fully submerge the scaffolds.</li>
	<li>Measure cell activity through standard techniques such as Alamar blue metabolic activity assay, PC12 neurite outgrowth assay or immunofluorescence.</li>
</ol>

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</ol>

The authors have nothing to disclose.

IPC fibers are formed by the interaction of two oppositely charged polyelectrolytes. The process utilizes the extraction of the complex from the interface of the polyelectrolytes, facilitating a self-assembly process for stable fiber formation. The mechanism of IPC fiber formation ensures that any biomolecule added into a similarly charged polyelectrolyte can be incorporated during the complexation process.10,11 Conversely, addition of a biomolecule into the oppositely charged polyelectrolyte will result in in...

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.1 For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.2 However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.3 Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix. 
Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors2,7 even cells8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic2 and hydrophilic polymers7 to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells. 
In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

<table><tbody><tr><td>Pullulan&amp;nbsp;</td><td>Hayashibara Inc Okayama Japan</td><td></td><td>Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.</td></tr><tr><td>Dextran</td><td>Sigma Aldrich</td><td>D1037</td><td>MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.</td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma Aldrich</td><td>S5761</td><td>Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.&amp;nbsp;</td></tr><tr><td>Sodium Trimetaphosphate</td><td>Sigma Aldrich</td><td>T5508</td><td>This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.</td></tr><tr><td>Chitosan</td><td>Sigma Aldrich</td><td>448877</td><td>MW 190-310 kDa. Acetylation degree of 75% to 85%. Purification of chitosan is required to create stable IPC fibers.</td></tr><tr><td>Acetic Acid</td><td>Merck</td><td></td><td>This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.&amp;nbsp;</td></tr><tr><td>Alginic acid sodium salt from brown algae, low viscosity</td><td>Sigma Aldrich</td><td>A2158</td><td>Dissolve in water overnight. Filter through sterile 0.2&amp;nbsp;&amp;micro;m syringe filter before use. Store at 4 &amp;deg;C.</td></tr><tr><td>Bovine Serum Albumin</td><td>Sinopharm Chemical Reagent</td><td></td><td>Dissolve in sterile PBS and filter using 0.2 &amp;micro;m syringe filter before use.&amp;nbsp;</td></tr><tr><td>BCA assay kit</td><td>Pierce</td><td>23225</td><td>This kit was used to measure BSA release from PD-IPC scaffolds.&amp;nbsp;</td></tr><tr><td>Human Recombinant Vascular Endothelial Growth Factor</td><td>R&amp;amp;D systems</td><td>293-VE</td><td>Dissolve growth factor in 0.2% heparin solution to a final concentration of 5&amp;nbsp;mg/ml.</td></tr><tr><td>Heparin Sodium Salt From Porcine</td><td>Sigma Aldrich</td><td>H3393</td><td>This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 &amp;micro;m syringe filter before use.&amp;nbsp;</td></tr><tr><td>Human Umbilical Vein Endothelial Cells (HUVEC)</td><td>Lonza</td><td>C2517A</td><td>This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.&amp;nbsp;</td></tr><tr><td>Alamar blue</td><td>Life Technologies</td><td>DAL1025</td><td>This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.&amp;nbsp;</td></tr><tr><td>ScanVac Coolsafe Lyophilizer</td><td>Labogene</td><td>7.001.200.060</td><td>This is a non-programmable freeze dryer that operates at -105 to -110&amp;nbsp;&amp;deg;C. This can be replaced by other standard lab lyophilizers.</td></tr><tr><td>Polycaprolactone (PCL)</td><td>Sigma Aldrich</td><td>181609</td><td>MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.</td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>V800151</td><td>This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.</td></tr><tr><td>Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)</td><td>Dow Corning</td><td>(240)4019862</td><td>The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.</td></tr><tr><td>Human Recombinant Beta-Nerve Growth Factor (NGF)</td><td>R&amp;amp;D systems</td><td>256-GF</td><td>Reconstituted in sterile DI water to a final concentration of 100&amp;nbsp;&amp;micro;g&amp;#8288;/&amp;#8288;ml. Aliquot and store in -20 &amp;deg;C until use.</td></tr><tr><td>Human Mesenchymal Stem Cells (hMSC)</td><td>Cambrex</td><td></td><td>This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.</td></tr><tr><td>Rat PC12 Pheochromocytoma Cells&amp;nbsp;</td><td>ATCC</td><td></td><td>This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.</td></tr><tr><td>Grade 93 filter paper</td><td>Whatman</td><td>Z699675</td><td>This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH&amp;nbsp;7.</td></tr><tr><td>Swing bucket centrifuge</td><td>Eppendorf</td><td>5810R</td><td>To be used during the purification of chitosan using 1,200 x g speed.</td></tr><tr><td>Motor with mandrel rotating at constant speed</td><td>Rhymebus</td><td>RM5E</td><td>The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.</td></tr><tr><td>Phosphate buffered saline</td><td>FirstBase</td><td></td><td>Sterilize through filtration (0.2 &amp;micro;m filter) and autoclave.&amp;nbsp;</td></tr><tr><td>10-mm diameter Tissue Culture Polystyrene Dish (TCPS)</td><td>Greiner</td><td></td><td>The TCPS dish is used for casting of pullulan frame.&amp;nbsp;</td></tr><tr><td>Human VEGF ELISA kit</td><td>R&amp;amp;D systems</td><td>DVE00</td><td>The ELISA kit is used for detection of VEGF in the release medium.</td></tr><tr><td>Human NGF ELISA kit</td><td>R&amp;amp;D systems</td><td>DY256</td><td>The ELISA kit is used for detection of NGF in the release medium.</td></tr><tr><td>Plastic Coated Adhesive Tape</td><td>Bel-Art</td><td>9040336</td><td>The adhesive tape is used to securely stick the alligator clip to the rotating mandrel</td></tr></tbody></table>

composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

In this article, we sought to create composite scaffolds with IPC fibers for the sustained release of various biomolecules. Characteristics of the biomolecules used in this study are found in Table 1. IPC fibers were first embedded into a hydrophilic PD hydrogel to create a PD-IPC composite scaffold (Figure 1B). Model molecule BSA was first tested to determine the feasibility of using a composite scaffold for controlled biomolecule release. BSA was incorporated into PD-IPC scaffolds with...

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Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

composite-scaffolds-interfacial-polyelectrolyte-fibers-for-temporally

Research

JoVE Journal

Bioengineering

8.3K Views.  National University of Singapore. Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Video: Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

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 ...

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.

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

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

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

Genetics

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

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 ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Biology

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

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